Site-directed mutagenesis of a possible type 1 copper ligand of bilirubin oxidase; a Met467Gln mutant shows stellacyanin-like properties.
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In our previous paper, we reported a mutant of recombinant Myrothecium verrucaria bilirubin oxidase, in which the Met467 residue was replaced by Gly [Shimizu, A. et al. (1999) Biochemistry 38, 3034-3042]. This mutant displayed a remarkable reduction in enzymatic activity and an evident decrease in the intensity of the absorption band around 600 nm (type 1 charge transfer transition). In this study, we report the preparation of three Met467 mutants (Met467Gln, Met467His, and Met467Arg) and characterize their enzymatic activities, midpoint potentials, and absorption and ESR spectra. Met467His and Met467Arg show no enzymatic activity and a great reduction in the intensity of the absorption band around 600 nm. Furthermore, their ESR spectra show no type 1 copper signal, but only a type 2 copper signal; however, oxidation by ferricyanide caused the type 1 copper signal to appear. On the other hand, Met467Gln as expressed shows both type 1 and type 2 copper signals in its ESR spectrum, the type 1 copper atom parameters being very different from usual blue copper proteins but very similar to those of stellacyanin. The enzymatic activity of the Met467Gln mutant for bilirubin is quite low (0.3%), but the activity for potassium ferrocyanide is similar (130%) to that of the wild type enzyme. These results indicate that Met467 is important for characterizing the features of the type 1 copper of bilirubin oxidase. (+info)
Inhibitory effect of Cordyceps sinensis on spontaneous liver metastasis of Lewis lung carcinoma and B16 melanoma cells in syngeneic mice.
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We investigated the effect of the water extract of Cordyceps sinensis (WECS) on liver metastasis of Lewis lung carcinoma (LLC) and B16 melanoma (B16) cells in mice. C57BL/6 mice were given a s.c. injection of LLC and B16 cells and sacrificed 20 and 26 days after tumor inoculation, respectively. WECS was daily administered p.o. to the mice in a dose of 100 mg/kg body weight (wt.) in the experiment of LLC and in a dose of 100 or 200 mg/kg body wt. in the experiment of B16 from one week before tumor inoculation to one day before the date of sacrifice. The tumor cells increased in the thigh in LLC-inoculated mice and in the footpad in B16-inoculated mice. The relative liver wt. of the tumor-inoculated mice significantly increased as compared to that of the normal mice due to the tumor metastasis, as verified by the hematoxylin-eosin staining pathological study in the LLC experiment. The relative liver wt. of the WECS-administered mice significantly decreased relative to that of the control mice in both the LLC and B16 experiments. WECS showed a strong cytotoxicity against LLC and B16 cells, while cordycepin (3'-deoxyadenosine), an active component of WECS, was not cytotoxic against these cells. These findings suggest that WECS has an anti-metastatic activity that is probably due to components other than cordycepin. (+info)
Activation of in vivo Kupffer cell function by oral administration of Cordyceps sinensis in rats.
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We investigated the effect of water extracts of Cordyceps sinensis (WECS) on Kupffer cell function in rats. Rats were received a single i.v. injection of a colloidal carbon solution and then the clearance rate from the blood were measured. The rats had been daily administered with WECS, p.o. at a dose of 200 mg/kg for 25 days until the day before the injection of colloidal carbon. The half-life of the colloidal carbon in the blood of rats administered WECS 200 mg/kg was significantly shorter than that of the control rats. This suggests that accelerated function of Kupffer cells is partially involved in the anti-metastatic action of WECS. (+info)
Interkingdom host jumping underground: phylogenetic analysis of entomoparasitic fungi of the genus cordyceps.
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Most members of the ascomycetous genus Cordyceps are endoparasitic fungi of insects and other arthropods, but about 20 of the 300 described species are parasitic to hart's truffles, Elaphomyces spp. In order to understand the evolution of host specificity and the process of interkingdom host jumping in Cordyceps, we investigated the phylogenetic relationships of 22 representatives, including 4 truffle parasites and 18 insect parasites, based on nuclear and mitochondrial rDNA sequences. Five monophyletic groups were identified in both nuclear and mitochondrial phylogenies. In three of the five clades, the members utilized hosts from the same insect group, suggesting that the endoparasite-host connections have been conserved to some extent. On the other hand, it was also shown that major host shifts between distantly related insects must have occurred repeatedly. Notably, phylogenetic analyses strongly suggested that parasites of hart's truffles originated from parasites of cicada nymphs during the evolution of the CORDYCEPS: The common habitats of cicada nymphs and hart's truffles, deep underground and associated with tree roots, suggest that the interkingdom host jumping from Animalia to Fungi might have been promoted by the overlapping ecological niche of the unrelated hosts. This finding provides an impressive case of a drastic host shift in favor of the host habitat hypothesis. (+info)
Cutaneous infection caused by Cylindrocarpon lichenicola in a patient with acute myelogenous leukemia.
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Cylindrocarpon lichenicola is a saprophytic soil fungus which has rarely been associated with human disease. We report the first case of localized invasive cutaneous infection caused by this fungus in a 53-year-old male from the rural midwestern United States with relapsed acute myelogenous leukemia. On admission for induction chemotherapy, the patient was noted to have an abrasive laceration between the fourth and fifth metacarpophalangeal joints and on the dorsum of the right hand, which progressed to frank ulceration following chemotherapy. A biopsy provided an initial diagnosis of an invasive fungal infection consistent with aspergillosis based on the histopathological appearance of the mold in tissue. Multiple positive fungal cultures which were obtained from the biopsied tissue were subsequently identified by microscopic and macroscopic characteristics to be C. lichenicola. The infection resolved following marrow regeneration, aggressive debridement of the affected tissue, and treatment with amphotericin B. This case extends the conditions associated with invasive disease caused by C. lichenicola. (+info)
Contribution of fungal loline alkaloids to protection from aphids in a grass-endophyte mutualism.
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Fungal endophytes provide grasses with enhanced protection from herbivory, drought, and pathogens. The loline alkaloids (saturated 1-aminopyrrolizidines with an oxygen bridge) are fungal metabolites often present in grasses with fungal endophytes of the genera Epichloe or Neotyphodium. We conducted a Mendelian genetic analysis to test for activity of lolines produced in plants against aphids feeding on those plants. Though most loline-producing endophytes are asexual, we found that a recently described sexual endophyte, Epichloe festucae, had heritable variation for loline alkaloid expression (Lol+) or nonexpression (Lol-). By analyzing segregation of these phenotypes and of linked DNA polymorphisms in crosses, we identified a single genetic locus controlling loline alkaloid expression in those E. festucae parents. We then tested segregating Lol+ and Lol- full-sibling fungal progeny for their ability to protect host plants from two aphid species, and observed that alkaloid expression cosegregated with activity against these insects. The in planta loline alkaloid levels correlated with levels of anti-aphid activity. These results suggested a key role of the loline alkaloids in protection of host plants from certain aphids, and represent, to our knowledge, the first Mendelian analysis demonstrating how a fungal factor contributes protection to plant-fungus mutualism. (+info)
White grain mycetoma caused by a Cylindrocarpon sp. in India.
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We describe a case of white grain eumycetoma of the foot of an Indian male caused by a slow-growing, poorly sporulating fungus that does not match any known agent of this infection. Histologic examination of a biopsy tissue specimen showed oval, lobular, white granules composed of hyaline, septate hyphae, and thick-walled chlamydospores. Culture of granules from a draining sinus yielded compact, very-slow-growing, poorly sporulating colonies producing a strong reddish brown pigment that diffused into the medium. The fungus was identified as a Cylindrocarpon sp. based on the development of rare cylindrical conidia borne from solitary phialides lacking collarettes, in addition to chlamydospores formed singly or in short chains. (+info)
Diheteropeptin, a new substance with TGF-beta-like activity, produced by a fungus, Diheterospora chlamydosporia. I. Production, isolation and biological activities.
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A new metabolite, diheteropeptin, was found in the culture broth of Diheterospora chlamydosporia Q58044 by screening for TGF-beta-like active substances. Diheteropeptin was extracted from the culture supernatant and purified by a series of chromatographies such as silica gel, gel filtration and HPLC. Diheteropeptin exhibited cytostatic activity in Mv1Lu cells with an IC50 value of 20.3 microM and inhibited histone deacetylase. (+info)