Physcion from marine-derived fungus Microsporum sp. induces apoptosis in human cervical carcinoma HeLa cells.

Recently, the relationship between apoptosis and cancer has been emphasized and the induction of apoptosis is recognized as one of the key mechanisms of anti-cancer agents. Marine-derived fungi are valuable sources of structurally diverse bioactive anticancer agents. In the present study, a marine-derived fungus, Microsporum sp. was cultured and an anthraquinone derivative, physcion (11.8 mg) was isolated from the culture broth extract (1710 mg). Physcion has shown cytotoxic effect on human cervical carcinoma HeLa cells and its apoptosis induction in HeLa cells was investigated by the expressions of p53, p21, Bax, Bcl-2, caspase-9, and caspase-3 proteins. The Western blot analysis has revealed that physcion could significantly induce cell apoptosis through down-regulating of Bcl-2 expression, up-regulating of Bax expression, and activating the caspase-3 pathway. Furthermore, physcion induced the formation of reactive oxygen species (ROS) in HeLa cells. Collectively, these results suggest that physcion could be a potential candidate in the field of anticancer drug discovery against human cervical cancer.

Melanogenesis in dermatophyte species in vitro and during infection.

Dermatophytes are keratinophilic fungi that are the most common cause of fungal skin infections worldwide. Melanin has been isolated from several important human fungal pathogens, and the polymeric pigment is now recognized as an important virulence determinant. This study investigated whether dermatophytes, including Trichophyton rubrum, Trichophyton mentagrophytes, Epidermophyton floccosum and Microsporum gypseum, produce melanin or melanin-like compounds in vitro and during infection. Digestion of the pigmented microconidia and macroconidia of dermatophytes with proteolytic enzymes, denaturant and hot concentrated acid yielded dark particles that retained the size and shape of the original fungal cells. Electron spin resonance spectroscopy revealed that particles derived from pigmented conidia contained a stable free radical signal, consistent with the pigments being a melanin. Immunofluorescence analysis demonstrated reactivity of a melanin-binding mAb with the pigmented conidia and hyphae, as well as the isolate particles. Laccase, an enzyme involved in melanization, was detected in the dermatophytes by an agar plate assay using 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) as the substrate. Skin scrapings from patients with dermatophytoses contained septate hyphae and arthrospores that were reactive with the melanin-binding mAb. These findings indicate that dermatophytes can produce melanin or melanin-like compounds in vitro and during infection. Based on what is known about the function of melanin as a virulence factor of other pathogenic fungi, this pigment may have a similar role in the pathogenesis of dermatophytic diseases.

A new radical scavenging anthracene glycoside, asperflavin ribofuranoside, and polyketides from a marine isolate of the fungus microsporum.

A new anthracene glycoside, asperflavin ribofuranoside (1), and the previously described polyketides, flavoglaucin (2), isodihydroauroglaucin (3), and citrinin (4) have been isolated from the marine-derived fungus Microsporum sp. The structure and absolute stereochemistry of a new compound (1) was assigned on the basis of physicochemical data. Compounds 1-3 exhibited a significant radical scavenging activity against 1,1-diphenyl-2-picrylhydrazyl (DPPH) with IC(50) values of 14.2, 11.3, and 11.5 microM, respectively, which are more potent than the positive control, ascorbic acid (IC(50), 20 microM). Compound 1 also showed a moderate antibacterial activity against the methicillin-resistant and multidrug-resistant Staphylococcus aureus (MRSA and MDRSA) with MIC value of 50 microg/ml.

Isolation of total RNA from dermatophytes.

We report a method for the preparation of total RNA from the anthropophilic dermatophyte Trichophyton rubrum. To generate large quantities of mycelia, the fungus was grown in liquid culture medium. The harvested mycelial mass was ground to a fine powder in liquid nitrogen and homogenized in guanidine isothiocyanate buffer followed by ultracentrifugation of the obtained suspension through a caesium chloride gradient. Analysis of the prepared RNA showed two prominent ribosomal RNA (rRNA) bands of about 3.36 and 1.82 kb. Northern blot hybridization with a beta-actin cDNA confirmed the high quality of the fungal mRNA. Successful isolation of RNA from two other dermatophyte species, namely Trichophyton mentagrophytes and Microsporum canis, demonstrated the general applicability of the described procedure.

The in vitro and in vivo production of a 31.5-kD keratinolytic subtilase from Microsporum canis and the clinical status in naturally infected cats.

BACKGROUND: Microsporum-canis-infected cats, especially the asymptomatic infected ones, are mainly responsible for the zoonotic disease. The important variability of the clinical signs in cats is poorly understood. Recently, a 31.5-kD keratinolytic subtilase was found to be a putative virulence factor. OBJECTIVE: To investigate the possible relationship between the clinical status of dermatophytic cats and the production of the keratinase. METHODS: Seven M. canis strains isolated either from clinically affected, asymptomatic infected or mechanical carrier cats were tested for the in vitro production of the enzyme. The immunohistochemical detection of the enzyme was also assessed in skin biopsies of 4 symptomatic and 7 asymptomatic naturally infected cats. RESULTS: All the strains produced in vitro a 31.5-kD keratinolytic subtilase. The enzyme was present in all but 1 of the infected cats. CONCLUSION: The production of the keratinase is not a factor directly responsible for the clinical picture seen in M.-canis-infected cats.

Solution conformation and dynamics of a fungal cell wall polysaccharide isolated from Microsporum gypseum.

The conformational and dynamical features of a branched mannan isolated from a fungal cell wall have been analysed by homo and heteronuclear NMR methods, employing different magnetic fields. 1H NMR cross relaxation times have been obtained for this polysaccharide and have been interpreted qualitatively using different motional models. 13C NMR relaxation parameters (T1, T2, NOE) have also been measured and interpreted using different approximations based on the Lipari and Szabo model free approach. The analysis of the data indicate the existence of important flexibility for the different linkages of the polysaccharide. Motions in the range of 4-6 ns contribute to the relaxation of the macromolecule, although faster internal motions in the 500 ps and 100 ps timescales are also present. These time scales indicate that segmental motions as well as internal motions around the glycosidic linkages are the major sources of relaxation for this molecule at 318 K. Molecular dynamics simulations have also been performed. The obtained results also indicate that the polysaccharide possess a substantial amount of conformational freedom.

Gypsetin, a new inhibitor of acyl-CoA:cholesterol acyltransferase produced by Nannizzia gypsea var. incurvata IFO 9228. I. Fermentation, isolation, physico-chemical properties and biological activity.

A novel inhibitor of acyl-CoA:cholesterol acyltransferase (ACAT), designated gypsetin, was isolated from the cultured broth of Nannizzia gypsea var. incurvata IFO 9228 by solvent extraction, silica gel chromatography and crystallization. Gypsetin inhibited rat liver microsomal ACAT activity competitively with respect to the substrate oleoyl-CoA with an apparent Ki value of 5.5 microM. In cultured macrophage J774 cells incubated with oxidized low density lipoprotein, gypsetin inhibited cholesteryl ester formation from [14C]oleate by 50% at a concentration of 0.65 microM without affecting cell surface binding, uptake and degradation of the lipoprotein.

Gypsetin, a new inhibitor of acyl-CoA: cholesterol acyltransferase produced by Nannizzia gypsea var. incurvata IFO 9228. II. Structure determination.

The elucidation of the structure of gypsetin, a new inhibitor of acyl-CoA: cholesterol acyltransferase, is described in this paper. By spectroscopic and X-ray crystallographic analyses, the structure of gypsetin has been determined to be 8a,16a-dihydroxy-5a,13a-bis[1,1-dimethylallyl]-[1]benzazolidine [3''' 2''':4'',5'']azolidino[1'',2'':4',5'][1,4]perhydrodiazin[1', 2':1, 5]azolidino[2,3-b]-[1]benzazolidine-7,15-dione.

Polyacrylamide gel electrophoresis patterns of some Microsporum species.

The paper reports observations made of various Microsporum species during a PAGE pattern study of the epidemiology of Microsporum canis. It is shown that M. audouinii can readily be distinguished from M. canis but that M. equinum is more difficult to separate. M. rivalierii was distinguishable from M. audouinii, confirming previous reports. M. distortum could not be separated from M. canis by PAGE gels of total protein or by isozyme patterns generated. There are particular problems with studies of that type on dermatophyte protein in that the intrinsic proteases degrade the preparations.