Ivor John Hodgkiss

Proteomic study of a model causative agent of harmful red tide, Prorocentrum triestinum I: Optimization of sample preparation methodologies for analyzing with two‐dimensional electrophoresis

A comprehensive study to find the optimal sample preparation conditions for two‐dimensional electrophoresis (2‐DE) analysis of Prorocentrum triestinum, a model causative agent of harmful algal blooms (HABs) was carried out. The four major sample preparation steps for 2‐DE: (a) cell disruption: i.e. sonication and homogenization with glass beads; (b) protein extraction : i.e. sequential and independent extraction procedures; (c) pre‐electrophoretic treatment: these included (i) treatment with RNAase/DNAase or benzonase; (ii) ultracentrifugation to sediment large macromolecules such as DNA; (iii) desalting and concentration by ultrafiltration through a Microcon centrifugal filter device (MWCO: 3000 daltons); and (iv) desalting by a micro BioSpin chromatography column (MWCO: 6000 daltons); and (d) rehydration buffers, reducing agents and sample application in the first dimension isoelectric focussing were studied. Our results showed that sonication is easy to perform and resulted in a higher protein yield. Among the four extraction buffers, the urea containing buffers resulted in the extraction of the highest amount of protein while tris(hydroxymethyl)aminomethane buffers and trichloroacetic acid (TCA)/acetone precipitation allowed detection of a higher number of protein species (i.e. protein spots). Desalting by BioSpin and ultrafiltration have improved the 2‐DE resolution of the water soluble fraction but have less effect on urea containing fractions. TCA/acetone precipitation was able to desalt all protein fractions independent of the extraction media, however extended exposure to this low pH medium has caused protein modification. Introduction of either DNase/RNase or benzonase treatment did not improve the discriminatory power of the 2‐DE but this treatment did yield 2‐DE with the clearest background. Proteolytic digestion was inhibited by addition of a protease inhibitor cocktail. Taken overall, a combination of sequential extraction and desalting by BioSpin chromatography for sample treatment before first dimension of 2‐DE gave best results based on its simplicity and minimal protein loss. Finally, triscarboxyethylphosphine (TCEP) has performed well as a reducing agent in both the rehydration and equilibration buffers. The rehydration buffer found to be best in this study was 8.0 M urea, 2% 3‐[(3‐cholamidoprphyldimethylamino]‐1‐propanesulfonate, 4 mM TCEP and 1% immobilized pH gradient buffer. Subsequently, we applied this finding and performed 2‐DE analysis on the soluble protein fractions extracted from light‐starved cultured algal cells (nonblooming) and cultured cells grown under optimal conditions (blooming). 2‐DE maps of these algal cultures were visibly different and many differentially expressed proteins were found.

Colonization patterns of wood-inhabiting fungi on baits in Hong kong rivers, with reference to the effects of organic pollution

The diversity of wood-inhabiting fungi was investigated by submerging woody baits at upstream and downstream sites of the Lam Tsuen and Tai Po Rivers in Hong Kong. The diversity of fungi in the Lam Tsuen River was also compared with that on natural woody substrates found in a previous study. There were differences in the species composition between the upstream and downstream sites, possibly reflecting natural variations along the river. The Tai Po River downstream was organically polluted, which appeared to have little effect on species diversity since more species were recorded. Organic pollution may, however, cause a shift in species composition. The fungal communities on baits and natural substrates in the Lam Tsuen River were similar, although a lower diversity was observed on baits. This may be related to the period of submergence and the fact that a single wood type was used. Cercophora spp. occurred frequently downstream in the Tai Po River, while the common species in the Lam Tsuen River were Aquaticola rhomboidea and Pseudoproboscispora aquatica. Further interpretation on the effects of organic pollution was limited because of single collection data but appropriate experimental designs – putting baits in unimpacted sites for assessing human impacts in streams – are suggested.

Three new species of Annulatascus (Ascomycetes) from Hong Kong freshwater habitats

Abstract Annulatascus joannae, A. lactus, and A. tropicalis are described and illustrated from decaying woody substrata in freshwater habitats in Hong Kong. Annulatascus joannae is distinguished by ellipsoidal and thick-walled ascospores whereas A. lacteus has milky ascomata and A. tropicalis has relatively large, fusiform, 1–3-septate ascospores. Annulatascus biatriisporus is reported as a new record in Hong Kong. A key to and a synoptic table of Annulatascus species are provided.

Three new Ophioceras species (Ascomycetes) from the tropics

Ophioceras guttulatum sp. nov.,O. hongkongense sp. nov. andO. palmae sp. nov. are described and illustrated from decaying terrestrial palms and woody substrates in freshwater habitats. They all have black perithecia with long necks, cylindrical asci with refractive apical rings and filiform ascospores.

Enzymatic studies of the extracellular mucilage of two aquatic hyphomycetes, Lemonniera aquatica and Mycocentrospora filiformis

The effect of three carbohydrate-digesting enzymes, β-glucuronidase, lyticase and α-mannosidase and three proteolytic enzymes, α-chymotrypsin, papain and pronase E, on the strength of conidial attachment ofLemonniera aquatica andMycoentrospora filiformis was determined using the LH_Fowler cell Adhesion Measurement Module. Carbohydrate-digesting enzyme treatments showed significant differences in number of attached and detached, conidia versus control samples; little or no effect was observed for the proteolytic enzymes. Scanning and transmission electron microscopy showed different degrees of mucilage digestion by the carbohydrate-digesting enzymes on the germ hyphae, hyphae subtending appressoria, and appressoria of the two species. The loss of mucilage integrity and decrease in mucilage thickness were more pronounced on the hyphal sheaths than on the appressorial sheaths. Lyticase caused the most severe damage to the mucilage and cytoplasm of both fungi, particularlyL. aquatica. β-Glucuronidase and α-mannosidase exhibited more effective mucilage digestion onM. filiformis than onL. aquatica. Results indicate that the mucilage of the two species is mainly polysaccharide, containing more β-1,3-glucans than β-glucuronide and α-mannosyl residues. Variability of mucilage composition exists between these species and also between different structures of the same fungus.

Characterization of the mucilage sheaths of Lemonniera aquatica by lectin-gold labelling

A pre-embedding lectin-gold labelling method was used to characterize the carbohydrate components in the mucilage ofLemonniera aquatica. A specific tissue processing protocol was developed, namely: a) primary fixation in 2% paraformaldehyde and 0.2% glutaraldehyde in PIPES buffer (pH 7.2) for 30 min; b) secondary fixation in 2% glutaraldehyde in the same buffer system for 1 h; c) post-fixation in 1% aqueous OsO4 for 1h; d) embedment in Möllenhaurs resin. The three gold conjugated lectins used were: concanavalin A, wheat germ agglutinin andLimax flavus agglutinin, allowing detection of their complementary saccharides, namely α-d-mannose/α-d-glucose,N-acetyl-d-glucosamine (GluNAc), andN-acetylneuraminic acid (NANA), respectively.N-Acetyl-d-glucosamine and NANA residues were the major components of germ tube mucilage with only a small amount of α-d-manose/α-d-glucose. However, NANA was restricted to the mucilage in the region of germ tube emergence from the conidial arm. The abundance of GluNAc and NANA residues on hyphae and appressoria was less than that on the germ tube. Conversely, α-d-mannose/α-d-glucose was more abundant in the appressorial mucilage. Variability of mucilage composition was found to exist between different structures of the germinated conidium and also between different regions of the same structure. Further, the conidial cell wall ofL. aquatica is not chitinous, and lacks NANA and α-d-mannose/α-d-gluocse.