Expression of <Emphasis Type="Italic">PEG11</Emphasis> and <Emphasis Type="Italic">PEG11AS</Emphasis> transcripts in normal and callipyge sheep

Background  

The callipyge mutation is located within an imprinted gene cluster on ovine chromosome 18. The callipyge trait exhibits polar overdominant inheritance due to the fact that only heterozygotes inheriting a mutant paternal allele (paternal heterozygotes) have a phenotype of muscle hypertrophy, reduced fat and a more compact skeleton. The mutation is a single A to G transition in an intergenic region that results in the increased expression of several genes within the imprinted cluster without changing their parent-of-origin allele-specific expression.     

Vascular endothelial growth factor (VEGF) induces remodeling and enhances TH2-mediated sensitization and inflammation in the lung

Exaggerated levels of VEGF (vascular endothelial growth factor) are present in persons with asthma, but the role(s) of VEGF in normal and asthmatic lungs has not been defined. We generated lung-targeted VEGF(165) transgenic mice and evaluated the role of VEGF in T-helper type 2 cell (T(H)2)-mediated inflammation. In these mice, VEGF induced, through IL-13-dependent and -independent pathways, an asthma-like phenotype with inflammation, parenchymal and vascular remodeling, edema, mucus metaplasia, myocyte hyperplasia and airway hyper-responsiveness. VEGF also enhanced respiratory antigen sensitization and T(H)2 inflammation and increased the number of activated DC2 dendritic cells. In antigen-induced inflammation, VEGF was produced by epithelial cells and preferentially by T(H)2 versus T(H)1 cells. In this setting, it had a critical role in T(H)2 inflammation, cytokine production and physiologic dysregulation. Thus, VEGF is a mediator of vascular and extravascular remodeling and inflammation that enhances antigen sensitization and is crucial in adaptive T(H)2 inflammation. VEGF regulation may be therapeutic in asthma and other T(H)2 disorders.     

Epidemiology,hypermutation, within-host evolution and the virulence of Neisseria meningitidis

Many so-called pathogenic bacteria such as Neisseria meningitidis, Haemophilus influenzae, Staphylococcus aureus and Streptococcus pneumoniae are far more likely to colonize and maintain populations in healthy individuals asymptomatically than to cause disease. Disease is a dead-end for these bacteria: virulence shortens the window of time during which transmission to new hosts can occur and the subpopulations of bacteria actually responsible for disease, like those in the blood or cerebral spinal fluid, are rarely transmitted to new hosts. Hence, the virulence factors underlying their occasional pathogenicity must evolve in response to selection for something other than making their hosts sick. What are those selective pressures? We address this general question of the evolution of virulence in the context of phase shifting in N. meningitidis, a mutational process that turns specific genes on and off, and, in particular, contingency loci that code for virulence determinants such as pili, lipopolysaccharides, capsular polysaccharides and outer membrane proteins. We use mathematical models of the epidemiology and the within-host infection dynamics of N. meningitidis to make the case that rapid phase shifting evolves as an adaptation for colonization of diverse hosts and that the virulence of this bacterium is an inadvertent consequence of short-sighted within-host evolution, which is exasperated by the increased mutation rates associated with phase shifting. We present evidence for and suggest experimental and retrospective tests of these hypotheses.     

Nuclear export of Vpr is required for efficient replication of human immunodeficiency virus type 1 in tissue macrophages

Retroviruses must gain access to the host cell nucleus for subsequent replication and viral propagation. Human immunodeficiency virus type 1 (HIV-1) and other primate lentiviruses are distinguished from the gammaretroviruses by their ability to infect nondividing cells such as macrophages, an important viral reservoir in vivo. Rather than requiring nuclear membrane breakdown during cell division, the HIV-1 preintegration complex (PIC) enters the nucleus by traversing the central aqueous channel of the limiting nuclear pore complex. The HIV-1 PIC contains three nucleophilic proteins, matrix, integrase, and Vpr, all of which have been implicated in nuclear targeting. The mechanism by which Vpr can display such nucleophilic properties and yet also be available for incorporation into virions assembling at the plasma membrane is unresolved. We recently characterized Vpr as a nucleocytoplasmic shuttling protein that contains two novel nuclear import signals and an exportin-1-dependent nuclear export signal (NES). We now demonstrate that mutation of this NES impairs the incorporation of Vpr into newly formed virions. Furthermore, we find that the Vpr NES is required for efficient HIV replication in tissue macrophages present in human spleens and tonsils. These findings underscore how the nucleocytoplasmic shuttling of Vpr not only contributes to nuclear import of the HIV-1 PIC but also enables Vpr to be present in the cytoplasm for incorporation into virions, leading to enhancement of viral spread within nondividing tissue macrophages.     

17-epiestriol,an estrogen metabolite,is more potent than estradiol in inhibiting vascular cell adhesion molecule 1 (VCAM-1) mRNA expression

17-beta estradiol (17-beta E(2)) attenuates the expression of vascular cell adhesion molecule 1 (VCAM-1) in vivo at physiological levels (pg/ml), whereas supraphysiological concentrations of 17-beta E(2) (ng/ml) are required in vitro. We assessed whether a metabolite of estrogen, which could only be generated in vivo, might be a more potent inhibitor of VCAM-1 expression and thereby explain this discrepancy. We report here that 17-epiestriol, an estrogen metabolite and a selective estrogen receptor (ER) beta agonist, is approximately 400x more potent than 17-beta E(2) in suppressing tumor necrosis factor (TNF) alpha-induced VCAM-1 mRNA as well as protein expression in human umbilical vein endothelial cells. Genistein, an ERbeta agonist, at low concentrations (1 and 10 nm) also suppressed TNFalpha-induced VCAM-1 mRNA expression. These actions of 17-epiestriol and genistein were significantly attenuated in the presence of the estrogen receptor antagonist ICI-182780. Other estrogenic compounds such as ethinyl estradiol and estrone did not have any effect on TNFalpha-induced VCAM-1 expression at the concentrations tested. We further show that, 1) 17-epiestriol induces the expression of endothelial nitric-oxide synthase mRNA and protein, 2) 17-epiestriol prevents TNFalpha-induced migration of NFkappaB into the nucleus, 3) N(G)-nitro-l-arginine methyl ester, an inhibitor of NO synthesis, abolishes 17-epiestriol-mediated inhibition of TNFalpha-induced VCAM-1 expression and migration of NFkappaB from the cytoplasm to the nucleus. Our results indicate that 17-epiestriol is more potent than 17-beta E(2) in suppressing TNFalpha-induced VCAM-1 expression and that this action is modulated at least in part through NO.     

Molecular dissection of the interaction between the small G proteins Rac1 and RhoA and protein kinase C-related kinase 1 (PRK1)

PRK1 is a serine/threonine kinase that belongs to the protein kinase C superfamily. It can be activated either by members of the Rho family of small G proteins, by proteolysis, or by interaction with lipids. Here we investigate the binding of PRK1 to RhoA and Rac1, two members of the Rho family. We demonstrate that PRK1 binds with a similar affinity to RhoA and Rac1. We present the solution structure of the second HR1 domain from the regulatory N-terminal region of PRK1, and we show that it forms an anti-parallel coiled-coil. In addition, we have used NMR to map the binding contacts of the HR1b domain with Rac1. These are compared with the contacts known to form between HR1a and RhoA. We have used mutagenesis to define the residues in Rac that are important for binding to HR1b. Surprisingly, as well as residues adjacent to Switch I, in Switch II, and in helix alpha5, it appears that the C-terminal stretch of basic amino acids in Rac is required for a high affinity interaction with HR1b.     

Electrostatic guidance of glycosyl cation migration along the reaction coordinate of uracil DNA glycosylase

The DNA repair enzyme uracil DNA glycosylase has been crystallized with a cationic 1-aza-2'-deoxyribose-containing DNA that mimics the ultimate transition state of the reaction in which the water nucleophile attacks the anomeric center of the oxacarbenium ion-uracil anion reaction intermediate. Comparison with substrate and product structures, and the previous structure of the intermediate determined by kinetic isotope effects, reveals an exquisite example of geometric strain, least atomic motion, and electrophile migration in biological catalysis. This structure provides a rare opportunity to reconstruct the detailed structural transformations that occur along an enzymatic reaction coordinate.     

Finding the molecular basis of quantitative traits: successes and pitfalls

Understanding the molecular basis of quantitative genetic variation is a principal goal for biomedicine. Although the complex genetic architecture of quantitative traits has so far largely frustrated attempts to identify genes in humans by standard linkage methodologies, quantitative trait loci (QTL) have been mapped in plants, insects and rodents. However, identifying the molecular bases of QTL remains a challenge. Here, we discuss why this is and how new experimental strategies and analytical techniques, combined with the fruits of the genome projects, are beginning to identify candidate genes for QTL studies in several model organisms.     

Myocardial function in rat genetic models of low and high aerobic running capacity

We recently evaluated treadmill aerobic running capacity in 11 inbred strains of rats and found that isolated working left ventricular function correlated (r = 0.86) with aerobic running capacity. Among these 11 strains the Buffalo (BUF) hearts produced the lowest and the DA hearts the highest isolated cardiac output. The goal of this study was to investigate the components of cardiac function (i.e., coronary flow, heart rates, stroke volume, contractile dynamics, and cross-bridge cycling) to characterize further the BUF and DA inbred strains as potential models of contrasting myocardial performance. Cardiac performance was assessed using the Langendorff-Neely working heart preparation. Isolated DA hearts were superior (P < 0.05) to the BUF hearts for cardiac output (63%), stroke volume (60%), aortic +dP/dt (47%), and aortic -dP/dt (46%). The mean alpha/beta-myosin heavy chain (MHC) isoform ratio for DA hearts was 21-fold higher relative to BUF hearts. At the steady-state mRNA level, DA hearts had a fivefold higher alpha/beta-ratio than the BUF hearts. The mean rate of ATP hydrolysis by MHCs was 64% greater in DA compared with BUF ventricles. These data demonstrate that the BUF and DA strains can serve as genetic models of contrasting low and high cardiac function.