Homeostatic signals do not drive post-thymic T cell maturation

Recent thymic emigrants, the youngest T cells in the lymphoid periphery, undergo a 3 week-long period of functional and phenotypic maturation before being incorporated into the pool of mature, na?ve T cells. Previous studies indicate that this maturation requires T cell exit from the thymus and access to secondary lymphoid organs, but is MHC-independent. We now show that post-thymic T cell maturation is independent of homeostatic and costimulatory pathways, requiring neither signals delivered by IL-7 nor CD80/86. Furthermore, while CCR7/CCL19,21-regulated homing of recent thymic emigrants to the T cell zones within the secondary lymphoid organs is not required for post-thymic T cell maturation, an intact dendritic cell compartment modulates this process. It is thus clear that, unlike T cell development and homeostasis, post-thymic maturation is focused not on interrogating the T cell receptor or the cell's responsiveness to homeostatic or costimulatory signals, but on some as yet unrecognized property.     

Surrogate genetics and metabolic profiling for characterization of human disease alleles

Cystathionine-β-synthase (CBS) deficiency is a human genetic disease causing homocystinuria, thrombosis, mental retardation, and a suite of other devastating manifestations. Early detection coupled with dietary modification greatly reduces pathology, but the response to treatment differs with the allele of CBS. A better understanding of the relationship between allelic variants and protein function will improve both diagnosis and treatment. To this end, we tested the function of 84 CBS alleles previously sequenced from patients with homocystinuria by ortholog replacement in Saccharomyces cerevisiae. Within this clinically associated set, 15% of variant alleles were indistinguishable from the predominant CBS allele in function, suggesting enzymatic activity was retained. An additional 37% of the alleles were partially functional or could be rescued by cofactor supplementation in the growth medium. This large class included alleles rescued by elevated levels of the cofactor vitamin B6, but also alleles rescued by elevated heme, a second CBS cofactor. Measurement of the metabolite levels in CBS-substituted yeast grown with different B6 levels using LC-MS revealed changes in metabolism that propagated beyond the substrate and product of CBS. Production of the critical antioxidant glutathione through the CBS pathway was greatly decreased when CBS function was restricted through genetic, cofactor, or substrate restriction, a metabolic consequence with implications for treatment.     

Backbone and side-chain assignment of the lipidated and non-lipidated forms of the meningococcal outer membrane protein LP2086

LP2086 is a lipidated outer membrane protein from Neisseria meningitidis that elicits bactericidal antibodies and represents a promising vaccine candidate against meningococcal infections. Here we report the backbone and side-chain assignment for two forms of LP2086: non-lipidated in aqueous buffer and the lipidated protein in micellar solution. Ethical standards  All experiments described comply with the current laws of the United States of America. The authors declare that they have no conflict of interest     

Mitochondrial Targeted Coenzyme Q,Superoxide, and Fuel Selectivity in Endothelial Cells

Background

Previously, we reported that the “antioxidant” compound “mitoQ” (mitochondrial-targeted ubiquinol/ubiquinone) actually increased superoxide production by bovine aortic endothelial (BAE) cell mitochondria incubated with complex I but not complex II substrates.

Methods and Results

To further define the site of action of the targeted coenzyme Q compound, we extended these studies to include different substrate and inhibitor conditions. In addition, we assessed the effects of mitoquinone on mitochondrial respiration, measured respiration and mitochondrial membrane potential in intact cells, and tested the intriguing hypothesis that mitoquinone might impart fuel selectivity in intact BAE cells. In mitochondria respiring on differing concentrations of complex I substrates, mitoquinone and rotenone had interactive effects on ROS consistent with redox cycling at multiple sites within complex I. Mitoquinone increased respiration in isolated mitochondria respiring on complex I but not complex II substrates. Mitoquinone also increased oxygen consumption by intact BAE cells. Moreover, when added to intact cells at 50 to 1000 nM, mitoquinone increased glucose oxidation and reduced fat oxidation, at doses that did not alter membrane potential or induce cell toxicity. Although high dose mitoquinone reduced mitochondrial membrane potential, the positively charged mitochondrial-targeted cation, decyltriphenylphosphonium (mitoquinone without the coenzyme Q moiety), decreased membrane potential more than mitoquinone, but did not alter fuel selectivity. Therefore, non-specific effects of the positive charge were not responsible and the quinone moiety is required for altered nutrient selectivity.

Conclusions

In summary, the interactive effects of mitoquinone and rotenone are consistent with redox cycling at more than one site within complex I. In addition, mitoquinone has substrate dependent effects on mitochondrial respiration, increases repiration by intact cells, and alters fuel selectivity favoring glucose over fatty acid oxidation at the intact cell level.     

Chromosomal expression of the Haemophilus influenzae Hap autotransporter allows fine-tuned regulation of adhesive potential via inhibition of intermolecular autoproteolysis

The Haemophilus influenzae Hap autotransporter is a nonpilus adhesin that promotes adherence to respiratory epithelial cells and selected extracellular matrix proteins and facilitates bacterial aggregation and microcolony formation. Hap consists of a 45-kDa outer membrane translocator domain called Hap(beta) and a 110-kDa extracellular passenger domain called Hap(S). All adhesive activity resides within Hap(S), which also contains protease activity and directs its own secretion from the bacterial cell surface via intermolecular autoproteolysis. In the present study, we sought to determine the relationship between the magnitude of Hap expression, the efficiency of Hap autoproteolysis, and the level of Hap-mediated adherence and aggregation. We found that a minimum threshold of Hap precursor was required for autoproteolysis and that this threshold approximated expression of Hap from a chromosomal allele, as occurs in H. influenzae clinical isolates. Chromosomal expression of wild-type Hap was sufficient to promote significant adherence to epithelial cells and extracellular matrix proteins, and adherence was enhanced substantially by inhibition of autoproteolysis. In contrast, chromosomal expression of Hap was sufficient to promote bacterial aggregation only when autoproteolysis was inhibited, indicating that the threshold for Hap-mediated aggregation is above the threshold for autoproteolysis. These results highlight the critical role of autoproteolysis and an intermolecular mechanism of cleavage in controlling the diverse adhesive activities of Hap.     

Effect of lead acetate on the in vitro engulfment and killing capability of toad (Bufo arenarum) neutrophils

Lead is an element of risk for the environment and human health and has harmful effects that may exceed those of other inorganic toxicants. The immune system is one of the targets of lead. Its immunomodulatory actions depend on the level of exposure, and it has been demonstrated that environmental amounts of the metal alter immune function. Very little information is available regarding the effect of the metal on different aspects of the immune system of lower vertebrates, in particular of amphibians. The aim of this study was to investigate the effect of sublethal lead (as acetate) on the function of polymorphonuclear cells of Bufo arenarum. The results revealed that phagocytic and lytic functions of the adherent blood cells collected from sublethal lead-injected toads and incubated with suspensions of Candida pseudotropicalis were affected negatively. The decrease of the phagocytic activity was correlated with increased blood lead levels (P < 0.0001). Additional information referred to the total and differential leukocyte counts was presented; the only difference found was in the number of blast-like cells that resulted augmented in the samples of lead-injected toads. It was concluded that the evaluation of these parameters might be a reliable tool for the biological monitoring of the immune status of amphibians.