R. Hugh Dunstan

ERYTHROCYTE ANTIOXIDANT SYSTEMS PROTECT CULTURED ENDOTHELIAL CELLS AGAINST OXIDANT DAMAGE.

A study was undertaken to assess the ability of the erythrocyte to protect other tissues against oxidative damage. Radiolabelled (51Cr) human umbilical vein endothelial cells (HUVEC) were incubated with erythrocytes and neutrophils activated with phorbol myristate acetate (PMA). Damage to the endothelial cells was indicated by release of radioactivity into the suspending medium. We found that the co‐incubation of HUVEC with an increasing range of erythrocyte concentrations resulted in a dose‐dependent reduction in the release of radioactivity. When the ability of superoxide to cross the erythrocyte membrane or the glutathione systems was inhibited, the extent of endothelial cell damage increased. Inhibition of the catalase system did not affect results. It was concluded that the erythrocytes afforded some protection against oxidative damage to the endothelial cells by taking up and deactivating the superoxide ions. This protection depends upon intact erythrocyte antioxidant systems. These data support the hypothesis that erythrocytes can provide antioxidant protection to other tissues in vivo.

Effect of irradiance on fatty acid, carotenoid, total protein composition and growth of Microcystis aeruginosa

Abstract The cyanobacterium, Microcystis aeruginosa , was grown under low, standard and high irradiance intensities (20, 40 and 70 μmol m −1 s −1 ) to determine whether irradiance affected pigment, lipid and protein composition, growth yields and the total dry weights of the cell cultures. The components detected in the saponified lipid extracts included C 14 to C 20 fatty acids with various levels of saturation, odd chain-length fatty acids and long-chain alcohols. 16:0 was the main fatty acid detected in all samples and its percentage abundance was significantly higher in cells from the lower and standard irradiance intensities compared with the high irradiance cells. Conversely, the proportions of the 14:0, 15:0, 17:0, 18:0 and 20:0 fatty acids were higher in the high irradiance cells compared with the low and standard irradiance cells. Polyunsaturated fatty acid concentrations were reduced and those of monounsaturated fatty acids were increased in the high irradiance cells compared with the low and standard irradiance cells. Chlorophyll a , zeaxanthin and β-carotene were the most abundant pigments detected. Cells exposed to the standard irradiance treatment had substantially higher amounts of carotenoid, chlorophyll a and total protein after 15 days of growth compared with cells exposed to either the low or high irradiance intensities. The ratios of the zeaxanthin, echinenone and β-carotene with respect to chlorophyll a from the high irradiance cells were approximately double those observed in the low and standard irradiance regimes. It was concluded that the changes in the fatty acid composition occurred as a cellular response to reduce the susceptibility of the cyanobacterial membranes to photo-oxidation.

Altered amino acid excretion in children with autism

Abstract Autism is a complex and life-long behavioural disorder of unknown aetiology. Recent reports have indicated the involvement of digestive tract dysfunction and possible complications from inadequate nutrition. In this study, 34 autistic children (12 untreated and 22 receiving therapeutic treatments related to digestive function and nutritional uptake) and 29 control subjects (all 5–15 years of age) were investigated to determine whether there were any anomalies in the urinary excretion of amino acids, glucose, sucrose, arabinose and tartaric acid using GC/FID and GC/MS analysis techniques. Significantly lower relative urinary levels of essential amino acids were revealed for both the untreated (mean ± SEM, 32.53 ± 3.09%) and treated (31.98 ± 2.87%) autistic children compared with the controls (37.87 ± 1.50%). There were no significant differences in measured excretions of sugars or tartaric acid. It was concluded that the untreated autistic children had evidence of altered metabolic homeostasis.

Oxidative stress and the fetotoxicity of alcohol consumption during pregnancy

Pregnant Quackenbush Special mice were exposed to ethanol under semiacute (3.0 g/kg body weight intragastrically, days 7 to 12 of pregnancy), and chronic conditions (15% ethanol in drinking water for 5 weeks before and during pregnancy) to assess whether embryo-fetotoxic actions of the drug involve oxidative stress effects. Effects were monitored both in the maternal system and embryo. Alcohol compromised the maternal system by increasing the generation of lipid peroxides in the liver. It also decreased glutathione and vitamin E levels, and glutathione peroxidase and superoxide dismutase activities in this organ. Glutathione peroxidase activity in the maternal blood decreased. Only minor alcohol-induced changes occurred in the uterine endometrium, including decreased xanthine oxidase and increased gamma-glutamyl transpeptidase. Similarly, only few changes were induced in day-12 embryos by alcohol. In this case, glutathione content and xanthine oxidase activity decreased while glutathione reductase activity increased following exposure to the chronic regime. With the possible exception of the maternal liver where evidence of oxidative damage was detected, these results do not reflect substantial changes in the antioxidant defences of either the pregnant mouse or embryo. However, the changes may contribute to the growth retarding and other fetotoxic effects of alcohol when they are totalled into the multifactorial actions of the drug.

Effect of high irradiance and iron on volatile odour compounds in the cyanobacterium Microcystis aeruginosa

The cyanobacterium, Microcystis aeruginosa was exposed to direct sunlight for 3, 6 or 9 h in media containing either low or high concentrations of iron, in order to determine any effects on the composition of volatile odour compounds (VOCs) released under photooxidative conditions. The most abundant VOCs detected included aliphatic hydrocarbons (C15-C21), naphthalene and the terpenoid compounds, beta-cyclocitral, and beta-ionone. Exposure to sunlight and low iron concentrations resulted in a decrease in beta-cyclocitral, beta-ionone, heptadecane and the total VOCs concentration after 9 h with respect to the control cultures. Six VOCs detected in the low iron cells were not detected in any of the high iron cells. However, those VOCs present in the high iron cells, in general, occurred at higher concentrations than the equivalent low iron cells after exposure to the sunlight conditions. Consequently, it was concluded that exposure to both high irradiance and high iron concentrations influenced the VOCs composition in cyanobacteria and this was interpreted to represent a cellular change during the photooxidation-promoting conditions.

Hematologic and Urinary Excretion Anomalies in Patients with Chronic Fatigue Syndrome

Patients with chronic fatigue syndrome (CFS) have a broad and variable spectrum of signs and symptoms with variable onsets. This report outlines the results of a single-blind, cross-sectional research project that extensively investigated a large cohort of 100 CFS patients and 82 nonfatigued control subjects with the aim of performing a case-control evaluation of alterations in standard blood parameters and urinary amino and organic acid excretion profiles. Blood biochemistry and full blood counts were unremarkable and fell within normal laboratory ranges. However, the case-control comparison of the blood cell data revealed that CFS patients had a significant decrease in red cell distribution width and increases in mean platelet volume, neutrophil counts, and the neutrophil-lymphocyte ratio. Evaluation of the urine excretion parameters also revealed a number of anomalies. The overnight urine output and rate of amino acid excretion were both reduced in the CFS group (P < 0.01). Significant decreases in the urinary excretion of asparagine (P < 0.0001), phenylalanine (P < 0.003), the branch chain amino acids (P < 0.005), and succinic acid (P < 0.0001), as well as increases in 3-methylhistidine (P < 0.05) and tyrosine (P < 0.05) were observed. It was concluded that the urinary excretion and blood parameters data supported the hypothesis that alterations in physiologic homeostasis exist in CFS patients.

Effect of Low Temperature on Growth and Ultra-Structure of Staphylococcus spp

The effect of temperature fluctuation is an important factor in bacterial growth especially for pathogens such as the staphylococci that have to remain viable during potentially harsh and prolonged transfer conditions between hosts. The aim of this study was to investigate the response of S. aureus, S. epidermidis, and S. lugdunensis when exposed to low temperature (4°C) for prolonged periods, and how this factor affected their subsequent growth, colony morphology, cellular ultra-structure, and amino acid composition in the non-cytoplasmic hydrolysate fraction. Clinical isolates were grown under optimal conditions and then subjected to 4°C conditions for a period of 8 wks. Cold-stressed and reference control samples were assessed under transmission electron microscopy (TEM) to identify potential ultra-structural changes. To determine changes in amino acid composition, cells were fractured to remove the lipid and cytoplasmic components and the remaining structural components were hydrolysed. Amino acid profiles for the hydrolysis fraction were then analysed for changes by using principal component analysis (PCA). Exposure of the three staphylococci to prolonged low temperature stress resulted in the formation of increasing proportions of small colony variant (SCV) phenotypes. TEM revealed that SCV cells had significantly thicker and more diffuse cell-walls than their corresponding WT samples for both S. aureus and S. epidermidis, but the changes were not significant for S. lugdunensis. Substantial species-specific alterations in the amino acid composition of the structural hydrolysate fraction were also observed in the cold-treated cells. The data indicated that the staphylococci responded over prolonged periods of cold-stress treatment by transforming into SCV populations. The observed ultra-structural and amino acid changes were proposed to represent response mechanisms for staphylococcal survival amidst hostile conditions, thus maintaining the viability of the species until favourable conditions arise again.

Extensive bacterial diversity indicates the potential operation of a dynamic micro-ecology within domestic rainwater storage systems

The concept that domestic rainwater storage tanks may host sustainable microbial ecosystems has not previously been addressed. The bacterial diversity, cultivated from more than 80 samples from 22 tanks at various locations across eastern Australia, is presented here as prima facie evidence for the potential operation of a functional micro-ecology within rainwater storage systems. Cultivated isolates were found to comprise members of four major bacterial divisions; Proteobacteria, Firmicutes, Actinobacteria and Bacteroidetes, including more than 200 species from 80 different genera. The pattern of abundance distribution was typical of that observed in most natural communities, comprising a small number of abundant taxa and a multitude of rare taxa, while the specific composition resembled that previously described in a number of natural aquatic systems. Although Proteobacteria from alpha, beta and gamma sub-classes were dominant, a set of core taxa comprising representative genera from all four phyla could be identified. Coliform and other species specifically associated with faecal material comprised <15% of the species identified, and represented <1.5% of total average abundance. The composition of the cultivated populations and scope of diversity present, suggested that rainwater tanks may support functional ecosystems comprising complex communities of environmental bacteria, which may have beneficial implications for the quality of harvested rainwater.

Phenotypic Variants of Staphylococci and Their Underlying Population Distributions Following Exposure to Stress

This study investigated whether alterations in environmental conditions would induce the formation of small colony variant phenotypes (SCV) with associated changes in cell morphology and ultra-structure in S. aureus, s. epidermidis, and S. lugdunensis. Wild-type clinical isolates were exposed to low temperature (4°C), antibiotic stress (penicillin G and vancomycin; 0-10,000 µg mL-1), pH stress (pH 3-9) and osmotic challenge (NaCl concentrations of 0-20%). Changes in cell diameter, cell-wall thickness, and population distribution changes (n ≥ 300) were assessed via scanning and transmission electron microscopy (SEM and TEM), and compared to control populations. Our analyses found that prolonged exposure to all treatments resulted in the subsequent formation of SCV phenotypes. Observed SCVs manifested as minute colonies with reduced haemolysis and pigmentation (NaCl, pH and 4°C treatments), or complete lack thereof (antibiotic treatments). SEM comparison analyses revealed significantly smaller cell sizes for SCV populations except in S. aureus and S. epidermidis 10% NaCl, and S. epidermidis 4°C (p<0.05). Shifts in population distribution patterns were also observed with distinct sub-populations of smaller cells appearing for S. epidermidis, and S. lugdunensis. TEM analyses revealed significantly thicker cell-walls in all treatments and species except S. lugdunensis exposed to 4°C. These findings suggest that staphylococci adapted to environmental stresses by altering their cell size and wall thickness which could represent the formation of altered phenotypes which facilitate survival under harsh conditions. The phenotypic response was governed by the type of prevailing environmental stress regime leading to appropriate alterations in ultra-structure and size, suggesting downstream changes in gene expression, the proteome, and metabolome.

Metabolomic and proteomic responses of Staphylococcus aureus to prolonged cold stress

UNLABELLED The high pathogenicity of Staphylococcus aureus is thought to be due to its extraordinary capacity to rapidly adapt to changes in environmental conditions. This study was carried out to investigate whether the cytoplasmic profiles of metabolites and proteins of S. aureus were altered in response to prolonged exposure to cold stress. Metabolic profiling and proteomics were used to characterise alterations in cytoplasmic proteins and metabolites in cells from the mid-exponential phase of growth under ideal conditions at 37°C and compared with equivalent cells exposed to prolonged cold stress for 2 weeks at 4°C. Principle component analysis (PCA) of the metabolomic and proteomic data indicated that, at the mid-exponential phase of growth, prolonged cold stress conditions generated cells with different metabolite and protein profiles compared with those grown at 37°C. Nine ribosomal proteins and citric acid were substantially elevated in the cytoplasmic fractions from the cells adapted to cold-stress but most amino acids showed a reduction in their concentration in cold-stressed samples. The data provided strong evidence supporting the hypothesis that specific changes in metabolic homeostasis and protein composition were critical to the adaptive processes required for survival under cold stress. BIOLOGICAL SIGNIFICANCE Work in our laboratory has shown that prolonged exposure of S. aureus to cold stress can result in the formation of small colony variants (SCVs) associated with significant alterations in the cell wall composition. Further studies revealed that S. aureus altered cell size and cell wall thickness in response to exposure to cold temperatures, alterations in pH and exposure to antibiotics. The current study has utilised the prolonged exposure to cold stress as a model system to explore changes in the proteome and associated metabolic homeostasis following environmental challenges. The study provides an improved understanding of how S. aureus adapts to the changing environment whilst in transition between human hosts. The results indicated an unexpected production of 9 ribosomal proteins and citric acid in response to cold stress suggesting specific survival roles for these proteins and citric acid as an adaptation mechanism for empowering survival under these conditions.