Compensatory Hemagglutinin Mutations Alter Antigenic Properties of Influenza Viruses

Influenza viruses routinely acquire mutations in antigenic sites on the globular head of the hemagglutinin (HA) protein. Since these antigenic sites are near the receptor binding pocket of HA, many antigenic mutations simultaneously alter the receptor binding properties of HA. We previously reported that a K165E mutation in the Sa antigenic site of A/Puerto Rico/8/34 (PR8) HA is associated with secondary neuraminidase (NA) mutations that decrease NA activity. Here, using reverse genetics, we show that the K165E HA mutation dramatically decreases HA binding to sialic acid receptors on cell surfaces. We sequentially passaged reverse-genetics-derived PR8 viruses with the K165E antigenic HA mutation in fertilized chicken eggs, and to our surprise, viruses with secondary NA mutations did not emerge. Instead, viruses with secondary HA mutations emerged in 3 independent passaging experiments, and each of these mutations increased HA binding to sialic acid receptors. Importantly, these compensatory HA mutations were located in the Ca antigenic site and prevented binding of Ca-specific monoclonal antibodies. Taken together, these data indicate that HA antigenic mutations that alter receptor binding avidity can be compensated for by secondary HA or NA mutations. Antigenic diversification of influenza viruses can therefore occur irrespective of direct antibody pressure, since compensatory HA mutations can be located in distinct antibody binding sites.     

Interaction of p190RhoGAP with C-terminal Domain of p120-catenin Modulates Endothelial Cytoskeleton and Permeability

p120-catenin is a multidomain intracellular protein, which mediates a number of cellular functions, including stabilization of cell-cell transmembrane cadherin complexes as well as regulation of actin dynamics associated with barrier function, lamellipodia formation, and cell migration via modulation of the activities of small GTPAses. One mechanism involves p120 catenin interaction with Rho GTPase activating protein (p190RhoGAP), leading to p190RhoGAP recruitment to cell periphery and local inhibition of Rho activity. In this study, we have identified a stretch of 23 amino acids within the C-terminal domain of p120 catenin as the minimal sequence responsible for the recruitment of p190RhoGAP (herein referred to as CRAD; catenin-RhoGAP association domain). Expression of the p120-catenin truncated mutant lacking the CRAD in endothelial cells attenuated effects of barrier protective oxidized phospholipid, OxPAPC. This effect was accompanied by inhibition of membrane translocation of p190RhoGAP, increased Rho signaling, as well as suppressed activation of Rac1 and its cytoskeletal effectors PAK1 (p21-activated kinase 1) and cortactin. Expression of p120 catenin-truncated mutant lacking CRAD also delayed the recovery process after thrombin-induced endothelial barrier disruption. Concomitantly, RhoA activation and downstream signaling were sustained for a longer period of time, whereas Rac signaling was inhibited. These data demonstrate a critical role for p120-catenin (amino acids 820–843) domain in the p120-catenin·p190RhoGAP signaling complex assembly, membrane targeting, and stimulation of p190RhoGAP activity toward inhibition of the Rho pathway and reciprocal up-regulation of Rac signaling critical for endothelial barrier regulation.     

Fatty acid remodeling in cellular glycerophospholipids following the activation of human T cells

Changes in fatty acid (FA) and glycerophospholipid (GPL) metabolism associated with cell cycle entry are not fully understood. In this study FA-GPL remodeling was investigated in resting and proliferating primary human T cells. Significant changes were measured in the composition and distribution of FAs in GPLs following receptor activation of human T cells. The FA distribution of proliferating T cells was very similar to that of the human Jurkat T cell line and when the stimulus was removed from proliferating T cells, they stopped proliferating and the FA distribution largely reverted back to that of resting T cells. The cellular content of saturated and monounsaturated FAs was significantly increased in proliferating cells, which was associated with an induction of FA synthase and stearoyl-CoA desaturase-1 gene expression. Additionally, cellular arachidonate was redistributed in GPLs in a distinct pattern that was unlike any other FAs. This redistribution was associated with an induction of CoA-dependent and CoA-independent remodeling. Accordingly, significant changes in the expression of several acyl-CoA synthetases, lysophospholipid acyltransferases, and phospholipase A₂ were measured. Overall, these results suggest that metabolic pathways are activated in proliferating T cells that may represent fundamental changes associated with human cell proliferation.     

Identification of Biochemically Distinct Properties of the Small Ubiquitin-related Modifier (SUMO) Conjugation Pathway in Plasmodium falciparum

Small ubiquitin-related modifiers (SUMOs) are post-translationally conjugated to other proteins and are thereby essential regulators of a wide range of cellular processes. Sumoylation, and enzymes of the sumoylation pathway, are conserved in the malaria causing parasite, Plasmodium falciparum. However, the specific functions of sumoylation in P. falciparum, and the degree of functional conservation between enzymes of the human and P. falciparum sumoylation pathways, have not been characterized. Here, we demonstrate that sumoylation levels peak during midstages of the intra-erythrocyte developmental cycle, concomitant with hemoglobin consumption and elevated oxidative stress. In vitro studies revealed that P. falciparum E1- and E2-conjugating enzymes interact effectively to recognize and modify RanGAP1, a model mammalian SUMO substrate. However, in heterologous reactions, P. falciparum E1 and E2 enzymes failed to interact with cognate human E2 and E1 partners, respectively, to modify RanGAP1. Structural analysis, binding studies, and functional assays revealed divergent amino acid residues within the E1-E2 binding interface that define organism-specific enzyme interactions. Our studies identify sumoylation as a potentially important regulator of oxidative stress response during the P. falciparum intra-erythrocyte developmental cycle, and define E1 and E2 interactions as a promising target for development of parasite-specific inhibitors of sumoylation and parasite replication.     

VACCINATION AND REDUCED COHORT DURATION CAN DRIVE VIRULENCE EVOLUTION: MAREK’S DISEASE VIRUS AND INDUSTRIALIZED AGRICULTURE

Marek’s disease virus (MDV), a commercially important disease of poultry, has become substantially more virulent over the last 60 years. This evolution was presumably a consequence of changes in virus ecology associated with the intensification of the poultry industry. Here, we assess whether vaccination or reduced host life span could have generated natural selection, which favored more virulent strains. Using previously published experimental data, we estimated viral fitness under a range of cohort durations and vaccine treatments on broiler farms. We found that viral fitness maximized at intermediate virulence, as a result of a trade‐off between virulence and transmission previously reported. Our results suggest that vaccination, acting on this trade‐off, could have led to the evolution of increased virulence. By keeping the host alive, vaccination prolongs infectious periods of virulent strains. Improvements in host genetics and nutrition, which reduced broiler life spans below 50 days, could have also increased the virulence of the circulating MDV strains because shortened cohort duration reduces the impact of host death on viral fitness. These results illustrate the dramatic impact anthropogenic change can potentially have on pathogen virulence.     

A Membrane-Translocating Peptide Penetrates into Bilayers without Significant Bilayer Perturbations

Using a high throughput screen, we have identified a family of 12-residue long peptides that spontaneously translocate across membranes. These peptides function by a poorly understood mechanism that is very different from that of the well-known, highly cationic cell penetrating peptides such as the tat peptide from HIV. The newly discovered translocating peptides can carry polar cargoes across synthetic bilayers and across cellular membranes quickly and spontaneously without disrupting the membrane. Here we report on the biophysical characterization of a representative translocating peptide from the selected family, TP2, as well as a negative control peptide, ONEG, from the same library. We measured the binding of the two peptides to lipid bilayers, their secondary structure propensities, their dispositions in bilayers by neutron diffraction, and the response of the bilayer to the peptides. Compared to the negative control, TP2 has a greater propensity for membrane partitioning, although it still binds only weakly, and a higher propensity for secondary structure. Perhaps most revealing, TP2 has the ability to penetrate deep into the bilayer without causing significant bilayer perturbations, a property that may help explain its ability to translocate without bilayer permeabilization.     

Degradation of benzene, toluene, and xylene isomers by a bacterial consortium obtained from rhizosphere soil of Cyperus sp. grown in a petroleum-contaminated area

Increasing contamination of soil and groundwater with benzene, toluene, and xylene (BTX) due to activities of the chemical and oil refinery industry has caused serious environmental damage. Efficient methods are required to isolate and degrade them. Microorganisms associated with rhizosphere soil are considered efficient agents to remediate hydrocarbon contamination. In this study, we obtained a stabilized bacterial consortium from the rhizosphere soil of Cyperus sp. grown in a petroleum-contaminated field in Southern Mexico. This consortium was able to completely degrade BTX in 14 days. Bacteria isolated from the consortium were identified by 16S rRNA gene sequence analysis as Ralstonia insidiosa, Cellulomonas hominis, Burkholderia kururiensis, and Serratia marcescens. The BTX-degradation capacity of the bacterial consortium was confirmed by the detection of genes pheA, todC1, and xylM, which encoded phenol hydroxylase, toluene 1,2-dioxygenase, and xylene monooxygenase, respectively. Our results demonstrate feasibility of BTX biodegradation by indigenous bacteria that might be used for soil remediation in Southern Mexico.     

The design of a novel series of muscarinic receptor antagonists leading to AZD8683, a potential inhaled treatment for COPD

A novel series of muscarinic receptor antagonists was developed, with the aim of identifying a compound with high M₃ receptor potency and a reduced risk of dose-limiting side effects with potential for the treatment of COPD.Initial compound modifications led to a novel cycloheptyl series, which was improved by focusing on a quinuclidine sub-series. A wide range of N-substituents was evaluated to determine the optimal substituent providing a high M₃ receptor potency, high intrinsic clearance and high human plasma protein binding. Compounds achieving in vitro study criteria were selected for in vivo evaluation. Pharmacokinetic half-lives, inhibition of bronchoconstriction and duration of action, as well as systemic side effects, induced by the compounds were assessed in guinea-pig models.Compounds with a long duration of action and good therapeutic index were identified and AZD8683 was selected for progression to the clinic.