Conformational flexibility in crystal structures of human 11beta-hydroxysteroid dehydrogenase type I provide insights into glucocorticoid interconversion and enzyme regulation

Human 11beta-hydroxysteroid dehydrogenase type I (11beta-HSD1) is an ER-localized membrane protein that catalyzes the interconversion of cortisone and cortisol. In adipose tissue, excessive cortisol production through 11beta-HSD1 activity has been implicated in the pathogenesis of type II diabetes and obesity. We report here biophysical, kinetic, mutagenesis, and structural data on two ternary complexes of 11beta-HSD1. The combined results reveal flexible active site interactions relevant to glucocorticoid recognition and demonstrate how four 11beta-HSD1 C termini converge to form an as yet uncharacterized tetramerization motif. A C-terminal Pro-Cys motif is localized at the center of the tetramer and forms reversible enzyme disulfides that alter enzyme activity. Conformational flexibility at the tetramerization interface is coupled to structural changes at the enzyme active site suggesting how the central Pro-Cys motif may regulate enzyme activity. Together, the crystallographic and biophysical data provide a structural framework for understanding 11beta-HSD1 activities and will ultimately facilitate the development of specific inhibitors.     

Structural basis for iron binding and release by a novel class of periplasmic iron-binding proteins found in gram-negative pathogens

We have determined the 1.35- and 1.45-A structures, respectively, of closed and open iron-loaded forms of Mannheimia haemolytica ferric ion-binding protein A. M. haemolytica is the causative agent in the economically important and fatal disease of cattle termed shipping fever. The periplasmic iron-binding protein of this gram-negative bacterium, which has homologous counterparts in many other pathogenic species, performs a key role in iron acquisition from mammalian host serum iron transport proteins and is essential for the survival of the pathogen within the host. The ferric (Fe(3+)) ion in the closed structure is bound by a novel asymmetric constellation of four ligands, including a synergistic carbonate anion. The open structure is ligated by three tyrosyl residues and a dynamically disordered solvent-exposed anion. Our results clearly implicate the synergistic anion as the primary mediator of global protein conformation and provide detailed insights into the molecular mechanisms of iron binding and release in the periplasm.     

(3)H]Adenosine uptake in brainstem membranes of CD-1 mice lacking the adenosine A(2a) receptor

Previous studies in our laboratory have demonstrated a decrease in [(3)H]nitrobenzylthioinosine binding sites in the brainstem of adenosine A(2a) receptor knockout mice, particularly in the brain nuclei involved in central control of cardiovascular function [Brain Research 877 (2000) 160]. The present study aimed to correlate this decrease, shown using autoradiography, with a functional change using a previously described method of [(3)H]adenosine uptake in a membrane preparation from the brainstem of wildtype CD - 1 and homozygous mutant mice lacking the adenosine A(2a) receptor. A statistically significant decrease was shown in the mean V(MAX) value obtained from homozygous mutant preparations (4.7 +/- 1.3 fmol/mg protein/20 s, P < 0.05, n = 4) compared to that obtained from wildtype controls (51.6 +/- 4.2 fmol/mg protein/20 s, n = 4). Competition studies using nucleoside uptake inhibitors showed a statistically significant increase in the log IC(50) values for dipyridamole (Wildtype: -4.3 +/- 0.2, Homozygous mutant: -8.3 +/- 0.4, n=5, P < 0.05) and dilazep (Wildtype: -3.9 +/- 0.8, Homozygous mutant: -8.3 +/- 0.8, n=5, P < 0.05) in the preparations using homozygous mutant tissue. The present study, in conjunction with the results of previous studies [Brain Research 877 (2000) 160], indicates that components of purinergic neurotransmission system have apparently adjusted in compensation for the lack of the A(2a) receptor.     

Phylogeographic history and gene flow among giant Galápagos tortoises on southern Isabela Island

Volcanic islands represent excellent models with which to study the effect of vicariance on colonization and dispersal, particularly when the evolution of genetic diversity mirrors the sequence of geological events that led to island formation. Phylogeographic inference, however, can be particularly challenging for recent dispersal events within islands, where the antagonistic effects of land bridge formation and vicariance can affect movements of organisms with limited dispersal ability. We investigated levels of genetic divergence and recovered signatures of dispersal events for 631 Galápagos giant tortoises across the volcanoes of Sierra Negra and Cerro Azul on the island of Isabela. These volcanoes are among the most recent formations in the Galápagos (<0.7 million years), and previous studies based on genetic and morphological data could not recover a consistent pattern of lineage sorting. We integrated nested clade analysis of mitochondrial DNA control region sequences, to infer historical patterns of colonization, and a novel Bayesian multilocus genotyping method for recovering evidence of recent migration across volcanoes using eleven microsatellite loci. These genetic studies illuminate taxonomic distinctions as well as provide guidance to possible repatriation programs aimed at countering the rapid population declines of these spectacular animals.     

Opposing roles for TRAF1 in the alternative versus classical NF-κB pathway in T cells

T cells lacking TRAF1 hyperproliferate in response to T cell receptor signaling but have impaired signaling downstream of specific TNFR family members such as 4-1BB. Here we resolve this paradox by showing that while TRAF1 is required for maximal activation of the classical NF-κB pathway downstream of 4-1BB in primary T cells, TRAF1 also restricts the constitutive activation of NIK in anti-CD3-activated T cells. Activation of the alternative NF-κB pathway is restricted in unstimulated cells by a cIAP1/2:TRAF2:TRAF3:NIK complex. Using knockdown of NIK by siRNA we show that in activated CD8 T cells TRAF1 is also involved in this process and that constitutive activation of the alternative NF-κB pathway is responsible for costimulation independent hyperproliferation and excess cytokine production in TRAF1-deficient CD8 T cells compared with WT CD8 T cells. The T cell costimulatory molecule 4-1BB critically regulates the survival of activated and memory CD8 T cells. We demonstrate that stimulation through 4-1BB induces cIAP1-dependent TRAF3 degradation and activation of the alternative NF-κB pathway. We also show that while both TRAF1 and cIAP1 have non-redundant roles in suppressing the alternative NF-κB pathway in T cells activated in the absence of costimulation, activation of the classical NF-κB pathway downstream of 4-1BB requires TRAF1, whereas cIAP1 plays a redundant role with cIAP2. Collectively these results demonstrate that TRAF1 plays a critical role in regulating T cell activation both through restricting the costimulation independent activation of NIK in activated T cells and by promoting the 4-1BB-induced classical NF-κB pathway.     

Enhanced polyhydroxybutyrate production in transgenic sugarcane

Polyhydroxybutyrate (PHB) is a bacterial polyester that has properties similar to some petrochemically produced plastics. Plant-based production has the potential to make this biorenewable plastic highly competitive with petrochemical-based plastics. We previously reported that transgenic sugarcane produced PHB at levels as high as 1.8% leaf dry weight without penalty to biomass accumulation, suggesting scope for improving PHB production in this species. In this study, we used different plant and viral promoters, in combination with multigene or single-gene constructs to increase PHB levels. Promoters tested included the maize and rice polyubiquitin promoters, the maize chlorophyll A/B-binding protein promoter and a Cavendish banana streak badnavirus promoter. At the seedling stage, the highest levels of polymer were produced in sugarcane plants when the Cavendish banana streak badnavirus promoter was used. However, in all cases, this promoter underwent silencing as the plants matured. The rice Ubi promoter enabled the production of PHB at levels similar to the maize Ubi promoter. The maize chlorophyll A/B-binding protein promoter enabled the production of PHB to levels as high as 4.8% of the leaf dry weight, which is approximately 2.5 times higher than previously reported levels in sugarcane. This is the first time that this promoter has been tested in sugarcane. The highest PHB-producing lines showed phenotypic differences to the wild-type parent, including reduced biomass and slight chlorosis.     

Spatial and Temporal Analysis of Contact Rates in Female White-Tailed Deer

Abstract: White-tailed deer (Odocoileus virginianus) are important game mammals and potential reservoirs of diseases of domestic livestock; thus, diseases of deer are of great concern to wildlife managers. Contact, either direct or indirect, is necessary for disease transmission, but we know little about the ecological contexts that promote intrasexual contact among deer. Using pair-wise direct contacts estimated from Global Positioning System collar locations and joint utilization distributions (JUDs), we assessed habitats in which contacts occur to test whether direct contact rates among female white-tailed deer in different social groups differs among land-cover types. We also tested whether contact rates differed among seasons, lunar phases, and times of day. We obtained locations from 27 female deer for periods of 0.5–17 months during 2002–2006. We designated any simultaneous pair of locations for 2 deer <25 m apart as a direct contact. For each season, we used compositional analysis to compare land-cover types where 2 deer had contact to available land-cover weighted by their JUD. We used mixed-model logistic regression to test for effects of season, lunar phase, and time of day on contact rates. Contact rates during the gestation season were greater than expected from random use in forest and grassland cover, whereas contact rates during the fawning period were greater in agricultural fields than in other land-cover types. Contact rates were greatest during the rut and lowest in summer. Diel patterns of contact rates varied with season, and contact rates were elevated during full moon compared to other lunar periods. Both spatial and temporal analyses suggest that contact between female deer in different social groups occurs mainly during feeding, which highlights the potential impact of food distribution and habitat on contact rates among deer. By using methods to associate contacts and land-cover, we have created beneficial tools for more elaborate and detailed studies of disease transmission. Our methods can offer information necessary to develop spatially realistic models of disease transmission in deer.