Optimization of crystals of an inhibitory antibody of urokinase plasminogen activator receptor (uPAR) with hydrogen peroxide and low protein concentration

Optimization of protein crystal formation is often a necessary step leading to diffraction-quality crystals to enable collection of a full X-ray data set. Typical protein crystal optimization involves screening different components, e.g., pH, precipitants, and additives of the precipitant solution. Here we present an example using an inhibitory antibody of urokinase plasminogen activator receptor (uPAR) where such procedures did not yield diffracting crystals. In contrast, it was the treatment of the protein with hydrogen peroxide incubation and the protein concentration reduction that were found to be key factors in obtaining diffracting crystals. Final crystals diffracted to 1.75 A, and belong to orthorhombic P2(1)2(1)2(1) space group with unit cell parameters a = 37.162 A, b = 84.474 A, c = 134.030 A, and contain one molecule of Fab fragment of anti-uro kinase receptor antibody in the asymmetric unit.     

Comparative analysis of cutaneous evaporative water loss in frogs demonstrates correlation with ecological habits

Most frog species show little resistance to evaporative water loss (EWL), but some arboreal species are known to have very high resistances. We measured EWL and cutaneous resistance to evaporation (Rc) in 25 species of frogs from northern Australia, including 17 species in the family Hylidae, six species in the Myobatrachidae, and one each in the Bufonidae and the Microhylidae. These species display a variety of ecological habits, including aquatic, terrestrial, and arboreal specialisations, with the complete range of habits displayed within just the one hylid genus, Litoria. The 25 species measured in this study have resistances that range from Rc=0 to 63.1. These include low values indistinguishable from a free water surface to high values typical of "waterproof" anuran species. There was a strong correlation between ecological habit and Rc, even taking phylogenetic relationships into account; arboreal species had the highest resistance, aquatic species tended to have little or no resistance, and terrestrial species tended to have resistance between those of arboreal and aquatic frogs. For one species, Litoria rubella, we found no significant changes in EWL along a 1,500-km aridity gradient. This study represents the strongest evidence to date of a link between ecological habits and cutaneous resistance to water loss among species of frogs.     

Host limits to accurate human growth hormone production in multiple plant systems

Human growth hormone (hGH) is not only a valuable recombinant therapeutic protein for hormone deficiency indications, but is also an extensively characterized molecule both from recombinant bacterial systems and as circulating in humans. We describe the characterization of hGH produced in three different plant systems: tobacco cell culture, soy seed, and maize seed. The data indicate highest production in the maize seed system, with continued productivity over multiple generations, and when bred to a new host genotype for improved productivity. Purification indicated significant material of the correct structure from both plant cell culture and maize seed, with maize seed also showing correct activity relative to that produced by Escherichia coli. However, all systems showed some proteolyzed hGH, with data from gel electrophoresis, mass spectrometry, and peptide mapping localizing to a region of the protein also prone to cleavage in some other systems. Together, the data indicate the dependence of recombinant protein accumulation on posttranslational processes in different host systems.     

The Y deletion gr/gr and susceptibility to testicular germ cell tumor

Testicular germ cell tumor (TGCT) is the most common cancer in young men. Despite a considerable familial component to TGCT risk, no genetic change that confers increased risk has been substantiated to date. The human Y chromosome carries a number of genes specifically involved in male germ cell development, and deletion of the AZFc region at Yq11 is the most common known genetic cause of infertility. Recently, a 1.6-Mb deletion of the Y chromosome that removes part of the AZFc region—known as the “gr/gr” deletion—has been associated with infertility. In epidemiological studies, male infertility has shown an association with TGCT that is out of proportion with what can be explained by tumor effects. Thus, we hypothesized that the gr/gr deletion may be associated with TGCT. Using logistic modeling, we analyzed this deletion in a large series of TGCT cases with and without a family history of TGCT. The gr/gr deletion was present in 3.0% (13/431) of TGCT cases with a family history, 2% (28/1,376) of TGCT cases without a family history, and 1.3% (33/2,599) of unaffected males. Presence of the gr/gr deletion was associated with a twofold increased risk of TGCT (adjusted odds ratio [aOR] 2.1; 95% confidence interval [CI] 1.3–3.6; P = .005) and a threefold increased risk of TGCT among patients with a positive family history (aOR 3.2; 95% CI 1.5–6.7; P = .0027). The gr/gr deletion was more strongly associated with seminoma (aOR 3.0; 95% CI 1.6–5.4; P = .0004) than with nonseminoma TGCT (aOR 1.5; 95% CI 0.72–3.0; P = .29). These data indicate that the Y microdeletion gr/gr is a rare, low-penetrance allele that confers susceptibility to TGCT.     

Long-term control of Mycobacterium tuberculosis infection is mediated by dynamic immune responses

The primary goal of this study was to determine how chronic exposure to Ag influences the functionality of Mycobacterium tuberculosis-specific T cell responses. The frequency of IFN-gamma-producing effector CD4(+) and CD8(+) T cells dynamically changed during persistent M. tuberculosis infection. CD8(+) T cells used differential effector functions during acute and chronic phases of the immune response, where CD8(+) T cells produced negligible amounts of IFN-gamma early in infection, but switched to cytokine production during the chronic stage of infection. Using limiting dilution analysis, CD8(+) T cells isolated during the initial phase of infection demonstrated lytic potential, but this waned in the chronic stage. The apparent loss of cytotoxic activity was not associated with the lack of perforin. Ag dose could potentially govern the functional program of CD8(+) T cells. Collectively, these results depict a host immune response mounted against M. tuberculosis of a significantly more dynamic nature than previously recognized.