Novel Fusicoccins R and S, and the fusicoccin S aglycon (phomopsiol) from Phomopsis amygdali niigata 2-A, and their seed germination-stimulating activity in the presence of abscisic acid

Our search for new 3-hydroxyfusicoccins structurally related to cotylenin A from a culture of Phomopsis amygdali Niigata 2-A resulted in the isolation of novel 3-hydroxy fusicoccins, called fusicoccins R and S, and the fusicoccin S aglycon, called phomopsiol, together with known 3alpha-hydroxyfusicoccin J. The structure of phomopsiol was identified as that of O-demethyl-3-epicotylenol based on spectroscopic evidence. The structures of fusicoccins R and S were also determined to be those of 3'-deacetyl-3alpha-hydroxyfusicoccin A and 3beta-hydroxy-3-epifusicoccin H. The lettuce seed germination-stimulating activity of fusicoccins R and S, phomopsiol and 3alpha-hydroxyfusicoccin J was examined in the presence of ABA; fusicoccin R and 3alpha-hydroxyfusicoccin J were highly active, while fusicoccin S and phomopsiol were inactive. The possible biosynthetic relationships among these novel fusicoccins having a 3alpha- or 3beta-hydroxy group in their diterpene moiety are briefly discussed.     

Fusicoccins P and Q,and 3-Epifusicoccins H and Q,New Polar Fusicoccins from Isolate Niigata 2-A of a Peach Fusicoccum Canker Fungus

Our search for new polar fusicoccins biosynthetically related to fusicoccin A from the culture filtrate of isolate Niigata 2-A of a peach Fusicoccum canker fungus resulted in the isolation of new fusicoccins named fusicoccins P and Q, and 3-epifusicoccins H and Q, together with 3′-deacetylfusicoccin A and 16-O-demethyl-3-epifusicoccin J. The structures of fusicoccins P and Q, and of 3-epifusicoccin Q were determined to be those of deisopentenylfusicoccin J, 12α-hydroxyfusicoccin H and 12α-hydroxy-3-epifusicoccin H, respectively, by NMR spectrometry and chemical derivation from known fusicoccins. 3-Epifusicoccin H was identified by comparing its 400 MHz NMR spectra with those of fusicoccin H. The lettuce seed germination-stimulating activity of these new fusicoccins was examined in the presence of ABA: fusicoccin P was highly active, while 3-epifusicoccins H and Q were slightly active, and fusicoccins H and Q were almost inactive. Possible biosynthetic pathways incorporating these new fusicoccins and 3-epifusicoccins from geranylgeranyl diphosphate to 3′-deacetlyfusicoccin A and 16-O-demethyl-3-epifusicoccin J are discussed.     

Fusicoccins P and Q,and 3-epifusicoccins H and Q,new polar fusicoccins from isolate Niigata 2-A of a peach Fusicoccum canker fungus

Our search for new polar fusicoccins biosynthetically related to fusicoccin A from the culture filtrate of isolate Niigata 2-A of a peach Fusicoccum canker fungus resulted in the isolation of new fusicoccins named fusicoccins P and Q, and 3-epifusicoccins H and Q, together with 3'-deacetylfusicoccin A and 16-O-demethyl-3-epifusicoccin J. The structures of fusicoccins P and Q, and of 3-epifusicoccin Q were determined to be those of deisopentenylfusicoccin J, 12alpha-hydroxyfusicoccin H and 12alpha-hydroxy-3-epifusicoccin H, respectively, by NMR spectrometry and chemical derivation from known fusicoccins. 3-Epifusicoccin H was identified by comparing its 400 MHz NMR spectra with those of fusicoccin H. The lettuce seed germination-stimulating activity of these new fusicoccins was examined in the presence of ABA: fusicoccin P was highly active, while 3-epifusicoccins H and Q were slightly active, and fusicoccins H and Q were almost inactive. Possible biosynthetic pathways incorporating these new fusicoccins and 3-epifusicoccins from geranylgeranyl diphosphate to 3'-deacetlyfusicoccin A and 16-O-demethyl-3-epifusicoccin J are discussed.     

Comparison of the biological activity of fusicoccin in higher plants with its binding to plasma membranes

The concentration dependences of the binding of fusicoccins (FCs) A, B, C, D, J and H to plasma membranes isolated from maize (Zea mays L.) roots have been studied in parallel with the effects of these compounds on elongation and ⁸⁶Rb transport in detached maize roots. The dissociation constants obtained showed a good correlation between the affinity of the FCs for the plasmalemma and their biological activity. However, the range of physiologically active FC concentrations proved to be about two orders of magnitude higher than that calculated from the dissociation constants. It was also shown that Vicia faba L. mesophyll protoplasts, unlike isolated plasma membranes, have two FC-binding sites, one with a K _D similar to that of the isolated plasmalemma while the other has a substantially higher K _D , apparently corresponding to the physiologically active state of the FC-binding proteins.Abbreviation FC fusicoccin     

Control of the production of individual fusicoccins at different dissolved oxygen concentrations

The regulatory effect of different concentrations of dissolved oxygen on the production of fusicoccins by the fungus Fusicoccum amygdali Del. was studied. The maximum output of total fusicoccins was obtained by using a profiled dissolved oxygen tension (DOT) regime, in which the DOT was maintained at 15–20% during the biomass growth phase and at 5–8% during the fusicoccins production phase. In comparison with the profiled regime, the maintenance of DOT at 15–20% during the whole fermentation shortened the fusicoccins production phase. The fermentation performance at a low DOT (5–8%) inhibited both the accumulation of biomass and the production of fusicoccins. At high DOT (40–50%), an accelerated accumulation of the biomass with an expressed autolysis of mycelia took place, and the production of fusicoccins was lowered. The qualitative composition of individual fusicoccins varied substantially at different DOTs. Fusicoccins, A, C, D, J, H, 16-O-demethyl-J, detretpentenylfusicoccin and some minor fusicoccin metabolites were found in the fermentation broth using the method of liquid secondary ion mass spectrometry. It was established that the profiled DOT regime (15–20% to 5–8%) provided both the maximum concentration of fusicoccins and an enhanced accumulation of the main metabolite – fusicoccin A (FC A). The performance of the fermentation at a DOT of 15–20% decreased the content of FC A by 2–6% in comparison with the profiled DOT regime, and increased the content of fusicoccin C to 14–20% of the total fusicoccins. Fermentation at DOT of 5–8% was characterized by the highest content of the precursors of FC A, the less oxidized fusicoccins H and J, the contents of which were in range 7–12% and 16–17% of total fusicoccins, respectively.     

A comparison of the seed dormancy characteristics of Spartina patens and Spartina alterniflora (Poaceae)

Seed dormancy characteristics of Spartina alterniflora were delineated previously by Plyler and Carrick (American Journal of Botany, vol. 80, pp. 752–756, 1993). This study was undertaken in order to determine whether or not the dormancy characteristics of S. patens are similar. As in the previous study, the site of a dormancy mechanism was determined by assessing the germinability of surgically altered dormant seeds. Likewise, the effects of three growth-regulating substances (abscisic acid, fusicoccin, and gibberellic acid), prechilling, and light were studied by assessing the germinability of appropriately treated dormant seeds. Surgical injury to the endosperm, and to a lesser extent the scutellum, produced significant germination in S. patens, whereas only injury to the scutellum produced germination in S. alterniflora. Exogenously applied abscisic acid was highly effective in maintaining dormancy in injured seeds of both species. Gibberellic acid and fusicoccin were ineffective in promoting germination in S. patens, but fusicoccin was highly effective in breaking dormancy in 5. alterniflora. Prechilling was effective in breaking dormancy in S. patens but not in S. alterniflora. In S. patens, treatments that broke dormancy were only successful when seeds were exposed to light during the germination period. It is concluded that the dissimilarities in the dormancy mechanisms may be manifestations of the different environmental adaptations these closely related species display.     

Costusoside-I and costusoside-J,two new furostanol saponins from the seeds of Costus speciosus

The structure of costusoside I and costusoside J have been established as 3-O-{β-d-glucopyranosyl (1 → 2)-α-l-rhamnopyranosyl (1 → 2) [α-l-rhamnopyranosyl (1 → 4)]-β-d-glucopyranosyl}-26-O-(β-d-glucopyranosyl)-22α-methoxy 25 R)-furost-5-en-3β, 26-diol and its 22-hydroxy compound respectively, isolated fron the seeds of Costus speciosus.     

Studies on the Transformation of Steroids by Microorganisms

Microbiological conversions of Reichstein’s substance S (4-pregnene-17α,21-diol-3,20-dione) and hydrocortisone to their corresponding 20β-hydroxy derivatives were achieved by means of numerous strains of Streptomyces such as S. diastaticus (ATCC 3315), S. flavogriseus (H-4449), S. albus (ATCC 3351) etc., and it became apparent that 20-carbonyl reduction is the, wide-spread type of transformation in the Streptomyces species.

Moreover, several interesting strains having both l-dehydrogenating and 20-carbonyl reducing activities were detected. For instance, when Reichstcin’s substance S was used as substrate 1,4-pregnadiene-17α,21-diol-3,20-dione, 4-pregnene-17α,20β,21-triol-3-one and 1,4-pregnadiene-17α,20β,21-triol-3-one were isolated simultaneously using S. flaveolus (D-551), s. roseochromogenes (O-36) etc. These strains also exhibited similar transformation patterns in the use of hydrocortisone.     

Studies on the Microbiological Transformation of Steroids

The microbiological reduction of the 20-carbonyl group of steroids has been investigated. Candida pulcherrima IFO 0964 and Sporotrichum gougeroti IFO 5982 converted the following substrates into the corresponding 20β-hydroxy derivatives (yields of the products are indicated in parentheses): Reichstein’s Compound S (60~70%) and 17α,21-dihydroxypregna-l,4-diene- 3,20-dione (40~80%). Rhodotorula glutinis IFO 0395 converted the following substrates into the corresponding 20α-hydroxy derivatives: Reichstein’s Compound S (65%), 17 α,21-dihydroxy- pregna-l,4-diene-3,20-dione (80%), llβ,l7α-dihydroxypregn-4-ene-3,20-dione (45%) and 17α, 19,21 -trihydroxypregn-4-ene-3,20-dione (10%).     

(+)-Menthol and its hydroxy derivatives,novel fungal monoterpenols from the fusicoccin-producing fungi,Phomopsis amygdali F6a and Niigata 2

In our search for new fusicoccins of unique diterpene glucosides from Phomopsis amygdali, we found that a fragrant substance was formed in the early stage of fusicoccin fermentation. This fragrant constituent was isolated and identified as (+)-menthol, which is a novel fungal metabolite as the enantiomer of well-known peppermint (-)-menthol. (+)-7-Hydroxymenthol and new (+)-(6S)-hydroxymenthol were also isolated and identified as fungal metabolites. In addition, p-menthanetriol, which has been reported as the first fungal monoterpene from the fungus, was also isolated. The possible biosynthetic relationship of these metabolites is discussed.     

Synthesis of (S)- and (R)-3-hydroxy acids using cells or purified (S)-3-hydroxycarboxylate oxidoreductase from Clostridium tyrobutyricum and the NADP(H) regeneration system of Clostridium thermoaceticum

A purified and partially characterized novel NADP⁺-dependent oxidoreductase from Clostridium tyrobutyricum DSM 1460 was applied for the preparative reduction of several 3-oxo acids to (S)-3-hydroxy acids. (R)-3-Hydroxybutyrate was prepared by the same enzyme selectively dehydrogenating the S enantiomer of (R,S)-3-hydroxybutyrate. The enantiomeric purity of the (S)- and (R)-3-hydroxy acids was at least 98% enantiomeric excess (e.e). NADPH for reductions and NADP⁺ for dehydrogenations were regenerated by applying artificial mediator accepting pyridine nucleotide oxidoreductases in the form of a crude extract of C. thermoaceticum cells. For NADP⁺ regeneration also the system 2-oxoglutarate/glutamate dehydrogenase was used for comparison. Instead of the purified (S)-3-hydroxycarboxylate oxidoreductase, resting cells of C. tyrobutyricum were also applied for reductions and dehydrogenations with substrate concentrations of 200–400 mM leading to products with e.e. values above 96%.Dedicated to Prof. H.G. Floss on the occasion of his 60th birthday     

Six New Polyhydroxysteroidal Glycosides,Anthenosides S1 – S6, from the Starfish Anthenea sibogae

Six new polyhydroxysteroidal glycosides, anthenosides S1  –  S6 ( 1  –  6 ), along with a mixture of two previously known related glycosides, 7 and 8 , were isolated from the methanolic extract of the starfish Anthenea sibogae. The structures of 1  –  6 were established by NMR and HR‐ESI‐MS techniques as well as by chemical transformations. All new compounds have a 5α‐cholest‐8(14)‐ene‐3α,6β,7β,16α‐tetrahydroxysteroidal nucleus and differ from majority of starfish glycosides in positions of carbohydrate moieties at C(7) and C(16) ( 1  –  4 , 6 ) or only at C(16) ( 5 ). The 4‐O‐methyl‐β‐d ‐glucopyranose residue ( 2 ) and Δ²⁴‐cholestane side chain ( 3 ) have not been found earlier in the starfish steroidal glycosides. The mixture of 7 and 8 slightly inhibited the proliferation of human breast cancer T‐47D cells and decreased the colony size in the colony formation assay.     

Microbial Production of Plant Gibberellins and Related Compounds from ent-Kaurene Derivatives in Gibberella fujikuroi

ent-15α-Hydroxykaurenoic acid (8) was synthesized and fed to a mycelium suspension of Gibberella fujikuroi in the presence of 1-n-decylimidazole, a gibberellin biosynthesis inhibitor. The metabolites included 15β-hydroxy GA₂₄, GA₄₅ (GA of Pyrus communis), 15β-hydroxy GA₁₅ and 15β-hydroxy GA₂₅. Microbial production of 12α-hydroxy GAs from ent-12β-hydroxykaurene is also described.     

Alleviation of Salinity-Induced Dormancy in Perennial Grasses

All seeds of Aeluropus lagopoides and Urochondra setulosa germinated under non-saline conditions except for Sporobolus ioclados which showed only 40 % germination. Increase in salinity substantially inhibited germination and few seeds germinated at 400 mM NaCl. Germination at 200 mM NaCl was alleviated in U. setulosa by the application of gibberellic acid and fusicoccin, in A. lagopoides by thiourea, betaine, kinetin, fusicoccin and ethephon, and in S. ioclados by gibberellin and ethephon. High salinity (400 mM NaCl) induced germination inhibition was alleviated by proline, kinetin, fusicoccin and ethephon only in A. lagopoides.     

Biliary bile acids in birds of the Cotingidae family: taurine-conjugated (24R,25R)-3α,7α,24-trihydroxy-5β-cholestan-27-oic acid and two epimers (25R and 25S) of 3α,7α-dihydroxy-5β-cholestan-27-oic acid

Three C(27) bile acids were found to be major biliary bile acids in the capuchinbird (Perissocephalus tricolor) and bare-throated bellbird (Procnias nudicollis), both members of the Cotingidae family of the order Passeriformes. The individual bile acids were isolated by preparative RP-HPLC, and their structures were established by RP-HPLC, LC/ESI-MS/MS and NMR as well as by a comparison of their chromatographic properties with those of authentic reference standards of their 12α-hydroxy derivatives. The most abundant bile acid present in the capuchinbird bile was the taurine conjugate of C(27) (24R,25R)-3α,7α,24-trihydroxy-5β-cholestan-27-oic acid, a diastereomer not previously identified as a natural bile acid. The four diastereomers of taurine-conjugated (24ξ,25ξ)-3α,7α,24-trihydroxy-5β-cholestan-27-oic acid could be distinguished by NMR and were resolved by RP-HPLC. The RRT of the diastereomers (with taurocholic acid as 1.0) were found to be increased in the following order: (24R,25R)<(24S,25R)<(24S,25S)<(24R,25S). Two epimers (25R and 25S) of C(27) 3α,7α-dihydroxy-5β-cholestan-27-oic acid were also present (as the taurine conjugates) in both bird species. Epimers of the two compounds could be distinguished by their NMR spectra and resolved by RP-HPLC with the (25S)-epimer eluting before the (25R)-epimer. Characterization of the taurine-conjugated (24R,25R)-3α,7α,24-trihydroxy-5β-cholestan-27-oic acid and two epimers (25R and 25S) of 3α,7α-dihydroxy-5β-cholestan-27-oic acid should facilitate their detection in peroxisomal disease and inborn errors of bile acid biosynthesis.     

The synthesis of cholestane and furostan saponin analogues and the determination of sapogenin's absolute configuration at C-22

A facile and efficient way for the synthesis of cholestane and furostan saponin analogues was established and adopted for the first time. Following this strategy, starting from diosgenin, three novel cholestane saponin analogues: (22S,25R)-3β,22,26-trihydroxy-cholest-5-ene-16-one 22-O-[O-α-l-rhamnopyranosyl-(1 → 2)-β-d-glucopyranoside] 11, (25R)-3β,16β,26-trihydroxy-cholest-5-ene-22-one 16-O-[O-α-l-rhamnopyranosyl-(1 → 2)-α-d-glucopyranoside] 14 and (25R)-3β,16β,26-trihydroxy-cholest-5-ene-22-one 16-O-[O-α-l-rhamnopyranosyl-(1 → 2)-β-d-glucopyranoside] 17, three novel furostan saponin analogues: (22S,25R)-furost-5-ene-3β,22,26-triol 22-O-(α-d-glucopyranoside) 23, (22R,25R)-furost-5-ene-3β,22,26-triol 22-O-(α-d-glucopyranoside) 24 and (22S,25R)-furost-5-ene-3β,22,26-triol 22-O-[O-α-l-rhamnopyranosyl-(1 → 2)-α-d-glucopyranoside] 26, were synthesized ultimately. The structures of all the synthesized analogues were confirmed by spectroscopic methods. The S-chirality at C-22 of cholestane was confirmed by Mosher's method. The absolute configuration at C-22 of furostan saponin analogues was distinguished by conformational analysis combined with the NMR spectroscopy. The cytotoxicities of the synthetic analogues toward four types of tumor cells were shown also.     

Evidence that the partial resistance of the Arabidopsis 5-2 mutant to fusicoccin depends on a decreased capacity of the H+ pump to respond to activating factors

Measurements of H⁺ extrusion activity K⁺ influx, and Es bm in 3-d-old seedlings of the 5-2 mutant of Arabidopsis thaliana (which is partially insensitive to fusicoccin) showed the following, (i) The reduced response of 5-2 to fusicoccin (FC) does not depend on the penetration of FC to its site of action, or on decreased affinity of the FC receptor, (ii) The reduced response of H⁺ and K⁺ transport to FC does not depend on an impairment of the K⁺ absorption system, (iii) The mutation can influence the H⁺ extrusion system independently of the presence of FC. In the presence of factors other than FC known to activate the plasma membrane H⁺-ATPase (e.g. a cytosol-acidifying treatment), the response in 5-2 is about 50% lower than in wt. (iv) When both genotypes grow in optimal conditions, the rate of fresh weight increase and stem elongation is higher in wt than 5-2. These data indicate that the 5-2 mutation affects some intrinsic component of the H⁺-extrusion machinery, the limiting effect of which becomes considerable when either the physiological or the experimental conditions induce a high level of proton pump activity. An alteration either of the ATPase itself or of a factor controlling its activity is compatible with our observations.     

Ferulic acid is a putative surrender signal to stimulate programmed cell death in grapevines after infection with Neofusicoccum parvum

An apoplectic breakdown from grapevine trunk diseases (GTDs) has become a serious challenge to viticulture as a consequence of drought stress. We hypothesize that fungal aggressiveness is controlled by a chemical communication between the host and colonizing fungus. We introduce the new concept of a ‘plant surrender signal’ accumulating in host plants under stress and facilitating the aggressive behaviour of the strain Neofusicoccum parvum (Bt-67) causing Botryosphaeriaceae-related dieback in grapevines. Using a cell-based experimental system (Vitis cells) and bioactivity-guided fractionation, we identify trans-ferulic acid, a monolignol precursor, as a ‘surrender signal’. We show that this signal specifically activates the secretion of the fungal phytotoxin fusicoccin A aglycone. We show further that this phytotoxin, mediated by 14-3-3 proteins, activates programmed cell death in Vitis cells. We arrive at a model showing a chemical communication facilitating fusicoccin A secretion that drives necrotrophic behaviour during Botryosphaeriaceae–Vitis interaction through trans-ferulic acid. We thus hypothesize that channelling the phenylpropanoid pathway from this lignin precursor to the trans-resveratrol phytoalexin could be a target for future therapy.     

Factors affecting the binding between fusicoccin and plasma membranes from maize roots

Plasma membranes have been purified from roots of maize (Zea mays L.) using a two-phase aqueous polymer system, dextran-polyethylene glycol. The plant material was homogenized in the presence of a mixture of natural protease inhibitors from potato (Solanum tuberosum L.); these inhibitors have been shown to be more effective than phenylmethylsulfonyl fluoride in suppressing the endogenous proteases in maize roots. Inhibition of proteolysis in the homogenization medium markedly increased (about tenfold) the number of lowaffinity binding sites for fusicoccin (FC). In addition, storage of plasma membranes at −20° C decreased both the number of the low-affinity sites and their dissociation constant (K_D); this effect was in all probability caused by lipid peroxidation. The presence of EDTA throughout isolation and storage of the plasma membranes stabilized the parameters of FC binding to the membranes. The kinetics of binding of [³H]dihydroFC and the competition between [³H]dihydroFC and FCs A, C, J, and H were determined for the low-affinity sites. It was found that (i) the rate constant of association between FC and the low-affinity binding sites is about two orders of magnitude lower than that for the high-affinity sites; (ii) different FCs can be arranged in the order of decreasing avidity for the low-affinity FCbinding site: FC A>FC C>FC J>FC H. The authors are indebted to Dr. L.M. Krasnopolskaya (Institute of Agricultural Biotechnology, Moscow, Russia) for fusicoccins A, C, J, and H, and to Dr. A.V. Galkin (Institute of Agricultural Biotechnology, Moscow, Russia) for valuable comments and ren dering the paper into English.     

Applied Spatial Data Analysis with R. by BIVAND, R. S., PEBESMA, E. J., and GOMEZ-RUBIO, V.

Applied Spatial Data Analysis with R (R. S. Bivand, E. J. Pebesma, and V. Gomez‐Rubio) Jay M. Ver Hoef Bayesian Disease Mapping: Hierarchical Modeling in Spatial Epidemiology (A. B. Lawson) J. Law Disease Surveillance: A Public Health Informatics Approach (J. S. Lombardo and D. L. Buckeridge, editors) Iris Pigeot Survival Analysis for Epidemiologic and Medical Research (S. Selvin) M. G. Valsecchi Survival and Event History Analysis: A Process Point of View (O. O. Aalen, O. Borgan, and H. K. Gjessing) Patricia Grambsch Nonlinear Dimensionality Reduction (J. A. Lee and M. Verleysen) Haonan Wang Model Selection and Model Averaging (G. Claeskens and N. L. Hjort) Thomas M. Loughin Meta‐Analysis of Binary Data Using Profile Likelihood (D. Böhning, R. Kuhnert, and S. Rattanasiri) Eloise Kaizar Wavelet Methods in Statistics with R (G. P. Nason) Jeffrey S. Morris Adaptive Design Theory and Implementation Using SAS and R (M. Chang) Feifang Hu Ecological Models and Data in R (B. M. Bolker) Rachel M. Fewster Applied Multiway Data Analysis (P. M. Kroonenberg) Renato Coppi Brief Reports by the Editor Sampling of Populations: Methods and Applications, 4th edition (P. S. Levy and S. Lemeshow) Applied Survival Analysis: Regression Modeling of Time‐to‐Event Data, 2nd edition (D. W. Hosmer, S. Lemeshow, and S. May) SAS for Data Analysis: Intermediate Statistical Methods (M. G. Marasinghe and W. J. Kennedy) Advances in Mathematical and Statistical Modeling (B. C. Arnold, N. Balakrishnan, J. M. Sarabia, and R. Mínguez, editors) An Introduction to Generalized Linear Models, 3rd edition (A. J. Dobson and A. G. Barnett) Design and Analysis of Bioavailability and Bioequivalence Studies, 3rd edition (S.‐C. Chow and J.‐P. Liu)