Lack of reactive oxygen species breaks T cell tolerance to collagen type II and allows development of arthritis in mice

The view on reactive oxygen species (ROS) in inflammation is currently shifting from being considered damaging toward having a more complex role in regulating inflammatory reactions. We recently demonstrated a role of ROS in regulation of animal models for the autoimmune disease rheumatoid arthritis. Low levels of ROS production, due to a mutation in the Ncf1 gene coding for the Ncf1 (alias p47(phox)) subunit of the NADPH oxidase complex, was shown to be associated with increased autoimmunity and arthritis severity in both rats and mice. To further investigate the role of ROS in autoimmunity, we studied transgenic mice expressing collagen type II (CII) with a mutation (D266E) in the immunodominant epitope that mimics the rat and human CII (i.e., mutated mouse collagen or MMC). This mutation results in a stronger binding of the epitope to the MHC class II molecule and leads to more pronounced tolerance and resistance to arthritis induced with rat CII. When the Ncf1 mutation was bred into these mice, tolerance was broken, resulting in enhanced T cell autoreactivity, high titers of anti-CII Abs, and development of severe arthritis. These findings highlight the importance of a sufficient ROS production in maintenance of tolerance to self-Ags, a central mechanism in autoimmune diseases such as rheumatoid arthritis. This is important as we, for the first time, can follow the effect of ROS on molecular mechanisms where T cells are responsible for either protection or promotion of arthritis depending on the level of oxygen species produced.     

Identification of a Fungal 1,8-Cineole Synthase from Hypoxylon sp. with Specificity Determinants in Common with the Plant Synthases

Terpenes are an important and diverse class of secondary metabolites widely produced by fungi. Volatile compound screening of a fungal endophyte collection revealed a number of isolates in the family Xylariaceae, producing a series of terpene molecules, including 1,8-cineole. This compound is a commercially important component of eucalyptus oil used in pharmaceutical applications and has been explored as a potential biofuel additive. The genes that produce terpene molecules, such as 1,8-cineole, have been little explored in fungi, providing an opportunity to explore the biosynthetic origin of these compounds. Through genome sequencing of cineole-producing isolate E7406B, we were able to identify 11 new terpene synthase genes. Expressing a subset of these genes in Escherichia coli allowed identification of the hyp3 gene, responsible for 1,8-cineole biosynthesis, the first monoterpene synthase discovered in fungi. In a striking example of convergent evolution, mutational analysis of this terpene synthase revealed an active site asparagine critical for water capture and specificity during cineole synthesis, the same mechanism used in an unrelated plant homologue. These studies have provided insight into the evolutionary relationship of fungal terpene synthases to those in plants and bacteria and further established fungi as a relatively untapped source of this important and diverse class of compounds.     

Metabolic variation in natural populations of wild yeast

Ecological diversification depends on the extent of genetic variation and on the pattern of covariation with respect to ecological opportunities. We investigated the pattern of utilization of carbon substrates in wild populations of budding yeast Saccharomyces paradoxus. All isolates grew well on a core diet of about 10 substrates, and most were also able to grow on a much larger ancillary diet comprising most of the 190 substrates we tested. There was substantial genetic variation within each population for some substrates. We found geographical variation of substrate use at continental, regional, and local scales. Isolates from Europe and North America could be distinguished on the basis of the pattern of yield across substrates. Two geographical races at the North American sites also differed in the pattern of substrate utilization. Substrate utilization patterns were also geographically correlated at local spatial scales. Pairwise genetic correlations between substrates were predominantly positive, reflecting overall variation in metabolic performance, but there was a consistent negative correlation between categories of substrates in two cases: between the core diet and the ancillary diet, and between pentose and hexose sugars. Such negative correlations in the utilization of substrate from different categories may indicate either intrinsic physiological trade‐offs for the uptake and utilization of substrates from different categories, or the accumulation of conditionally neutral mutations. Divergence in substrate use accompanies genetic divergence at all spatial scales in S. paradoxus and may contribute to race formation and speciation.     

Measuring the fitness of symbiotic rhizobia

The legume-rhizobia symbiosis is an important model system for research on the evolution of cooperation and conflict. A key strength of this system is that the fitness consequences of greater or lesser investment in cooperative behaviors can be measured for each partner. Most empirical studies have characterized the fitness of symbiotic rhizobia exclusively by their numbers within nodules, often estimated using nodule size as a proxy. Here we show that the relationship between nodule size and rhizobial numbers can differ drastically between strains of the same species. We further show that differences in accumulation of the storage polyester poly-3-hydroxybutyrate (PHB), which can support future reproduction, can be large enough that even direct measurements of rhizobial numbers alone can lead to qualitatively incorrect conclusions. Both results come from a comparison of strains differing in production of the ethylene-inhibitor rhizobitoxine (Rtx). A broader study (using three legume-rhizobia species pairs) showed that PHB/cell cannot be reliably estimated from its correlation with rhizobia/nodule or nodule size. Differences in PHB between strains or treatments will not always make major contributions to differences in fitness, but situation-specific data are needed before PHB can safely be neglected.     

The assay design used for measurement of therapeutic antibody concentrations can affect pharmacokinetic parameters: Case studies

To interpret pharmacokinetic (PK) data of biotherapeutics, it is critical to understand which drug species is being measured by the PK assay. For therapeutic antibodies, it is generally accepted that “free” circulating antibodies are the pharmacologically active form needed to determine the PK/ pharmacodynamic (PD) relationship, safety margin calculations, and dose projections from animals to humans and the eventual characterization of the exposure in the clinic. However, “total” drug may be important in evaluating the dynamic interaction between the drug and the target, as well as the total drug exposure. In the absence of or with low amounts of soluble ligand /shed receptor, total and free drug species are often equivalent and their detection is less sensitive to assay formats or reagent choices. In contrast, in the presence of a significant amount of ligand, assay design and characterization of assay reagents are critical to understanding the PK profiles. Here, we present case studies where different assay formats affected measured PK profiles and data interpretation. The results from reagent characterizations provide a potential explanation for the observed discrepancies and highlight the importance of reagent characterization in understanding which drug species are being measured to accurately interpret PK parameters.     

Enhanced repair of cyclobutane pyrimidine dimers and improved UV resistance in photolyase transgenic mice

During evolution, placental mammals appear to have lost cyclobutane pyrimidine dimer (CPD) photolyase, an enzyme that efficiently removes UV-induced CPDs from DNA in a light-dependent manner. As a consequence, they have to rely solely on the more complex, and for this lesion less efficient, nucleotide excision repair pathway. To assess the contribution of poor repair of CPDs to various biological effects of UV, we generated mice expressing a marsupial CPD photolyase transgene. Expression from the ubiquitous beta-actin promoter allowed rapid repair of CPDs in epidermis and dermis. UV-exposed cultured dermal fibroblasts from these mice displayed superior survival when treated with photoreactivating light. Moreover, photoreactivation of CPDs in intact skin dramatically reduced acute UV effects like erythema (sunburn), hyperplasia and apoptosis. Mice expressing the photolyase from keratin 14 promoter photo reactivate CPDs in basal and early differentiating keratinocytes only. Strikingly, in these animals, the anti-apoptotic effect appears to extend to other skin compartments, suggesting the presence of intercellular apoptotic signals. Thus, providing mice with CPD photolyase significantly improves repair and uncovers the biological effects of CPD lesions.