β-glucosidase induction and repression in the cellulolytic fungus,Trichoderma reesei

β-Glucosidase inTrichoderma reesei (QM 6a) can be induced by methyl-β-glucoside and less effectively by gentiobiose; other glycosides tested, including cellobiose, did not induce this enzyme. Sophorose (a β-1,2 dimer of glucose) at sub-micromolar concentrations repressed β-glucosidase, repression being only partially reversed after sophorose was removed. β-Glucosidase induction has no well-defined pH optimum, although in citrate buffer it is sharply enhanced around pH 3. The optimum temperature for induction is 28°C (at pH 3.0) and response to inducer concentration is hyperbolic. β-Glucosidase (1) is tightly associated with mycelium, (2) is produced with no detectable lag between the time inducer is absorbed and induction starts, and (3) is produced constitutively at low levels. The low, constitutive activity of β-glucosidase is more than adequate to theoretically account for respiration of the fungus on cellobiose.     

Pestalofones A–E,bioactive cyclohexanone derivatives from the plant endophytic fungus Pestalotiopsis fici

Pestalofones A–E (1–5), five new cyclohexanone derivatives, have been isolated from cultures of the plant endophytic fungus Pestalotiopsis fici, along with the known compounds, isosulochrin (6), isosulochrin dehydrate (7), and iso-A82775C (8). The structures of 1–5 were determined by NMR spectroscopy, and the absolute configuration of 1 was assigned using the modified Mosher method. Compounds 1, 2, and 5 displayed inhibitory effects on HIV-1 replication in C8166 cells, whereas 3 and 5 showed significant antifungal activity against Aspergillus fumigatus.     

Japonicones A–D,bioactive dimeric sesquiterpenes from Inula japonica Thunb.

Four new dimeric sesquiterpene lactones japonicones A?D (1?4), comprised by eudesmane and guaiane sesquiterpenes, were isolated from the aerial part of Inula japonica Thunb. The structures and stereochemistry of 1?4 were elucidated by use of 2D NMR spectroscopic techniques, X-ray crystallography and modified Mosher method. Japonicone A (1) showed the most potent cytotoxicities against four tumor cell lines, A549, LOVO, CEM and MDA-MB-435.     

Asperidines A–C,pyrrolidine and piperidine derivatives from the soil-derived fungus Aspergillus sclerotiorum PSU-RSPG178

One new pyrrolidine derivative, asperidine A (1), and two new piperidine derivatives, asperidines B (2) and C (3), were isolated from the soil-derived fungus Aspergillus sclerotiorum PSU-RSPG178 together with two known alkaloids. Compound 3 possessed an unprecedented 7-oxa-1-azabicyclo[3.2.1]octane skeleton with four chiral centers. Their structures were determined by spectroscopic evidence. The absolute configurations of compounds 2 and 3 were established using Mosher’s method and further confirmed for compound 3 by X-ray crystallographic data. Compound 2 dose-dependently inhibited the CFTR-mediated chloride secretion in T84 cells with an IC₅₀ value of 0.96?μM whereas 3 displayed the same activity with the IC₅₀ value of 58.62?μM. Compounds 2 and 3 also significantly reduced intracellular ROS under both normal and H₂O₂-treated conditions compared with their respective controls in a dose-dependent manner without cytotoxic effect on Caco-2 cells. In addition, compound 3 was inactive against noncancerous Vero cells whereas compound 2 was considered to be inactive with the IC₅₀ value of >10?μM.     

Euryspongins A–C,three new unique sesquiterpenes from a marine sponge Euryspongia sp.

Three new unique sesquiterpenes, euryspongins A–C (1–3), were isolated from a marine sponge Euryspongia sp. collected at Iriomote Island, Okinawa, Japan. Compound 1 possessed a bicyclic furanosesquiterpene structure with six- and eight-membered rings, whereas compounds 2 and 3 had an α,β-unsaturated-γ-lactone ring instead of the furan ring in 1. Only five natural products in this class have been reported, and compounds 1–3 are the sixth–eighth examples of natural products. Compounds 1–3 had no inhibition effect against PTP1B, an important target enzyme for the treatment of diabetes, while the dehydro derivative of 1 [dehydroeuryspongin A (4)] exhibited inhibitory activity (IC₅₀ = 3.6 μM).     

Phenol derivatives from the cold-seep fungus Aspergillus sydowii 10–31

Two bisabolane-type sesquiterpenoid phenols inclusive of the known (7 S)-flavilane A (1) and the new (7 S)− 4-iodo-flavilane A (2), along with a new tetraphenyl ether, bisviolaceol II (3), were isolated from the cold-seep fungus Aspergillus sydowii 10–31. Their structures were identified through spectroscopic methods, such as MS, IR, and NMR, and the absolute configurations of 1 and 2 were ascertained by comparison of ECD curves. Compounds 1 and 2 represent rarely encountered sulfur-bearing sesquiterpenes in nature, and the iodination in 2 makes it unique in particular. Additionally, 3 is the first tetraphenyl ether derivative from the fungal species A. sydowii. Compounds 1-3 inhibited the growth of several Vibrio bacteria and harmful microalgae.     

Quiquelignan A–H,eight new lignoids from the rattan palm Calamus quiquesetinervius and their antiradical,anti-inflammatory and antiplatelet aggregation activities

Eight new lignin derivatives, termed quiquelignan A–H (1–8), comprising three tricin-type flavonolignans (1–3) and five 8-O-4′ neolignans (4–8), were isolated from the ethanol extract of Calamus quiquesetinervius stems. Structural elucidation of the new isolates was accomplished on the basis of spectroscopic data. Compounds 1–8 showed strong-to-moderate antioxidant activity against the hydroxy radical (OH). Among them, compound 5 showed significantly higher hydroxy radical scavenging activity (IC₅₀ 4.4 μg/mL). Compounds 2–4 and 6–8 dose-dependently suppressed the LPS-stimulated production of nitric oxide (NO) in RAW 264.7 cells. The anti-inflammatory potency of 4 and 6 was 2.7–4.5-fold higher compared with quercetin. Compounds 2–4, 6 and 8 also exhibited mild collagen-antagonistic activity, but were inactive with respect to thrombin-induced platelet aggregation.     

Thaixylogranins A–H: eight new limonoids from the Thai mangrove,Xylocarpus granatum

Eight new limonoids named thaixylogranins A–H (1–8), including seven mexicanolides (1–3 and 5–8), were isolated from the seeds of a Thai mangrove, Xylocarpus granatum. The structures of these limonoids were established on the basis of HRESIMS and NMR spectroscopic data. Compounds 1–4 possess an 8α, 30α-epoxy ring, whereas compounds 5−8 have a C8C30 bond. Compounds 1–8 exhibited weak cytotoxicities against the MDA-MB-231 cell with IC₅₀ values of 49.4, 58.3, 53.6, 61.1, 57.9, 44.6, 40.6, and 38.5 μM, respectively.     

Trichoderone, a novel cytotoxic cyclopentenone and cholesta-7, 22-diene-3β, 5α, 6β-triol, with new activities from the marine-derived fungus Trichoderma sp.

The historical paradigm of the deep ocean as a biological ‘desert’ has shifted to one of a ‘rainforest’ owing to the isolation of many novel microbes and their associated bioactive compounds. To explore the potential of the bioactive compounds in our marine microbial natural product library, we screened it for the selective cytotoxicity of six different cancer cell lines to human normal lung fibroblast cell line HLF. The crude extract from a marine-derived fungal strain showed notable selectivity against cancer cell lines. For a bioactivity-guided fractionation and purification, a novel cyclopentenone, (−)-(4R ^*, 5S ^*)-3-ethyl-4,5-dihydroxycyclopent-2-enone (1, trichoderone), and a known compound with new activity, cholesta-7,22- diene-3β,5α,6β-triol (2), were identified from a marine Trichoderma sp. that was isolated from the deep sea sediment of the South China Sea. Their structures were determined by NMR and MS data analyses. Trichoderone (1) displayed potent cytotoxicity against a panel of six cancer cell lines, whereas it did not show much cytotoxicity against normal human lung fibroblast cell line HLF even at a concentration of 7.02 mM. The selectivity index (SI) value for 1 was greater than 100. To the best of our knowledge, both compounds were isolated from marine fungi for the first time. They also exhibited bioactivities against HIV protease and Taq DNA polymerase. Optimization of the compounds would shed new light on treating cancer and infectious diseases.     

Alpneumines A–H,new anti-melanogenic indole alkaloids from Alstonia pneumatophora

Eight new indole alkaloids, alpneumines A–H (1–8) were isolated from the Malaysian Alstonia pneumatophora (Apocynaceae) and their structures were determined by MS and 2D NMR spectroscopic methods. Alpneumines E and G (5 and 7), vincamine, and apovincamine showed anti-melanogenesis in B16 mouse melanoma cells.     

Sinopestalotiollides A–D,cytotoxic diphenyl ether derivatives from plant endophytic fungus Pestalotiopsis palmarum

Four new diphenyl ether derivatives, sinopestalotiollides A–D (1–4), one new natural α-pyrone product (11), as well as twelve known compounds (5–1?7), were obtained from the ethyl acetate extract of the endophytic fungus Pestalotiopsis palmarum isolated from the leaves of medicinal plant Sinomenium acutum (Thunb.) Rehd et Wils. The structures were elucidated by HR-ESI-MS and NMR spectrometry data. Bioassay experiments revealed that compounds 1–4 and 11 exhibited strong to weak cytotoxicities against three human tumor cell lines Hela, HCT116 and A549.     

A new β-glucosidase gene from the zygomycete fungus Rhizomucor miehei

In this study, a β-glucosidase coding gene (bgl) of the zygomycete fungus Rhizomucor miehei has been cloned and characterized. The gene comprises a total of 2,826 bp including the coding sequence of a 717 amino acids length putative protein and 10 introns dispersed in the whole coding region. The putative N-and C-terminal catalytic domains (aa 68 to aa 274 and aa 358–601, respectively) were identified; the two domains are connected with a 84-amino-acids linker. The catalytic region showed an extensive sequence homology with other fungal β-glucosidases classified as family 3 glycoside hydrolases. The isolated Rhizomucor gene was expressed in the related fungus Mucor circinelloides. Transformant Mucor strains maintained the introduced plasmid in an autoreplicative manner and showed significantly higher cellobiase activity than the recipient strain.     

Interactions between a soil fungus,Trichoderma harzianum,and IIb metals—adsorption to mycelium and production of complexing metabolites

Fungi are capable of accumulating metals and, in soil, such accumulation may influence metal speciation and transport. The interactions between a common soil fungus, Trichoderma harzianum, and IIb elements were studied in the present investigation. The accumulation of the metals zinc, cadmium and mercury by starved and non-starved mycelium at different pH was determined by a batch technique using radioactive tracers; uptake of the metals was found to be large, with respective distribution coefficients of about 10^3.5, 10^2.5 and 10^4.0 for zinc, cadmium and mercury, respectively. Metal accumulation by a starved system was largely independent of pH in the range 3–9, where in a non-starved system an increased accumulation of zinc (at 10^– m) was observed at low pH (3–5). Potentiometric titrations performed on the two systems revealed significant differences in acid capacities, i.e. values close to zero for the starved system and 500–800 meq kg^– for the non-starved system. The maximum metal uptake was at least 50 mmol kg^– at pH 6.5 (calculated from adsorption isotherms). The present findings suggests that in the non-starved system a metabolite is produced and then released when the pH is within a certain range.     

α-Pyrones,secondary metabolites from fungus Cephalotrichum microsporum and their bioactivities

Cephalotrichum microsporum (SYP-F 7763) was a fungus isolated from the rhizosphere soil of traditional Chinese medicine Panax notoginseng. The EtOAc extract of Cephalotrichum microsporum cultivated on sterilized moistened-rice medium was separated by various chromatographic techniques, which yielded 11 metabolites (1–11) of this fungus. On the basis of the widely spectroscopic data, the chemical structures of isolated metabolites were determined, most of which were α-pyrones, including 5 compounds (4–7, and 10) unreported. In the anti-bacterial bioassay, compound 1 displayed significant inhibitory effects on three pathogenic bacteria, MR S. aureus, S. aureus, and B. cereus. α-Pyrones 2, 3, and 5–7 also displayed moderate inhibitory effects on MR S. aureus, S. aureus, and B. subtilis, which could be the major anti-bacterial constituents of Cephalotrichum microsporum. Additionally, compounds 1, 4, and 5 displayed significant cytotoxicity on five human cancer cell lines, with the IC₅₀ values < 20 μM, which are more effective than positive control 5-fluorouracil. Therefore, α-pyrones were important secondary metabolites of Cephalotrichum microsporum, which displayed anti-bacterial and anti-tumor activities.     

Formosusin A,a novel specific inhibitor of mammalian DNA polymerase β from the fungus Paecilomyces formosus

Variotin (1) and three novel compounds, formosusin A (2), B (3), and C (4), were isolated from the cultures of the fungus Paecilomyces formosus, and their structures were determined by spectroscopic analyses. Compound 2 is (6Z,8E,10E)-variotin, a new cis-olefin analog of compound 1. Compound 2 selectively inhibited the activity of mammalian DNA polymerase β (pol β) in vitro, with an IC₅₀ of 35.6 μM. By contrast, compounds 1, 3, and 4 did not influence the activity of pol β. These four compounds showed no effect on the activities of other 10 mammalian pols (i.e., pols α, γ, δ, ε, η, ι, κ, λ, and μ, and terminal deoxynucleotidyl transferase). These compounds also did not inhibit the activities of fish, insect, plant, and prokaryotic pols and other DNA metabolic enzymes tested. These results suggested that compound 2 could be a selective inhibitor of mammalian pol β. The compound 2-induced inhibition of rat pol β activity was competitive and non-competitive with respect to the DNA template–primer substrate and the dNTP substrate, respectively. On the basis of these results, the relationship between the three-dimensional structure and pol β inhibitory mechanism of compound 2 is discussed.     

Partial purification and properties of a β-N-acetylglucosaminidase from the fungus Sclerotinia fructigena

1. As cultures of the fungus Sclerotinia fructigena autolysed, the filtrates contained increasing quantities of a beta-N-acetylglucosaminidase. 2. The enzyme was purified up to 42-fold by a combination of isoelectric focusing and gel filtration. 3. It ran as a single band in cellulose acetate strip electrophoresis and in isoelectric focusing (pI3.76). 4. The enzyme did not readily hydrolyse chitin or a glycopeptide with terminal N-acetylglucosamine residues, but rapidly degraded the N-acetylglucosamine dimer NN'-diacetylchitobiose; the monomer was readily utilized by the fungus as a nitrogen source. The K(m) value for hydrolysis of p-nitrophenyl beta-2-acetamido-2-deoxy-d-glucopyranoside at 37 degrees C was 2.0mm. The Sclerotinia enzyme was generally less susceptible to inhibition by 2-acetamido-2-deoxygluconolactone and other related sugars than the corresponding enzyme from other sources. Inhibition by excess of substrate was observed. 5. The culture filtrate also contained N-acetylgalactosaminidase activity; conflicting evidence was obtained as to whether the same enzyme was responsible for both hexosaminidase activities.     

17β-hydroxysteroid dehydrogenase from the fungus Cochlioboluslunatus: structural and functional aspects

17β-Hydroxysteroid dehydrogenase (17β-HSD) activity has been described in all filamentous fungi tested, but until now only one 17β-HSD from Cochlioboluslunatus (17β-HSDcl) was sequenced. We examined the evolutionary relationship among 17β-HSDcl, fungal reductases, versicolorin reductase (Ver1), trihydroxynaphthalene reductase (THNR), and other homologous proteins. In the phylogenetic tree 17β-HSDcl formed a separate branch with Ver1, while THNRs reside in another branch, indicating that 17β-HSDcl could have similar function as Ver1. The structural relationship was investigated by comparing a model structure of 17β-HSDcl to several known crystal structures of the short chain dehydrogenase/reductase (SDR) family. A similarity was observed to structures of bacterial 7α-HSD and plant tropinone reductase (TR). Additionally, substrate specificity revealed that among the substrates tested the 17β-HSDcl preferentially catalyzed reductions of steroid substrates with a 3-keto group, Δ⁴ or 5α, such as: 4-estrene-3,17-dione and 5α-androstane-3,17-dione.     

Production, purification and characterization of a constitutive intracellular α-galactosidase from the thermophilic fungus Humicola sp

The thermophilic fungus Humicola sp constitutively produces intracellular α-galactosidase (1.33 U mg⁻¹ protein) within 48 h at 45°C in shaken flasks, when grown in a medium containing 7% wheat bran extract as a carbon source and 0.5% yeast extract as a nitrogen source. The enzyme has been purified to homogeneity by ultrafiltration, ethanol precipitation, DEAE cellulose and Sephacryl S-300 chromatography with a 124-fold increase in specific activity and 29.5% recovery. The molecular weight of the enzyme is 371.5 kDa by gel filtration on Sephacryl S-300 and 87.1 kDa by SDS-polyacrylamide gel electrophoresis. The enzyme has an optimum temperature of 65°C and an optimum pH of 5.0. Humicola α-galactosidase is a glycoprotein with 8.3% carbohydrate content and is acidic in nature with a pI of 4.0. The K _m S for p-nitrophenyl-α-D-galactopyranoside, O-nitrophenyl-α-D-galactopyranoside, raffinose and stachyose are 0.279, 0.40, 1.45 and 1.42 mM respectively. The enzyme activity was strongly inhibited by Ag⁺ and Hg²⁺. D-Galactose inhibited α-galactosidase competitively and the inhibition constant (K _i) for galactose was 11 mM. Received 28 January 1999/ Accepted in revised form 07 April 1999