Characterization of virus-like particles associated with the human faecal and caecal microbiota

This work represents an investigation into the presence, abundance and diversity of virus-like particles (VLPs) associated with human faecal and caecal samples. Various methodologies for the recovery of VLPs from faeces were tested and optimized, including successful down-stream processing of such samples for the purpose of an in-depth electron microscopic analysis, pulsed-field gel electrophoresis and efficient DNA recovery. The applicability of the developed VLP characterization method beyond the use of faecal samples was then verified using samples obtained from human caecal fluid.

Effects of oxidative stress on the cardiac myocyte proteome: modifications to peroxiredoxins and small heat shock proteins

Endogenous oxidative stress is a likely cause of cardiac myocyte death in vivo. We examined the early (0-2 h) changes in the proteome of isolated cardiac myocytes from neonatal rats exposed to H2O2 (0.1 mM), focussing on proteins with apparent molecular masses of between 20 and 30 kDa. Proteins were separated by two-dimensional gel electrophoresis (2DGE), located by silver-staining and identified by mass spectrometry. Incorporation of [35S]methionine or 32Pi was also studied. For selected proteins, transcript abundance was examined by reverse transcriptase-polymerase chain reaction. Of the 38 protein spots in the region, 23 were identified. Two families showed changes in 2DGE migration or abundance with H2O2 treatment: the peroxiredoxins and two small heat shock protein (Hsp) family members: heat shock 27 kDa protein 1 (Hsp25) and alphaB-crystallin. Peroxiredoxins shifted to lower pI values and this was probably attributable to 'over-oxidation' of active site Cys-residues. Hsp25 also shifted to lower pI values but this was attributable to phosphorylation. alphaB-crystallin migration was unchanged but its abundance decreased. Transcripts encoding peroxiredoxins 2 and 5 increased significantly. In addition, 10 further proteins were identified. For two (glutathione S-transferase pi, translationally-controlled tumour protein), we could not find any previous references indicating their occurrence in cardiac myocytes. We conclude that exposure of cardiac myocytes to oxidative stress causes post-translational modification in two protein families involved in cytoprotection. These changes may be potentially useful diagnostically. In the short term, oxidative stress causes few detectable changes in global protein abundance as assessed by silver-staining.

The effect of simulated flooding post-anthesis on pre-harvest sprouting and subsequent seed longevity in contrasting rice cultivars

Unpredictable flooding is a major constraint to rice production. It can occur at any growth stage. The effect of simulated flooding post-anthesis on yield and subsequent seed quality of pot-grown rice (Oryza sativa L.) plants was investigated in glasshouses and controlled-environment growth cabinets. Submergence post-anthesis (9-40 DAA) for 3 or 5 days reduced seed weight of japonica rice cv. Gleva, with considerable pre-harvest sprouting (up to 53%). The latter was greater the later in seed development and maturation that flooding occurred. Sprouted seed had poor ability to survive desiccation or germinate normally upon rehydration, whereas the effects of flooding on the subsequent air-dry seed storage longevity (p50) of the non-sprouted seed fraction was negligible. The indica rice cvs IR64 and IR64Sub1 (introgression of submergence tolerance gene Submergence1A-1) were both far more tolerant to flooding post-anthesis than cv. Gleva: four days’ submergence of these two near-isogenic cultivars at 10-40 DAA resulted less than 1% sprouted seeds. The presence of the Sub1A-1 allele in cv. IR64Sub1 was verified by gel electrophoresis and DNA sequencing. It had no harmful effect on loss in seed viability during storage compared with IR64 in both control and flooded environments. Moreover, the germinability and changes in dormancy during seed development and maturation were very similar to IR64. The efficiency of using chemical spray to increase seed dormancy was investigated in the pre-harvest sprouting susceptible rice cv. Gleva. Foliar application of molybdenum at 100 mg L-1 reduced sprouted seeds by 15-21% following 4 days’ submergence at 20-30 DAA. Analyses confirmed that the treatment did result in molybdenum uptake by the plants, and also tended to increase seed abscisic acid concentration. The latter was reduced by submergence and declined exponentially during grain ripening. The selection of submergence-tolerant varieties was more successful than application of molybdenum in reducing pre-harvest sprouting.