Poisoning by Coprinus atramentarius.

The ink cap--Coprinus atramentarius (Bulliard ex Fries) Fries--is responsible for poisoning when ingested with alcohol. The investigation of the "Coprinus syndrome," although a minor poisoning incident, stimulated numerous research programs because the results were expected to yield a novel drug useful during the treatment of alcoholism. This work led to the identification of the active principle--coprine--and to an explanation of its mode of action; nevertheless, detailed toxicology investigations have shown that the mutagenic and gonadotoxic properties of this compound made it unsuitable for therapeutic use. Our current knowledge of the poisoning, the chemistry of the toxin, and its mode of action are here reviewed.     

Meiosis in Coprinus

Chromosome pairing takes two steps: members of the homologous pair become aligned first and then zip up throughout their length. Initial contact may begin from one end or from both ends of the chromosomes. Prom late paehytene to diplotene, the chromosomes are somewhat elongated and exhibit lampbrush characteristics. This is followed by a diffuse stage in which the chromosomes are most indistinct. The centrosome divides at this time. The chromosome numbers are as follows: Coprinus lagopus, n=12; C. micaceus, n=12, C. comatus, n=14; and C. atramentarius, n=16.     

Improved fruiting of Coprinus atramentarius using cold-shock treatment during growth

Coprinus atramentarius was grown on two commercial composts at a constant 20°C or with a cold shock (25°C20°C) after 10 days. Cold shock was required for it to form fruiting bodies.The authors are with the University of Western Sydney, Hawkesbury, School of Horticulture, Locked Bag 1, Richmond NSW 2753, Australia     

Silver staining of meiotic chromosomes in the fungus,Coprinus cinereus

We have taken advantage of the synchronous meiotic process in the basidiomycete Coprinus cinereus to develop a simple and rapid method to selectively stain meiotic chromosomes and nucleoli in this fungus without prior removal of the cell wall. Electron microscopic examination of these silver-stained chromosomes indicated that the lateral elements of the synaptonemal complexes were prominently stained, and terminal attachment plaques were apparent. We found that a translocation quadrivalent could be recognized easily in the light microscope using these methods. The procedures appear suitable for the characterization of chromosome rearrangements in this small genome, and should facilitate cytogenetic analysis in this fungus.     

Dark Dependence of Meiosis at Elevated Temperatures in the Basidiomycete Coprinus lagopus

Under continuous-light regimen Coprinus lagopus can enter and complete meiosis at 25 C, but is unable to do so at 35 C. The temperature-sensitive and the dark-dependent period is 10 hr before karyogamy. It is conjectured that the basidia are programmed for the initiation of meiosis during that 10-hr period.     

DNA polymerase of a basidiomycete fungus, Coprinus cinereus.

DNA polymerase activity was studied in Coprinus cinereus, a basidiomycete fungus. Only one from of the enzyme could be demonstrated, whether by affinity or ion-exchange chromatography; this enzyme had a molecular weight of 185000 on Sephadex G-200, and was inhibited by mercaptoethanol. Coprinus, a representative of the most advanced type of the filamentous fungi, resembles other eukaryotic micro-organisms in its lack of a mammalian beta-type DNA polymerase. The properties of the polymerase are compared with those of two other fungi, and found to resemble most closely the yeast polymerase A in Mg2+ requirements and template preference.     

Chloride channel-dependent copper acquisition of laccase in the basidiomycetous fungus Cryptococcus neoformans

The CLC chloride channel gene CLC-A of the pathogen yeast Cryptococcus neoformans was previously reported to be critical for multicopper laccase activity and growth at an elevated pH.This study reports that copper homeostasis was impaired in the clc-a mutant.This was demonstrated by the substantial decrease of the intracellular quantity of copper under copper-limited growth as determined by flame atomic absorption spectrometry.CLC-A is a critical factor in copper homeostasis which is required for copper acquisition of laccase in C.neoformans.     

Involvement of Ca(2+)/calmodulin-dependent protein kinases in mycelial growth of the basidiomycetous mushroom, Coprinus cinereus

Although multifunctional Ca(2+)/calmodulin-dependent protein kinases (CaM-kinases) are widely distributed in animal cells, the occurrence of CaM-kinases in the basidiomycetous mushroom has not previously been documented. When the extracts from various developmental stages from mycelia to the mature fruiting body of Coprinus cinereus were analyzed by Western blotting using Multi-PK antibodies, which had been generated to detect a wide variety of protein serine/threonine kinases (Ser/Thr kinases), a variety of stage-specific Ser/Thr kinases was detected. Calmodulin (CaM) overlay assay using digoxigenin-labeled CaM detected protein bands of 65 kDa, 58 kDa, 46 kDa, 42 kDa, and 38 kDa only in the presence of CaCl(2), suggesting that these bands were CaM-binding proteins. When the CaM-binding fraction was prepared from mycelial extract of C. cinereus by CaM-Sepharose and analyzed with Multi-PK antibodies, two major immunoreactive bands corresponding to 65 kDa and 46 kDa were detected. CaM-binding fraction, thus obtained, exhibited Ca(2+)/CaM-dependent protein kinase activity toward protein substrates such as histones. These CaM-kinases were found to be highly expressed in the actively growing mycelia, but not in the resting mycelial cells. Mycelial growth was enhanced by the addition of CaCl(2) in the culture media, but inhibited by the addition of EGTA or trifluoperazine, a potent CaM inhibitor. This suggested that CaM-dependent enzymes including CaM-kinases play crucial roles in mycelial growth of basidiomycete C. cinereus.     

Alternative methods for genetic transformation of Pseudozyma antarctica, a basidiomycetous yeast-like fungus

Electroporation and Agrobacterium tumefaciens-mediated transformation (ATMT) were adapted and optimized for genetic transformation of the basidiomycetous yeast-like fungus Pseudozyma antarctica as alternatives to the cumbersome PEG/CaCl₂-mediated transformation of protoplasts. Electroporation yielded 100–200 transformants per μg of DNA per 10⁸ cells after 3 days on selective medium. For its part, ATMT yielded 60–160 transformants per 10⁶ input cfu after 5–10 days on a selective medium. Transformants obtained from both methods showed stable hygromycin resistance and strong expression of green fluorescent protein. Analysis of integration events revealed a limited number of predominantly tandem insertions in the genome of transformants, an improvement over PEG/CaCl₂-mediated transformation. Both protocols relied on intact conidia of P. antarctica as starting material and thus eliminated the need for cell wall-degrading or weakening agents such as lytic enzymes or chemicals. Other advantages over protoplast transformation included higher yield of transformants and shorter recovery time of transformed colonies on selective medium.     

Identification of the basidiomycetous fungus isolated from butt rot of the Japanese cypress

 To identify a basidiomycetous fungus isolated from butt rot of Chamaecyparis obtusa, Japanese cypress, its cultural features were examined, and sequences of its nuclear ribosomal 18S and ITS1–5.8S–ITS2 regions were analyzed. In culture, this fungus is characterized by the occurrence of chlamydospores, blastoconidium-like cells, and clavate-to-spathulate hyphal ends at the tips of aerial hyphae, and production of a small basidioma on the mycelial mat after 3 months of incubation. The morphological features of the basidioma are identical to those of Phlebia brevispora. Furthermore, molecular data of the sequences of these strains and P. brevispora showed a high level of similarity. These results appear to justify determining the present fungus as P. brevispora. This is the first report of this species for Japan and outside of southeastern USA. Received: March 11, 2002 / Accepted: September 20, 2002 Acknowledgments We thank Dr. Karen K. Nakasone, Center for Forest Mycology Research, Forest Products Laboratory, USDA Forest Service, for providing the fungal strains used in this study. Correspondence to:R. Kondo     

Molecular characterization,relatedness and antifungal susceptibility of the basidiomycetous hormographiella species and Coprinus cinereus from clinical and environmental sources

Hormographiella-like strains, isolated from different natural substrates and producing sclerotia and occasionally basidiomata of Coprinus cinereus, were compared morphologically and using molecular techniques with clinical strains of Hormographiella aspergillata and H. verticillata. Analysis of restriction fragment length polymorphisms of ribosomal and mitochondrial-like DNA confirmed interspecific differences between H. aspergillata and H. verticillata, supporting the morphological data, and helped demonstrate that H. aspergillata is the anamorph of C. cinereus. The latter was confirmed also by crossing tests. The analysis of the mtDNA restriction profiles revealed intraspecific variability in C. cinereus, which allowed differentiation of clinical and environmental strains. Due to the implication of C. cinereus and Hormographiella in human opportunistic infections, the antifungal susceptibility test is included. Results show that all strains were susceptible to miconazole, itraconazole and ketoconazole but not to flucytosine and fluconazol. Susceptibility against amphotericin B was variable; while H. verticillata was susceptible, four out of seven C. cinereus strains tested were resistant.     

Novel Ca2+/calmodulin-dependent protein kinase expressed in actively growing mycelia of the basidiomycetous mushroom Coprinus cinereus

We isolated cDNA clones for novel protein kinases by expression screening of a cDNA library from the basidiomycetous mushroom Coprinus cinereus. One of the isolated clones was found to encode a calmodulin (CaM)-binding protein consisting of 488 amino acid residues with a predicted molecular weight of 53,906, which we designated CoPK12. The amino acid sequence of the catalytic domain of CoPK12 showed 46% identity with those of rat Ca2+/CaM-dependent protein kinase (CaMK) I and CaMKIV. However, a striking difference between these kinases is that the critical Thr residue in the activating phosphorylation site of CaMKI/IV is replaced by a Glu residue at the identical position in CoPK12. As predicted from its primary sequence, CoPK12 was found to behave like an activated form of CaMKI phosphorylated by an upstream CaMK kinase, indicating that CoPK12 is a unique CaMK with different properties from those of the well-characterized CaMKI, II, and IV. CoPK12 was abundantly expressed in actively growing mycelia and phosphorylated various proteins, including endogenous substrates, in the presence of Ca2+/CaM. Treatment of mycelia of C. cinereus with KN-93, which was found to inhibit CoPK12, resulted in a significant reduction in growth rate of mycelia. These results suggest that CoPK12 is a new type of multifunctional CaMK expressed in C. cinereus, and that it may play an important role in the mycelial growth.