Optimization of submerged culture conditions for the production of angiotensin converting enzyme inhibitor from <Emphasis Type="Italic">Flammulina velutipes</Emphasis>

The angiotensin-converting enzyme (ACE) inhibitory effect was tested in the culture broth from submerged mycelial cultures of 20 basidiomycetes. The ACE inhibitory effect of culture broth from Flammulina velutipes strain 414 was the highest (52.8%), followed by Lentinus edodes strains 2 (44.4%) and 16 (41.3%). Nutritional requirements for the production of ACE inhibitory substance from F. velutipes were studied. Sucrose, ammonium acetate, and glutamic acid were chosen for the maximum production of ACE inhibitory substance. The optimal medium composition was (g/l): sucrose 20, ammonium acetate 5, glutamic acid 2, KH₂PO₄ 3, MgSO₄·7H₂O 0.8, and yeast extract 0.5. Under optimal culture conditions, the ACE inhibitory effect was more than 80%. Received 04 May 2002/ Accepted in revised form 11 June 2002     

Biotransformation of polychlorinated dibenzo-<Emphasis Type="Italic">p</Emphasis>-dioxin by <Emphasis Type="Italic">Coprinellus</Emphasis> species

A degradation experiment on dibenzo-p-dioxin (DD) and 2,7-dichlorodibenzo-p-dioxin (2,7-DCDD) was carried out using basidiomycetous fungi belonging to the genera Coprinus, Coprinellus, and Coprinopsis. Some species showed a high rate of decrease in DD for the 2-week test period. Among them, Coprinellus disseminatus showed the highest ability to decrease the DD level, close to 100% by the end of 2 weeks. Further examination showed that Coprinellus disseminatus and Coprinellus micaceus, belonging to the genus Coprinellus, were able to metabolize 2,7-DCDD to a monohydroxylated compound, probably mediated by the P450 system. The metabolism of chlorinated DD by fungi capable of living in soil conditions is reported here for the first time.     

Formation of diacylglyceryltrimethylhomoserines in the surface culture of the basidiomycete <Emphasis Type="Italic">Flammulina velutipes</Emphasis>

Betaine-type lipids—diacylglyceryltrimethylhomoserines (DGTS)—were revealed in the mycelium of the basidial fungus Flammulina velutipes obtained by surface cultivation on agarized malt extract. DGTS accumulation was shown to occur at the late stages of culture development under deficiency of a complex of nutrients, including nitrogen, phosphorus, potassium, and trace elements. Induction of the synthesis of betaine lipids in F. velutipes occurred against the background of a decreased rate of growth of the vegetative mycelium, formation of monilioid hyphae, and inhibition of fructification. The relationship between DGTS formation and the environmental factors (temperature, illumination) was studied. It was established that the most active DGTS accumulation occurred at 15°C in the dark.     

<Emphasis Type="Italic">Exobasidium dubium</Emphasis> and <Emphasis Type="Italic">E. miyabei</Emphasis> sp. nov. causing Exobasidium leaf blisters on <Emphasis Type="Italic">Rhododendron </Emphasis>spp. in Japan

 Two Exobasidium species causing Exobasidium leaf blister on Rhododendron spp. are described. An Exobasidium leaf blister on Rhododendron yedoense var. yedoense f. yedoense has been recognized in Hokkaido Prefecture, Japan, since the first report was issued in 1950. The causal fungus is identified with Exobasidium dubium from the morphology of its hymenial structure and mode of germination of the basidiospores. Another Exobasidium leaf blister on Rhododendron dauricum has been observed in Hokkaido Prefecture, Japan. In comparison with morphology based on hymenial structure and mode of germination of the basidiospores of the 100 validly described taxa, this fungus differs from those known taxa in the size of basidia and basidiospores, the numbers of sterigmata and septa of basidiospores, and the mode of germination of basidiospores. Thus, a new species, Exobasidium miyabei, is established and illustrated. Received: February 13, 2002 / Accepted: September 25, 2002  Present address: National Institute of Agrobiological Sciences, Tsukuba 305-8602, Japan Acknowledgments We profoundly appreciate the cooperation of Dr. V. Melnik in providing Russian papers and Dr. L. Vasilyeva for translating them into English. We thank Prof. H. Takahashi for loaning the materials in the Herbarium of the Hokkaido University Museum and Dr. W. Abe, Graduate School of Science, University of Hokkaido, for his kind help with the sampling of R. dauricum in Teshikaga, Hokkaido Prefecture. This study was supported in part by a Grant-in-Aid for Scientific Research (B) (No. 13460019), Japan Society for the Promotion of Science (JSPS). Contribution No. 171, Laboratory of Plant Parasitic Mycology, Institute of Agriculture and Forestry, University of Tsukuba. Correspondence to:M. Kakishima     

<Emphasis Type="Italic">Exobasidium symploci-japonicae</Emphasis> var. <Emphasis Type="Italic">carpogenum</Emphasis> var. nov. causing Exobasidium fruit deformation on <Emphasis Type="Italic">Symplocos lucida</Emphasis> in Japan

Exobasidium symploci-japonicae var. carpogenum, causing Exobasidium fruit deformation on Symplocos lucida collected in Fukuoka Prefecture, Japan, is newly described based on morphological observations of hymenial structure and mode of basidiospore germination. This new variety differs morphologically from the type variety, particularly in the septal number of basidiospores and in the shapes and sizes of conidia formed on the medium. Colonies of this new variety are also distinguishable from those of the type variety by yeast-like growth, morphology, and color of colonies.Contribution no.178, Laboratory of Plant Parasitic Mycology, Institute of Agriculture and Forestry, University of Tsukuba, Japan     

Electrophoretic karyotype of <Emphasis Type="Italic">Flammulina velutipes</Emphasis> and its variation among monokaryotic progenies

 The karyotype of Flammulina velutipes (Curt. : Fr.) Sing. was investigated using contour-clamped homogeneous electric fields (CHEF) gel electrophoresis. A parental dikaryotic stock, JA, was resolved into at least eight chromosomal DNA bands ranging from 1.4- to 4.9-megabase (Mb) pairs. Overall, little size variation was found among monokaryotic strains with a few major exceptions. Among 13 monokaryotic progenies examined, 11 strains were resolved into at least eight chromosomal DNA bands in a manner similar to the parent dikaryon, whereas the other 2 were resolved into at least seven chromosomes lacking the 2.1-Mb chromosome possessed in the former. A slightly larger size variation was found in a chromosome carrying ribosomal DNA. An estimated haploid genome size of this stock was 24.0 Mb or more. Received: October 11, 2001 / Accepted: November 11, 2002 Acknowledgments We thank Professor T. Morinaga, Hiroshima Prefectural University, and Dr. T. Arima for their technical advice regarding CHEF gel electrophoresis. Correspondence to:E. Tanesaka     

Isolation and characterization of melanin pigment from <Emphasis Type="Italic">Pleurotus cystidiosus</Emphasis> (telomorph of <Emphasis Type="Italic">Antromycopsis</Emphasis><Emphasis Type="Italic">macrocarpa</Emphasis>)

Melanins are enigmatic pigments that are produced by a wide variety of microorganisms including several species of bacteria and fungi. For more than 40 years, fungi have been known to produce pigments called melanins. Melanin pigment production by mushrooms was not intensively studied. The present study was carried out on isolation and characterization of melanin from an edible mushroom Pleurotus cystidiosus var. formosensis. The mushroom produced dark mucous mass of hyaline arthrospores on mycelium. The coremia exclusively produced dikaryotic arthrospores with the remnant of a clamp connection. Continuous cell extension and division in the coremium stipe supplied cells for arthroconidiation at the coremium apex, which is surrounded by a liquid droplet (coremioliquid). The black coloured coremea (conidia) were produced by Antromycopsis macrocarpa (anamorph of P. cystidiosus) when cultured on potato dextrose agar medium. The agar plate was incubated at continuous light illumination for high amount of pigment (coremea) production. The slimy layer of the coremea was extracted and partially purified by alkaline and acid treatment. The black pigment was confirmed as melanin based on UV, IR and EPR spectra apart from chemical analysis. This is the first report on characterization of melanin obtained from Pleurotus cystidiosus var. formosensis.     

Influence of culture conditions on glutathione production by <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

A strategy of experimental design using a fractional factorial design (FFD) and a central composite rotatable design (CCRD) were carried out with the aim to obtain the best conditions of temperature (20–30°C), agitation rate (100–300 rpm), initial pH (5.0–7.0), inoculum concentration (5–15%), and glucose concentration (30–70 g/l) for glutathione (GSH) production in shake-flask culture by Saccharomyces cerevisiae ATCC 7754. By a FFD (2^5–2), the agitation rate, temperature, and pH were found to be significant factors for GSH production. In CCRD (2²) was obtained a second-order model equation, and the percent of variation explained by the model was 95%. The results showed that the optimal culture conditions were agitation rate, 300 rpm; temperature, 20°C; initial pH, 5; glucose, 54 g/l; and inoculum concentration, 5%. The highest GSH concentration (154.5 mg/l) was obtained after 72 h of fermentation.     

<Emphasis Type="Italic">Ty</Emphasis>1<Emphasis Type="Italic">/copia</Emphasis>- and <Emphasis Type="Italic">Ty</Emphasis>3<Emphasis Type="Italic">/gypsy</Emphasis>-like DNA sequences in <Emphasis Type="Italic">Helianthus </Emphasis>species

Two repeated DNA sequences isolated from a partial genomic DNA library of Helianthus annuus, p HaS13 and p HaS211, were shown to represent portions of the int gene of a Ty3 /gypsy retroelement and of the RNase-Hgene of a Ty1 /copia retroelement, respectively. Southern blotting patterns obtained by hybridizing the two probes to BglII- or DraI-digested genomic DNA from different Helianthus species showed p HaS13 and p HaS211 were parts of dispersed repeats at least 8 and 7 kb in length, respectively, that were conserved in all species studied. Comparable hybridization patterns were obtained in all species with p HaS13. By contrast, the patterns obtained by hybridizing p HaS211 clearly differentiated annual species from perennials. The frequencies of p HaS13- and p HaS211-related sequences in different species were 4.3x10(4)-1.3x10(5) copies and 9.9x10(2)-8.1x10(3) copies per picogram of DNA, respectively. The frequency of p HaS13-related sequences varied widely within annual species, while no significant difference was observed among perennial species. Conversely, the frequency variation of p HaS211-related sequences was as large within annual species as within perennials. Sequences of both families were found to be dispersed along the length of all chromosomes in all species studied. However, Ty3 /gypsy-like sequences were localized preferentially at the centromeric regions, whereas Ty1/ copia-like sequences were less represented or absent around the centromeres and plentiful at the chromosome ends. These findings suggest that the two sequence families played a role in Helianthusgenome evolution and species divergence, evolved independently in the same genomic backgrounds and in annual or perennial species, and acquired different possible functions in the host genomes.     

Cloning of glyceraldehyde-3-phosphate dehydrogenase gene and use of the <Emphasis Type="Italic">gpd</Emphasis> promoter for transformation in <Emphasis Type="Italic">Flammulina velutipes</Emphasis>

The glyceraldehyde-3-phosphate dehydrogenase gene of Flammulina velutipes was isolated. The complete gpd sequence (from ATG to TAA) was 1,489 bp in length and contained nine introns. The locations of these nine introns were similar to those of other basidiomycetes, which might reflect the evolutionary divergence of these mushrooms. The F. velutipes gpd gene was found to encode a protein of 339 amino acids and its putative amino acid sequence revealed a high similarity to an analogous protein deriving from other basidiomycetes. Results of Southern blot analysis suggested that there existed only one copy of the gpd gene in the genome of F. velutipes and that there was one typical TATA box and two CAAT boxes located in the 5 flanking region. The F. velutipes gpd promoter was fused to a hygromycin B phosphotransferase gene (hph) derived from Escherichia coli as a selection marker. Using the resulting construction, hph was efficiently transformed into F. velutipes by basidiospore electroporation. No false-positive antibiotic-resistant cultures were detected by PCR amplification and the hygromycin resistance trait was maintained stably during mitotic cell division for 3 months. Southern analysis of transformants indicated the integration of gene might occur by non-homologous recombination. This rapid and convenient electroporation procedure offers new prospects for the genetic manipulation and a tool for tagging genes of this important edible mushroom species. Sequence data will appear in the DDBJ/EMBL/GenBank nucleotide sequence database under accession number AF515622.     

Revision of the <Emphasis Type="Italic">Serrulati</Emphasis> species of <Emphasis Type="Italic">Penstemon</Emphasis> section <Emphasis Type="Italic">Saccanthera</Emphasis> subsection <Emphasis Type="Italic">Serrulati</Emphasis>

A revision of Penstemon sect. Saccanthera subsect. Serrulati includes a new species (P. salmonensis), a new variety (P. triphyllus var. infernalis), and the elevation of a subspecies to species (P. curtiflorus), bringing the total number of species to eight, which are keyed and described, complete with nomenclature and type citations.     

<Emphasis Type="Italic">In vitro</Emphasis> shoot culture and antimicrobial activity of <Emphasis Type="Italic">Berberis buxifolia</Emphasis> Lam

Berberis buxifolia Lam., known as “Calafate”, is a plant native to Argentina that exhibits antimicrobial activity. This biological activity is attributed to the isoquinoline alkaloid berberine. The aim of this research was to test the antimicrobial properties of different extracts of this species, taking berberine as the reference molecule, and to examine if the expression of bacterial multidrug resistance (MDR) efflux pumps could be responsible for possible resistance mechanisms. To this end, a wild-type and a mutant strain of Staphylococcus aureus with a defective MDR efflux pump were used and the minimum inhibitory concentrations of the extracts were determined. The studies were carried out with infusions of in vivo shoots and “Calafate” commercial tea, as well as with the media derived from shoot cultures incubated with different plant growth regulators (thidiazuron, picloram, and jasmonic acid). As far as antimicrobial activity is concerned, all the extracts tested were significantly more effective than berberine standard. “Calafate” commercial tea and shoot tea had inhibitory concentrations similar to the one observed for ampicillin standard. The media from the shoot cultures, however, were significantly more effective than all the others, particularly the one derived from jasmonic acid, suggesting the presence of compounds that could be acting synergistically with berberine. There were no differences in antimicrobial activity against the wild-type and the mutant S. aureus; no definite conclusions could be drawn concerning the relationship between MDR pumps and possible pathogen resistance to extracts of B. buxifolia.     

<Emphasis Type="Italic">In vitro</Emphasis> culture studies on <Emphasis Type="Italic">Stevia rebaudiana</Emphasis>

Summary Shoot apex, nodal, and leaf explants of Stevia rebaudiana Bertoni can regenerate shoots when cultured on Murashige and Skoog (MS) medium supplemented with 6-benzyladenine (BA; 8.87 μM) and indole-3-acetic acid (5.71 μM). Rooting of the in vitro-derived shoots could be achieved following subculture onto auxin-containing medium. A survival rate of 70% was recorded at the hardening phase on the substrate cocopeat. The presence of the sweet diterpene glycosides, viz. stevioside and rebaudioside, was confirmed in the in vitro-derived tissues of Stevia using HPTLC techniques. Callus cultured on agar-solidified MS medium supplemented with BA (8.87 μM) and indole-3-butyric acid (9.80 μM) showed the highest sweetener content.     

<Emphasis Type="Italic">Galleria</Emphasis><Emphasis Type="Italic">mellonella</Emphasis> are Resistant to <Emphasis Type="Italic">Pneumocystis</Emphasis><Emphasis Type="Italic">murina</Emphasis> Infection

Studying Pneumocystis has proven to be a challenge from the perspective of propagating a significant amount of the pathogen in a facile manner. The study of several fungal pathogens has been aided by the use of invertebrate model hosts. Our efforts to infect the invertebrate larvae Galleria mellonella with Pneumocystis proved futile since P. murina neither caused disease nor was able to proliferate within G. mellonella. It did, however, show that the pathogen could be rapidly cleared from the host.     

<Emphasis Type="Italic">Agrobacterium</Emphasis><Emphasis Type="Italic">tumefaciens</Emphasis>-mediated transformation of callus cells of <Emphasis Type="Italic">Crataegus</Emphasis><Emphasis Type="Italic">aronia</Emphasis>

A genetic transformation system has been developed for callus cells of Crataegus aronia using Agrobacterium tumefaciens. Callus culture was established from internodal stem segments incubated on Murashige and Skoog (MS) medium supplemented with 5 mg l⁻¹ Indole-3-butyric acid (IBA) and 0.5 mg l⁻¹ 6-benzyladenine (BA). In order to optimize the callus culture system with respect to callus growth and coloration, different types and concentrations of plant growth regulators were tested. Results indicated that the best average fresh weight of red colored callus was obtained on MS medium supplemented with 2 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.5 mg l⁻¹ kinetin (Kin) (callus maintenance medium). Callus cells were co-cultivated with Agrobacterium harboring the binary plasmid pCAMBIA1302 carrying the mgfp5 and hygromycin phosphotransferase (hptII) genes conferring green fluorescent protein (GFP) activity and hygromycin resistance, respectively. Putative transgenic calli were obtained 4 weeks after incubation of the co-cultivated explants onto maintenance medium supplemented with 50 mg l⁻¹ hygromycin. Molecular analysis confirmed the integration of the transgenes in transformed callus. To our knowledge, this is the first time to report an Agrobacterium-mediated transformation system in Crataegus aronia.     

Role of Arabidopsis <Emphasis Type="Italic">ABF1</Emphasis>/<Emphasis Type="Italic">3</Emphasis>/<Emphasis Type="Italic">4</Emphasis> during <Emphasis Type="Italic">det1</Emphasis> germination in salt and osmotic stress conditions

Key message

Arabidopsis det1 mutants exhibit salt and osmotic stress resistant germination. This phenotype requires HY5, ABF1, ABF3, and ABF4.

Abstract

While DE-ETIOLATED 1 (DET1) is well known as a negative regulator of light development, here we describe how det1 mutants also exhibit altered responses to salt and osmotic stress, specifically salt and mannitol resistant germination. LONG HYPOCOTYL 5 (HY5) positively regulates both light and abscisic acid (ABA) signalling. We found that hy5 suppressed the det1 salt and mannitol resistant germination phenotype, thus, det1 stress resistant germination requires HY5. We then queried publically available microarray datasets to identify genes downstream of HY5 that were differentially expressed in det1 mutants. Our analysis revealed that ABA regulated genes, including ABA RESPONSIVE ELEMENT BINDING FACTOR 3 (ABF3), are downregulated in det1 seedlings. We found that ABF3 is induced by salt in wildtype seeds, while homologues ABF4 and ABF1 are repressed, and all three genes are underexpressed in det1 seeds. We then investigated the role of ABF3, ABF4, and ABF1 in det1 phenotypes. Double mutant analysis showed that abf3, abf4, and abf1 all suppress the det1 salt/osmotic stress resistant germination phenotype. In addition, abf1 suppressed det1 rapid water loss and open stomata phenotypes. Thus interactions between ABF genes contribute to det1 salt/osmotic stress response phenotypes.

     

The <Emphasis Type="Italic">Caenorhabditis</Emphasis><Emphasis Type="Italic">briggsae</Emphasis> genome contains active <Emphasis Type="Italic">CbmaT1</Emphasis> and <Emphasis Type="Italic">Tcb1</Emphasis> transposons

The maT clade of transposons is a group of transposable elements intermediate in sequence and predicted protein structure to mariner and Tc transposons, with a distribution thus far limited to a few invertebrate species. We present evidence, based on searches of publicly available databases, that the nematode Caenorhabditis briggsae has several maT-like transposons, which we have designated as CbmaT elements, dispersed throughout its genome. We also describe two additional transposon sequences that probably share their evolutionary history with the CbmaT transposons. One resembles a fold back variant of a CbmaT element, with long (380-bp) inverted terminal repeats (ITRs) that show a high degree (71%) of identity to CbmaT1. The other, which shares only the 26-bp ITR sequences with one of the CbmaT variants, is present in eight nearly identical copies, but does not have a transposase gene and may therefore be cross mobilised by a CbmaT transposase. Using PCR-based mobility assays, we show that CbmaT1 transposons are capable of excising from the C. briggsae genome. CbmaT1 excised approximately 500 times less frequently than Tcb1 in the reference strain AF16, but both CbmaT1 and Tcb1 excised at extremely high frequencies in the HK105 strain. The HK105 strain also exhibited a high frequency of spontaneous induction of unc-22 mutants, suggesting that it may be a mutator strain of C. briggsae.