Cyclohelminthols Y1-Y4 Metabolites Possessing Two Spirocyclopropanes in Their Structure.

Cyclohelminthols Y1-Y4 (1-4) were isolated from the culture broth of Helminthosporium velutinum yone96. These compounds are diastereomers to each other featuring 3-azabicyclo[3.1.0]hexane-6-spirocyclopentane linked with a cyclopentanespirocyclopropane framework. Their planar structures were established via the comparison of their spectra with the simpler analogue cyclohelminthol X as well as analysis of their HMBC spectra. Although the proton-deficient core frameworks of 1-4 prevented us from obtaining configurational information via conventional NMR analysis, their total structures involving the relative and absolute configurations were established using density functional theory (DFT)-based molecular modeling calculations. The present study demonstrates the effectiveness of the comparison between the theoretical and experimental δ13C values for stereochemical analysis by focusing on the carbons that show relatively large δ13C deviations among the isomers. The G-ring of these molecules most likely originates from the cyclopropanation of the C6C7 double bond with the carbene equivalent 6 derived from cyclohelminthol IV (7), which was isolated from the same producer fungus. Preliminary biological experiments revealed the potent cytotoxicity of the (6 S)-isomers against COLO201 cells, whereas the (6 R)-isomers exhibited weak activity. The antifungal assay with Cochiobolus miyabeanus showed a slightly different profile.

Characterization of a novel broad-spectrum antifungal protein from virus-infected Helminthosporium (Cochliobolus) victoriae.

A broad-spectrum anti-fungal protein of approximately 10 kDa, designated victoriocin, was purified from culture filtrates of a virus-infected isolate of the plant-pathogenic fungus Helminthosporium victoriae (teleomorph: Cochliobolus victoriae) by a multistep procedure involving ultrafiltration and reverse-phase high-performance liquid chromatography (RP-HPLC). Amino acid sequences, obtained by automated Edman degradation sequencing of RP-HPLC-purified victoriocin-derived peptides, were used to design primers for degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR) amplification from H. victoriae DNA and cDNA templates. An open reading frame coding for a victoriocin precursor of 183 amino acids with calculated molecular mass of approximately 20 kDa was amplified by PCR from H. victoriae genomic DNA but not from the control fungus Penicillium chrysogenum. Southern hybridization analysis confirmed the presence of the victoriocin gene in all H. victoriae strains tested. Sequence analysis indicated that victoriocin has a sequence motif similar to that found in scorpion short toxin/charybdotoxin and a consensus sequence similar to that found in defensins. Victoriocin, like some other antifungal proteins, including the totivirus-encoded killer proteins, is predicted to be expressed in vivo as a preprotoxin precursor consisting of a hydrophobic N-terminal secretion signal followed by a pro-region and terminating in a classical Kex2p endopeptidase cleavage site that generates the N terminus of the mature victoriocin. A putative cell wall protein of approximately 30 kDa (P30) co-purified with victoriocin from cultural filtrates. The potential role of P30 in the antifungal activity of H. victoriae culture filtrates is discussed.

Overexpression of the victoriocin gene in Helminthosporium (Cochliobolus) victoriae enhances the antifungal activity of culture filtrates.

We have previously reported the isolation and characterization of the broad-spectrum antifungal protein, victoriocin, from culture filtrates of a virus-infected isolate of the plant-pathogenic fungus Helminthosporium (teleomorph: Cochliobolus) victoriae. We predicted that the 10-kDa mature victoriocin is derived in vivo from a preprotoxin precursor that is processed by a signal peptidase and kexin-like endopeptidase. We also presented evidence that the victoriocin precursor is encoded by a host gene, designated the victoriocin (vin) gene. In the present study, an H. victoriae genomic DNA library was constructed in the cosmid vector pMLF-2, and a cosmid clone carrying the vin gene and flanking sequences was isolated and used to generate constructs for transformation of virus-free and virus-infected H. victoriae isolates with the vin gene. Culture filtrates of the virus-free vin transformants exhibited high levels of antifungal activity compared with that revealed by the nontransformed virus-free wild-type strain, which exhibited little or no antifungal activity. Moreover, transformation of the wild-type virus-infected H. victoriae strain with the vin gene resulted in still higher production of victoriocin and higher antifungal activity in the culture filtrates of the vin transformants compared with the virus-infected wild-type strain. As previously predicted, the presence in the vin transformants of the preprovictoriocin and its post-translationally generated products, the provictoriocin and the mature victoriocin, was clearly demonstrated. Processing of the victoriocin preprotoxin requires eukaryotic host factors because no processing occurred in an in vitro translation system or in bacteria. It is of interest that some of the virus-free isolates transformed with the vin gene exhibited some features of the virus-induced disease phenotype, including moderate stunting and sectoring. Present data suggests that victoriocin may play an indirect role in disease development. Taken together, these results indicate that victoriocin is the primary protein responsible for the antifungal activity in culture filtrates of virus-infected H. victoriae isolates and that virus infection upregulates the expression of victoriocin. Overproduction of victoriocin may give the slower-growing virus-infected fungal strains some competitive advantage by inhibiting the growth of other fungi.

Biomimetic enantioselective total synthesis of (-)-siccanin via the Pd-catalyzed asymmetric allylic alkylation (AAA) and sequential radical cyclizations.

(-)-Siccanin (1), a natural product possessing significant antifungal properties, was synthesized enantioselectively via a biomimetic route. This synthetic route features two sequential radical cyclizations: a Ti(III)-mediated radical cyclization of epoxyolefin 48 to construct the B-ring, and a Suarez reaction to establish the tetrahyrofuran ring. Chiral chroman moiety of siccanin was prepared based on our recent development of the Pd-catalyzed asymmetric allylic alkylation (AAA) of phenol trisubstituted allyl carbonates. Several other members of the siccanin family were also synthesized including siccanochromenes A (2), B (3), E (6), F (7), and the methyl ether of siccanochromene C (55). These studies may shed light on the biosynthesis of this novel family of compounds.

Biotransformation of beta-ketosulfides to produce chiral beta-hydroxysulfoxides.

The biotransformations of a series of substituted phenylthio-2-propanone and benzylthio-2-propanone were carried out using Helminthosporium sp. NRRL 4671, Mortierella isabellina ATCC 42613, or Rhodococcus erythropolis IGTS8. Several products gave microbial oxidation of sulfide to sulfoxide and reduction of carbonyl to secondary alcohol, producing beta-hydroxysulfoxides in medium to high enantiomeric and diastereomeric purities. Fungal biotransformations using Helminthosporium sp. and M. isabellina resulted in the opposite sulfoxide configurations of various beta-hydroxysulfoxide products.

Chronopathological aspects of disease incidence in rice (Oryza sativa L.).

Rice seedlings maintained under uncontrolled glasshouse conditions were inoculated with conidial suspensions of a fungal pathogen, Helminthosporium oryzae, at various times during the 24h. Significant increase in the percent germination and germ tube length of conidia were observed in the rice samples inoculated at 02:00 and 06:00h. The 24 h temporal variation in leaf temperature was positively correlated with variation in stomatal movements. The results indicate a 24 h rhythm in the behavior of the fungal pathogen on the host in relation to the conditions of the growing environment. In all the inoculated seedlings, the appearance of a large number of brown leaf spots was confined to the light span. Among the plants inoculated, earlier initiation of brown leaf spot appearance, maximum number of leaf spots, and highest disease severity were observed when plants were inoculated at 02:00h. There was a positive correlation between disease severity of the host and in vivo values of percent germination of conidia and germ tube length of the pathogen in plants inoculated between 02:00 and 06:00h. The findings of this study implicate that light intensity and temperature could play a predominant role in controlling disease susceptibility rhythms in plants.

Comparative molecular field analysis (CoMFA) for sulfoxidation reactions in Mortierella isabellina ATCC 42613 and Helminthosporium sp. NRRL 4671.

Previous models for mechanisms of enzymatic sulfoxidation have been somewhat limited by a lack of knowledge of the essential features of substrate-enzyme versus product-enzyme relationships. Computerized methods for modeling ligand-protein (substrate-enzyme) interactions can overcome some of these limitations. Specifically, CoMFA (comparative molecular field analysis) provided a useful general approach in which to evaluate substrate-enzyme and product-enzyme relationships. The present investigation examined the relationship between substrate and product structure in predicting enantioselective sulfoxidation reactions using CoMFA for two species of microorganisms that have been used as models for mammalian metabolism, Mortierella isabellina and Helminthosporium sp. The overall enantioselectivity observed was based on the composite stereoselectivity of sulfoxide formation, sulfone formation (from the sulfoxide), and sulfoxide reduction back to the achiral substrate (sulfide).

Levene tests of homogeneity of variance for general block and treatment designs.

This article develops a weighted least squares version of Levene's test of homogeneity of variance for a general design, available both for univariate and multivariate situations. When the design is balanced, the univariate and two common multivariate test statistics turn out to be proportional to the corresponding ordinary least squares test statistics obtained from an analysis of variance of the absolute values of the standardized mean-based residuals from the original analysis of the data. The constant of proportionality is simply a design-dependent multiplier (which does not necessarily tend to unity). Explicit results are presented for randomized block and Latin square designs and are illustrated for factorial treatment designs and split-plot experiments. The distribution of the univariate test statistic is close to a standard F-distribution, although it can be slightly underdispersed. For a complex design, the test assesses homogeneity of variance across blocks, treatments, or treatment factors and offers an objective interpretation of residual plots.

Enantioselective synthesis and pharmacological evaluation of a new type of verapamil analog with hypotensive and calcium antagonist activities.

PURPOSE: The syntheses and evaluation for cardiovascular activity in the rat of both enantiomers of a verapamil analog in which the cyano group has been replaced by hydroxyl. METHODS: (+)- and (-)-alpha-[3-[[2-(3,4-Dimethoxyphenyl)ethyl]methylamino]propyl]- 3,4-dimethoxy-alpha-(1-methyl ethyl)benzyl alcohol were prepared from chiral sulfoxides produced by microbial biotransformations using Mortierella isabellina ATCC 42613 or Helminthsporium species NRRL 4671, and were examined for hypotensive and calcium antagonist activity using anaesthetized normotensive rats and isolated rat aorta and atria. RESULTS: The analogs showed a pharmacological profile similar to that exhibited by verapamil, possessing a remarkable hypotensive activity, accompanied by a significant bradycardia, in anaesthetized normotensive rats. In vitro, these analogs displayed clear inhibitory effects: in isolated rat aorta they inhibited, in a concentration-dependent fashion, the contractions and 45Ca2+ uptake induced by norepinephrine and high KCl, and in isolated rat atria the analogs considerably decreased the rate of contraction (negative chronotropic effects). No significant differences between the quantitative cardiovascular effects produced by the two enantiomers of the verapamil analogs were observed. CONCLUSIONS: The results suggest that, like that of verapamil, the cardiovascular activity exhibited by the new compounds seems to be due, at least in part, to a blockage of transmembrane calcium channels present in vascular smooth muscle cells.

A fatty acid synthase gene in Cochliobolus carbonum required for production of HC-toxin, cyclo(D-prolyl-L-alanyl-D-alanyl-L-2-amino-9, 10-epoxi-8-oxodecanoyl).

The fungal maize pathogen Cochliobolus carbonum produces a phytotoxic and cytostatic cyclic peptide, HC-toxin, of structure cyclo(D-prolyl-L-alanyl-D-alanyl-L-Aeo), in which Aeo stands for 2-amino-9,10-epoxi-8-oxodecanoic acid. Here we report the isolation of a gene, TOXC, that is present only in HC-toxin-producing (Tox2+) fungal strains. TOXC is present in most Tox2+ strains in three functional copies, all of which are on the same chromosome as the gene encoding HC-toxin synthetase. When all copies of TOXC are mutated by targeted gene disruption, the fungus grows and sporulates normally in vitro but no longer makes HC-toxin and is not pathogenic, indicating that TOXC has a specific role in HC-toxin production and hence virulence. The TOXC mRNA is 6.5 kb and the predicted product has 2,080 amino acids and a molecular weight of 233,000. The primary amino acid sequence is highly similar (45 to 47% identity) to the beta subunit of fatty acid synthase from several lower eukaryotes, and contains, in the same order as in other beta subunits, domains predicted to encode acetyl transferase, enoyl reductase, dehydratase, and malonyl-palmityl transferase. The most plausible function of TOXC is to contribute to the synthesis of the decanoic acid backbone of Aeo.

A putative cyclic peptide efflux pump encoded by the TOXA gene of the plant-pathogenic fungus Cochliobolus carbonum.

Race 1 isolates of Cochliobolus carbonum are pathogenic on certain maize lines due to production of a host-selective cyclic tetrapeptide, HC-toxin. Flanking HTS1, which encodes the central enzyme in HC-toxin biosynthesis, a gene was identified and named TOXA. Like HTS1, TOXA occurred only in isolates of the fungus that make HC-toxin and was present as two linked copies in most toxin-producing isolates. HTS1 and TOXA were transcribed in the opposite orientation and their transcriptional start sites were 386 bp apart. The predicted product of TOXA was a 58 kDa hydrophobic protein with 10-13 membrane-spanning regions. The sequence was highly similar to several members of the major facilitator superfamily that confer resistance to tetracycline, methylenomycin, and other antibiotics. Although it was possible to mutate one copy or the other of TOXA by targeted gene disruption, numerous attempts to disrupt both copies in a single strain were unsuccessful, suggesting that TOXA is an essential gene in strains that synthesize HC-toxin. On the basis of its presence only in HC-toxin-producing strains, its proximity to HTS1 and its predicted amino acid sequence, we propose that TOXA encodes an HC-toxin efflux pump which contributes to self-protection against HC-toxin and/or the secretion of HC-toxin into the extracellular milieu.

Biotransformation of organic sulfides--IV. Formation of chiral benzyl alkyl and phenyl alkyl sulfoxides by Helminthosporium species NRRL 4671.

The fungus Helminthosporium species NRRL 4671 has been used for the biotransformation of a series of phenyl alkyl sulfides with alkyl groups ranging from methyl to n-hexyl, and benzyl alkyl sulfides with alkyl groups from methyl to n-nonyl. Several 2-phenylethyl and 3-phenylpropyl sulfides have also been examined as substrates, together with cyclohexyl methyl sulfide and 1- and 2-naphthyl methyl sulfides. For the majority of substrates, sulfoxide formation occurred in moderate yield and with predominant (S) chirality at sulfur; lesser amounts of sulfone product were also obtained in some cases. The data so obtained have been used to define the preparatively useful limits of S-oxidation of phenyl alkyl sulfides and benzyl alkyl sulfides by biotransformation using Helminthosporium.

Fungal biotransformation of organophosphines.

1. The biotransformations of triphenyl phosphite and of triphenyl-, tri-n.-butyl-, diethylphenyl-, and ethylmethylphenyl-phosphines by the fungi Mortierella isabellina ATCC 42613, Helminthosporium species NRRL 4671, and Aspergillus foetidus ATCC 10254, have been examined. 2. The triaryl and tri-n.-butyl substrates underwent oxidation at phosphorus in low yield, a process attributed largely to auto-oxidation under the experimental conditions used. 3. The alkyl aryl phosphines were enzymically oxidized at phosphorus, but concurrent auto-oxidation also occurred. 4. Incubation of ethylmethylphenyl phosphine with M. isabellina gave the corresponding S(-) phosphine oxide in a 92% yield with an enantiomeric purity of 13%.

Mycotoxin formation in moist 2-row and 6-row barley during granary storage.

Eleven-kilogram parcels of 2-row and 6-row barley initially at 18% moisture content were implanted in dry bulk oats in a farm granary in Manitoba for 60 weeks between August 1983 and October 1984. Temperature, moisture content, O2 and CO2 levels, fat acidity values, seed germination, microfloral incidence and abundance and the presence of major mycotoxins (aflatoxins, sterigmatocystin, ochratoxin A, citrinin, penicillinic acid, patulin) were monitored. Ochratoxin A reached maximum levels of 0.97 ppm by week 24 in the 6-row barley, and 0.05 ppm by week 28 in the 2-row; no other mycotoxins were detected. The effect of cultivar type was significant (P less than 0.01) with greater effects in the 6-row barley for the following parameters: fat acidity value, germination, incidence of Alternaria alternata, Penicillium spp. and Helminthosporium sativum, total fungal propagule count and ochratoxin A levels. The effect of time was significant (P less than 0.05) for all variables except oxygen, carbon dioxide, Aspergillus versicolor, and total fungal propagule count. The interaction between cultivar and time was significant (P less than 0.01) for Alternaria and Helminthosporium only.

Microbial transformations of pergolide to pergolide sulfoxide and pergolide sulfone.

Fifty-eight microorganisms were investigated for their ability to effect the biotransformation of the ergoline alkaloid pergolide. A majority of these organisms formed pergolide sulfoxide, and a Helminthosporium species was investigated in greater detail since it yielded significant amounts of pergolide sulfoxide. A preparative-scale transformation afforded material which was identified as the sulfoxide based on melting point, spectral, and chromatographic comparison with authentic material as well as its conversion to pergolide by reduction with triphenylphosphine. An analytical high-performance liquid chromatographic determination of the enzymatic versus spontaneous air-oxidation of pergolide in growing cultures and controls showed negligible air-oxidation and an approximately 40% enzymatic conversion of pergolide to the sulfoxide. Several organisms, including Aspergillus alliaceus formed a second metabolite, pergolide sulfone, which was identified on the basis of co-chromatographic data.

Convenient procedures for the biosynthesis, isolation, and isotope labeling of cytochalasins.

Efforts to improve small-scale yields of useful cytochalasins by fermentation resulted in selection of an enriched aflatoxin medium which increased yields by fivefold over those reported in the literature. With Helminthosporium dematoideum and Zygosporium masonii in stationary culture for 3 weeks, cytochalasins B and D were obtained in quantities approaching 700 and 500 mg/liter, respectively. It appears that the critical component in this growth medium is factors associated with whole wheat. By using these procedures, coupled with improvements in isolation, supplementation with two radioactive phenylalanine species readily produced [14C]- or [3H]cytochalasin B. Oxidation of carrier-free radioactive cytochalasin B to cytochasasin A readily provided this labeled congener as well. The isotopic ocnversion of precursor to crystalline products that met analytical criteria ranged from 2 to 4% of the administered radioactivity.

Amino acid content in relation to growth of Helminthosporium maydis Nishikado and Miyake.

During the growth period of thirty days, altogether thirteen amino acids showed their presence and activity in the developing culture of Helminthosporium maydis Nishikado and Miyake. Initiation of growth by the 5th day coincided with the appearance of profuse amounts of dl-alanine and serine, and moderate amounts of valine, threonine, and aspartic acid. The thirteen amino acids, which appeared in full complement by the 15th day, were actively engaged in accelerating mycelial accumulation. Beyond this growth period, there was a decline in the amino acids.

Varying spectrum of amino acids in relation to growth in Helminthosporium oryzae Breda de Haan.

Fifteen amino acids were detected in the developing mycelium of Helminthosporium oryzae Breda de Haan. During the first fortnight of mat accumulation, which was more rapid, a marked influence of amino acids on the growth pattern was discernible. A high level maintenance of dl-alanine l-cysteine, l-leucine/isoleucine, threonine, tyrosine, glutamic, and aspartic acids, as well as an increase in the amount of gamma-aminobutyric acid and proline during the first fortnight of culture development were effective in initiating growth and keeping it at a more rapid pace. Glutamine and methionine appeared in the latter half of fungal growth. During this slow pace of mycelial accumulation in the latter half period, there was a general and overall decline in the amount of most of the amino acids. The amino acids started disappearing from the mycelial extract from the 20th day onwards till the end of the 30th day. In the culture filtrate, aspartic acid and proline appeared during the rapid growth phase and dl-alanine and methionine during the slower phase.