The agr radiation: an early event in the evolution of staphylococci

Agr is a global regulatory system in the staphylococci, operating by a classical two-component signaling module and controlling the expression of most of the genes encoding extracellular virulence factors. As it is autoinduced by a peptide, encoded within the locus, that is the ligand for the signal receptor, it is a sensor of population density or a quorum sensor and is the only known quorum-sensing system in the genus. agr is conserved throughout the staphylococci but has diverged along lines that appear to parallel speciation and subspeciation within the genus. This divergence has given rise to a novel type of interstrain and interspecies cross-inhibition that represents a fundamental aspect of the organism's biology and may be a predominant feature of the evolutionary forces that have driven it. We present evidence, using a newly developed, luciferase-based agr typing scheme, that the evolutionary divergence of the agr system was an early event in the evolution of the staphylococci and long preceded the development of the nucleotide polymorphisms presently used for genotyping. These polymorphisms developed, for the most part, within different agr groups; mobile genetic elements appear also to have diffused recently and, with a few notable exceptions, have come to reside largely indiscriminately within the several agr groups.     

Alpha-tocopherol regulation of hepatic cytochrome P450s and ABC transporters in rats

To test the hypothesis that supra-elevated hepatic alpha-tocopherol concentrations would up-regulate mechanisms that result in increased hepatic alpha-tocopherol metabolism and excretion, rats received daily subcutaneous alpha-tocopherol injections (10 mg/100 g body wt) and then were sacrificed on Day 0 or 12 h following their previous injection on Days 3, 6, 9, 12, 15, and 18. Liver alpha-tocopherol concentrations increased from 12 +/- 1 nmol/g (mean +/- SE) to 819 +/- 74 (Day 3), decreased at Day 9 (486 +/- 67), and continued to decrease through Day 18 (338 +/- 37). alpha-Tocopherol metabolites and their intermediates increased and decreased similarly to alpha-tocopherol albeit at lower concentrations. There were no changes in known vitamin E regulatory proteins, i.e., hepatic alpha-tocopherol transfer protein or cytochrome P450 (CYP) 4F. In contrast, both CYP3A and CYP2B, key xenobiotic metabolizing enzymes, doubled by Day 6 and remained elevated, while P450 reductase increased more slowly. Consistent with the decrease in liver alpha-tocopherol concentrations, a protein involved in biliary xenobiotic excretion, p-glycoprotein, increased at Day 9, doubling by Day 15. Thus hepatic alpha-tocopherol concentrations altered hepatic proteins involved in metabolism and disposition of xenobiotic agents.     

A slipped-mispairing mutation in AgrA of laboratory strains and clinical isolates results in delayed activation of agr and failure to translate delta- and alpha-haemolysins

agr is a global regulator of staphylococcal virulence and other accessory gene functions, especially including the haemolysins. Lack of haemolysin production therefore generally represents a defect in agr function. An example of this is Staphylococcus aureus strain RN4220, a widely used laboratory strain that carries a nitrosoguanidine (MNNG)-induced mutation enabling it to accept DNA from Escherichia coli and other bacteria. We show here that the non-haemolytic phenotype of RN4220 is caused by an extra A residue in a run of seven As at the 3' end of agrA (agrA-8A). This causes a frameshift that results in the addition of three amino acyl residues to the C-terminal end of the protein. The 8A mutation does not inactivate the agr locus, but rather delays agr activation by 2-3 h, which results in failure to translate alpha- and delta-haemolysins, and hence, in a non-haemolytic phenotype. This mutation turned out not to be an adventitious consequence of MNNG mutagenesis, but rather had arisen in RN450, the immediate parent of RN4220. RN450 had become haemolytically heterogeneous in storage, and its non-haemolytic variants had the 8A mutation. The same mutation was also identified in a clinical isolate in which a non-haemolytic variant had arisen during the course of infection. Haemolytic activity in the mutant laboratory strains could be restored by the addition of auto-inducing peptide (AIP) early in growth, indicating that delayed production of RNAIII is responsible for the failure to translate alpha- and delta-haemolysins. Discovery of the 8A mutation has revealed the basis of the dissociation between agr activity and the non-haemolytic phenotype of RN4220, and has solved the long-standing mystery of the variable non-haemolytic phenotype of its immediate parent, RN450. The occurrence of this mutation in a clinical isolate indicates that it is not simply a laboratory phenomenon, and may represent a naturally occurring mechanism for the modulation of agr activity.     

A note on the activity cycle of captive white-fronted lemurs (Eulemur fulvus albifrons)

We studied the activity cycle of captive-born white-fronted lemurs (Eulemur fulvus albifrons) at Zurich Zoo with the aim to discuss current hypotheses on the evolution of cathemerality in lemurs. In contrast to their relatives in the wild, these lemurs were active exclusively during the day. If cathemerality is a strategy to increase food intake or to avoid predators, then nocturnal activity is not essential for captive animals and may be suppressed simply due to the absence of stimuli. This suggests that cathemerality includes a distinct element of flexibility regarding the distribution of diurnal and nocturnal activity, with the option to omit nightly activities.     

Poor lysosomal membrane integrity in proximal tubule cells of haptoglobin 2-2 genotype mice with diabetes mellitus

The haptoglobin (Hp) genotype is a major determinant of progression of nephropathy in individuals with diabetes mellitus (DM). The major function of the Hp protein is to bind and modulate the fate of extracorpuscular hemoglobin and its iron cargo. We have previously demonstrated an interaction between the Hp genotype and the DM on the accumulation of iron in renal proximal tubule cells. The primary objective of this study was to determine the intracellular localization of this iron in the proximal tubule cell and to assess its potential toxicity. Transmission electron microscopy demonstrated a marked accumulation of electron-dense deposits in the lysosomes of proximal tubules cells in Hp 2-2 DM mice. Energy-dispersive X-ray spectroscopy and electron energy loss spectroscopy were used to perform elemental analysis of these deposits and demonstrated that these deposits were iron rich. These deposits were associated with lysosomal membrane lipid peroxidation and loss of lysosomal membrane integrity. Vitamin E administration to Hp 2-2 DM mice resulted in a significant decrease in both intralysosomal iron-induced oxidation and lysosomal destabilization. Iron-induced renal tubular injury may play a major role in the development of diabetic nephropathy and may be a target for slowing the progression of renal disease.     

Role of L-selectin in physiological manifestations after burn and smoke inhalation injury in sheep.

The effects of a monoclonal antibody against L-selectin [leukocyte adhesion molecule (LAM)1-3] on microvascular fluid flux were determined in conscious sheep subjected to a combined injury of 40% third-degree burn and smoke inhalation. This combined injury induced a rapid increase in systemic prefemoral lymph flow (sQlymph) from the burned area and a delayed-onset increase in lung lymph flow. The initial increase in sQlymph was associated with an elevation of the lymph-to-plasma oncotic pressure ratio; consequently, it leads to a predominant increase in the systemic soft tissue permeability index (sPI). In an untreated control group, the increased sPI was sustained beyond 24 h after injury. Pretreatment with LAM1-3 resulted in earlier recovery from the increased sPI, although the initial responses in sQlymph and sPI were identical to those in the nontreatment group. The delayed-onset lung permeability changes were significantly attenuated by pretreatment with LAM1-3. These findings indicate that both leukocyte-dependent and -independent mechanisms are involved in the pathogenesis that occurs after combined injury with burn and smoke inhalation.     

Vitamin E: function and metabolism.

Although vitamin E has been known as an essential nutrient for reproduction since 1922, we are far from understanding the mechanisms of its physiological functions. Vitamin E is the term for a group of tocopherols and tocotrienols, of which alpha-tocopherol has the highest biological activity. Due to the potent antioxidant properties of tocopherols, the impact of alpha-tocopherol in the prevention of chronic diseases believed to be associated with oxidative stress has often been studied, and beneficial effects have been demonstrated. Recent observations that the alpha-tocopherol transfer protein in the liver specifically sorts out RRR-alpha-tocopherol from all incoming tocopherols for incorporation into plasma lipoproteins, and that alpha-tocopherol has signaling functions in vascular smooth muscle cells that cannot be exerted by other forms of tocopherol with similar antioxidative properties, have raised interest in the roles of vitamin E beyond its antioxidative function. Also, gamma-tocopherol might have functions apart from being an antioxidant. It is a nucleophile able to trap electrophilic mutagens in lipophilic compartments and generates a metabolite that facilitates natriuresis. The metabolism of vitamin E is equally unclear. Excess alpha-tocopherol is converted into alpha-CEHC and excreted in the urine. Other tocopherols, like gamma- and delta-tocopherol, are almost quantitatively degraded and excreted in the urine as the corresponding CEHCs. All rac alpha-tocopherol compared to RRR-alpha-tocopherol is preferentially degraded to alpha-CEHC. Thus, there must be a specific, molecular role of RRR-alpha-tocopherol that is regulated by a system that sorts, distributes, and degrades the different forms of vitamin E, but has not yet been identified. In this article we try to summarize current knowledge on the function of vitamin E, with emphasis on its antioxidant vs. other properties, the preference of the organism for RRR-alpha-tocopherol, and its metabolism to CEHCs.     

gamma-Tocopherol nebulization by a lipid aerosolization device improves pulmonary function in sheep with burn and smoke inhalation injury

Fire accident victims who sustain both thermal injury to skin and smoke inhalation have gross evidence of systemic and pulmonary oxidant damage and acute lung injury. We hypothesized that gamma-tocopherol (gT), a reactive O(2) and N(2) scavenger, when delivered into the airway, would attenuate lung injury induced by burn and smoke inhalation. Acute lung injury was induced in chronically prepared, anesthetized sheep by 40% total burn surface area, third-degree skin burn and smoke insufflation (48 breaths of cotton smoke, <40 degrees C). The study groups were: (1) Sham (not injured, flaxseed oil (FO)-nebulized, n=6); (2) SA-neb (injured, saline-nebulized, n=6); (3) FO-neb (injured, FO-nebulized, n=6); and (4) gT+FO-neb (injured, gT and FO-nebulized, n=6). Nebulization was started 1 h postinjury, and 24 ml of FO with or without gT (51 mg/ml) was delivered into airways over 47 h using our newly developed lipid aerosolization device (droplet size: 2.5-5 microm). The burn- and smoke inhalation-induced pathological changes seen in the saline group were attenuated by FO nebulization; gT addition further improved pulmonary function. Pulmonary gT delivery along with a FO source may be a novel effective treatment strategy in management of patients with acute lung injury.     

Alpha-tocopherol modulates genes involved in hepatic xenobiotic pathways in mice

Hepatic proteins involved in xenobiotic pathways (Phases I, II and III) are responsible for the metabolism and disposition of endogenous and exogenous compounds including dietary phytochemicals. To test the hypothesis that elevated alpha-tocopherol intakes alter gene expression of hepatic xenobiotic pathways, mice were fed diets supplemented with either 1000 IU (+E) or 35 IU (E) all-rac-alpha-tocopheryl acetate for 4 months; liver RNA was isolated, and gene expression was determined using both whole genome microarray and real-time quantitative polymerase chain reaction analyses. Hepatic alpha-tocopherol (173+/-18 vs. 21+/-1 nmol/g, mean+/-S.E.) and its metabolite (2,5,7,8-tetramethyl-2-(2'-carboxyethyl)-6-hydroxychroman, 0.232+/-0.046 vs. 0.031+/-0.019 nmol/g) concentrations were approximately eightfold higher following the +E dietary treatment. In +E relative to E mice, gene expression of Phase I enzymes, P450 oxidoreductase and cytochrome P450 3a11 increased 1.6- and 4.0-fold, respectively; two Phase II genes, sulfotransferase 2a and glutathione S-transferase mu 3, increased 10.8- and 1.9-fold respectively, and a Phase III biliary transporter, Abcb1a, doubled. Thus, consumption of high-level dietary alpha-tocopherol simultaneously coordinated Phase I, II and III gene expression. These data demonstrate that increased hepatic alpha-tocopherol modulates its own concentrations through increasing xenobiotic metabolism, a process that may alter metabolism of other foreign compounds, such as therapeutic drugs and phytochemicals, in humans.