R. B. Gardiner

Melanin synthesis by Sclerotinia sclerotiorum.

We confirmed that the melanin produced by Sclerotinia sclerotiorum is a dihydroxynaphthalene (DHN). The specific DHN melanogenesis inhibitor test that uses tricyclazole at low levels (typically2–5 ppm) to cause a confirmatory appearance of soluble red-brown inhibition products does not work when analyzing melanin synthesis in the sclerotia of S. sclerotiorum. We demonstrated the presence of scytalone dehydratase, an enzyme specific to DHN melanogenesis, in melanized sclerotia and melanized nonsclerotial mycelia and observed formation of mycelial nonsclerotial melanin when the fungus was grown on the surface of sterilized dialysis membrane or in rich organic media. Nonsclerotial melanized hyphae in wild type and mutant strains showed the typical excretion of pigmented inhibition products of the DHN pathway in the presence of tricyclazole, and one of these products, 2-hydroxyjuglone, was identified by thin layer chromatography and spectroscopy. We report basic conditions for sclerotial melanin degradation by the white rot fungus Phanerochaete chrysosporium.

Serological Studies on the Fimbriae of Yeasts and Yeastlike Species

Antisera prepared against pure fimbrial proteins of Ustilago violacea (antiserum U) and of Rhodotorula rubra (antiserum R) agglutinate cells carrying fimbriae of appropriate cross-reactivity. All except one of 24 species of smut fungi in the Ustilaginales agglutinated strongly with antiserum U and varied from no response to moderate agglutination with antiserum R. The exceptional species, Tilletia caries, has been shown to be afimbriate under tested conditions. A group of 42 strains of basidiomycetous yeasts varied in their response from no agglutination to either antiserum, agglutination with one antiserum, or agglutination with both antisera. Approximately one-quarter were afimbriate under the tested conditions. Fimbriated basidiomycetous yeasts, like the smut fungi, produce long flexuous fimbriae with a maximum length ca. 10-20 μm. Eleven ascomycetous yeasts produced a short fringe of fimbriae, usually ca. 0.5-1.0 μm long. Many of these species were agglutinated by one or both antisera, but none of five algal species responded to either antibody. No strains responded to control sera, and tests on a few species with purified antisera confirmed the results with crude sera. The agglutination response was correlated with visible fimbriation on all occasions, including studies with temperature-sensitive fimbriated strains. These results indicate that a family of relatively conserved proteins is common to many species of smut fungi, basidiomycetous yeasts, and ascomycetous yeasts.

In Situ Loading of Basic Fibroblast Growth Factor Within Porous Silica Nanoparticles for a Prolonged Release

Basic fibroblast growth factor (bFGF), a protein, plays a key role in wound healing and blood vessel regeneration. However, bFGF is easily degraded in biologic systems. Mesoporous silica nanoparticles (MSNs) with well-tailored porous structure have been used for hosting guest molecules for drug delivery. Here, we report an in situ route to load bFGF in MSNs for a prolonged release. The average diameter (d) of bFGF-loaded MSNs is 57 ± 8 nm produced by a water-in-oil microemulsion method. The in vitro releasing profile of bFGF from MSNs in phosphate buffer saline has been monitored for 20 days through a colorimetric enzyme linked immunosorbent assay. The loading efficiency of bFGF in MSNs is estimated at 72.5 ± 3%. In addition, the cytotoxicity test indicates that the MSNs are not toxic, even at a concentration of 50 μg/mL. It is expected that the in situ loading method makes the MSNs a new delivery system to deliver protein drugs, e.g. growth factors, to help blood vessel regeneration and potentiate greater angiogenesis.

Fungal fimbriae. IV. Composition and properties of fimbriae from Ustilago violacea

Abstract The fimbriae of Ustilago violacea consist of long protein fibrils of 7-nm diameter. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the fimbriae of this species and of several other species of Ustilago and Rhodotorula demonstrated that they are composed of a protein of 74, 000 Da which can spontaneously assemble into 7-nm fibrils. No carbohydrate moiety was detected. Fimbrial protein retained both its fibrillar structure and antigenicity when exposed to a variety of chemical treatments, and even when autoclaved. Concentrations of cations greater than 10−1M (monovalent cations) or 5 × 10−2M (divalent cations) resulted in a loss of fimbriae, while the effect of chelators suggested that calcium is important to the structural integrity of fimbriae. Ouchterlony tests using antisera prepared against the fimbriae of U. violacea and R. rubra indicated that while there are differences in the antigenic sites, the fimbrial protein of different basidiomycete species is highly conserved. Fimbrial protein did not react with several mammalian antisera directed against cytoskeletal proteins.

Fungal fimbriae: V. Protein A-gold immunocytochemical labeling of the fimbriae ofUstilago violacea

Abstract Protein A-gold immunocytochemical labeling of long protein appendages (fimbriae) on the surfaces of cells of Ustilago violacea is described. The specificity of the technique is confirmed by a series of serological and morphological controls. The technique allows specific identification and localization of fimbrial antigens in electron microscopic studies of a variety of fungi in both pathogenic and saprophytic situations.

Use of the Black Yeast Phaeococcomyces Fungal Melanin Model System for Preparation of 1,3,6,8‐Tetrahydroxynaphthalene and the Other Components of the DHN Fungal Melanin Pathway

Compounds of the dihydroxynaphthalene (DHN) fungal melanin pathway are in short supply worldwide, especially 1,3,6,8‐tetrahydroxynaphthalene, the chemically unstable starting point of the pathway, and are not commercially available. This is becoming an impediment to physiological survey work in the DHN pathway. We describe the use of our black yeast Phaeococcomyces DHN melanin model system to produce sufficient amounts of 1,3,6,8‐tetrahydroxynaphthalene for physiological studies, using a simple modified culture method that uses an oxygen‐scavenging growth medium containing cysteine and thioglycollate, which is also sealed with an oxygen‐excluding layer of the black yeast. The medium also contains the DHN pathway reductase enzyme inhibitor tricyclazole, which causes excretion of 1,3,6,8‐tetrahydroxynaphthalene and 1,3,8‐dihydroxynaphthalene. The presence of oxygen scavengers and the thick yeast growth prevents oxidation of the hydroxynaphthalenes. Most of the other components of the DHN fungal melanin pathway can be produced using the black yeast or using a scytalone dehydratase negative mutant of the black yeast that accumulates scytalone.

Extracellular protein fibrils in Chrysochromulina breviturrita (Prymnesiophyceae) and their serological relationship to fungal fimbriae

An extensive network of extracellular fibrils was revealed by negative staining in the greenish gold algal flagellate, Chrysochromulina breviturrita. These fibrils were of uniform diameter (4–5 nm), sometimes exceeding 5 μm in length. In addition there were short, narrower fibrils (2–3 nm) on the surface of the flagella. Six protein bands were isolated from spent culture medium by SDS-PAGE and one of 80,000 Da was found to polymerize after dialysis into 4–5 nm fibrils identical to those found on the cell surface. Two other proteins of 58,000 Da and 65,000 Da also formed 4–5 nm fibrils but these were either rare or of a shorter length and different appearance. An antiserum directed against the surface 7 nm fibrils (fimbriae) of fungi agglutinated cells of C. breviturrita and some other Prymnesiophyceae and Chrysophyceae, but did not agglutinate cells of algal species in other groups. Immunofluorescence and protein A gold labelling confirmed that antigens related to fungal fimbriae were present on the surface of cells of C. breviturrita. Only the 80,000 and 58,000 Da proteins labelled heavily following protein A gold labelling. Some individual 4–5 nm fibrils labelled with gold were observed in the material prepared from the 80,000 Da band. These results therefore establish that C. breviturrita produces a surface network of fibrils that are serologically related to the fimbriae of fungi, and suggest a previously unrecognized relationship between members of the Prymnesiophyceae, Chrysophyceae and fungal groups.

Impact of ventilation-induced lung injury on the structure and function of lamellar bodies

Alterations to the pulmonary surfactant system have been observed consistently in ventilation-induced lung injury (VILI) including composition changes and impairments in the surface tension reducing ability of the isolated extracellular surfactant. However, there is limited information about the effects of VILI on the intracellular form of surfactant, the lamellar body. It is hypothesized that VILI leads to alterations of lamellar body numbers and function. To test this hypothesis, rats were randomized to one of three groups, nonventilated controls, control ventilation, and high tidal volume ventilation (VILI). Following physiological assessment to confirm lung injury, isolated lamellar bodies were tested for surfactant function on a constrained sessile drop surfactometer. A separate cohort of animals was used to fix the lungs followed by examination of lamellar body numbers and morphology using transmission electron microscopy. The results showed an impaired ability of reducing surface tension for the lamellar bodies isolated from the VILI group as compared with the two other groups. The morphological assessment revealed that the number, and the relative area covered by, lamellar bodies were significantly decreased in animals with VILI animals as compared with the other groups. It is concluded that VILI causes significant alterations to lamellar bodies. It is speculated that increased secretion causes a depletion of lamellar bodies that cannot be compensated by de novo synthesis of surfactant in these injured lungs.

Local Release of Basic Fibroblast Growth Factor (bFGF) through Silica Nanoparticles-laden Biomimic Matrix

Basic fibroblast growth factor (bFGF), a protein, plays a key role in wound healing and blood vessel regeneration. However, most negative effects in vivo , or in vitro result from the over dosage of bFGF. Furthermore, it needs to keep the bFGF from protein denaturant. Thus, this study aims to develop a new delivery system based on silica nanoparticles (SiO 2 NPs) dispersed in collagen patch for delivery of the bFGF in a local and prolonged manner. In this research, SiO 2 NPs are used to encapsulate bFGF through a modified water-in-oil micro-emulsion. The bFGF-loaded nanoparticles afterwards are dispersed in the collagen-based matrix through a EDC cross-linking step. The in vitro release kinetics of SiO2 NPs - encapsulated bFGF with and without collagen matrix have been monitored through ELISA. In addition, the cytotoxicity of SiO 2 NPs is investigated by studying the viability of Human Umbilical Vein Endothelial Cells (HUVEC) under the different concentrations of SiO 2 NPs. It has found the average diameter ( d ) for SiO 2 NPs encapsulating bFGF is 45 ± 8 nm with a loading efficiency of 72.5±3%. The maximum concentration of bFGF locally released from SiO 2 NPs impregnated collagen matrix can be monitored after 30 days, while bFGF released from SiO 2 NPs can be detected in 20 days. The further prolonged releasing after the nanoparticle-encapsulated bFGF laden collagen matrix is possibly due to the interaction between the nanoparticles and collagen matrix. In addition, the biocompatibility of the SiO 2 NP has been investigated. We found that SiO 2 NPs at the concentration of 50 μg/ml can still keep the cell alive. The results indicate that the nanoparticle-laden collagen matrix can locally deliver growth factor in a prolonged manner. This new delivery system may benefit to blood vessel regeneration and potentiate greater angiogenesis.