Mechanistic insight of platelet apoptosis leading to non-surgical bleeding among heart failure patients supported by continuous-flow left ventricular assist devices

Vascular endothelial cells are highly sensitive to oxidative stress, and this is one of the mechanisms by which widespread endothelial dysfunction is induced in most cardiovascular diseases and disorders. However, how these cells can survive in oxidative stress environments remains unclear. Salidroside, a traditional Chinese medicine, has been shown to confer vascular protective effects. We aimed to understand the role of autophagy and its regulatory mechanisms by treating human umbilical vein endothelial cells (HUVECs) with salidroside under oxidative stress. HUVECs were treated with salidroside and exposed to hydrogen peroxide (H₂O₂). The results indicated that salidroside exerted cytoprotective effects in an H₂O₂-induced HUVEC injury model and suppressed H₂O₂-induced apoptosis of HUVECs. Pretreatment with 3-methyladenine (3-MA), an autophagy inhibitor, increased oxidative stress-induced HUVEC apoptosis, while the autophagy activator rapamycin induced anti-apoptosis effects in HUVECs. Salidroside increased autophagy and decreased apoptosis of HUVECs in a dose-dependent manner under oxidative stress. Moreover, 3-MA attenuated salidroside-induced HUVEC autophagy and promoted apoptosis, whereas rapamycin had no additional effects compared with salidroside alone. Salidroside upregulated AMPK phosphorylation but downregulated mTOR phosphorylation under oxidative stress; however, administration of compound C, an AMPK inhibitor, abrogated AMPK phosphorylation and increased mTOR phosphorylation and apoptosis compared with salidroside alone. These results suggest that autophagy is a protective mechanism in HUVECs under oxidative stress and that salidroside might promote autophagy through activation of the AMPK pathway and downregulation of mTOR pathway.     

Structural consequences of phosphorylation of two serine residues in the cytoplasmic domain of HIV-1 VpU.

The human immunodeficiency virus type 1 (HIV-1) protein U (VpU) is an accessory protein responsible for enhancement of viral particle release and down regulation of the T-lymphocyte coreceptor CD4. Direct binding between the cytoplasmic domains of CD4 and VpU as well as phosphorylation of serines 53 and 57 in the cytoplasmic domain of VpU plays a central role in CD4 downregulation. We investigated structural consequences of phosphorylation of the two serines using nuclear magnetic resonance spectroscopy. A uniformly 15N and 13C stable isotope-labeled 45-residue peptide comprising the cytoplasmic domain of VpU (VpUcyt) was recombinantly produced in E .coli. The peptide forms two helices (commonly referred to as helix 2 and 3) in the presence of membrane mimicking dodecylphosphocholine (DPC) micelles, which flank a flexible region containing the two phosphorylation sites. Phosphorylation does not cause any drastic structural changes in the secondary structure of VpUcyt. However, an N-terminal elongation of helix 3 and a slightly reduced helicity at the C-terminus of helix 2 are observed upon phosphorylation based on characteristic changes of 13Calpha and 13Cbeta chemical shifts. Phosphorylation also reduces the local mobility of the protein backbone in the loop region containing the phosphorylation sites according to heteronuclear 1H--15N nuclear Overhauser enhancement (NOE) data.     

The First Cellular Models Based on Frataxin Missense Mutations That Reproduce Spontaneously the Defects Associated with Friedreich Ataxia

Background

Friedreich ataxia (FRDA), the most common form of recessive ataxia, is due to reduced levels of frataxin, a highly conserved mitochondrial iron-chaperone involved in iron-sulfur cluster (ISC) biogenesis. Most patients are homozygous for a (GAA)_n expansion within the first intron of the frataxin gene. A few patients, either with typical or atypical clinical presentation, are compound heterozygous for the GAA expansion and a micromutation.

Methodology

We have developed a new strategy to generate murine cellular models for FRDA: cell lines carrying a frataxin conditional allele were used in combination with an EGFP-Cre recombinase to create murine cellular models depleted for endogenous frataxin and expressing missense-mutated human frataxin. We showed that complete absence of murine frataxin in fibroblasts inhibits cell division and leads to cell death. This lethal phenotype was rescued through transgenic expression of human wild type as well as mutant (hFXN^G130V and hFXN^I154F) frataxin. Interestingly, cells expressing the mutated frataxin presented a FRDA-like biochemical phenotype. Though both mutations affected mitochondrial ISC enzymes activities and mitochondria ultrastructure, the hFXN^I154F mutant presented a more severe phenotype with affected cytosolic and nuclear ISC enzyme activities, mitochondrial iron accumulation and an increased sensitivity to oxidative stress. The differential phenotype correlates with disease severity observed in FRDA patients.

Conclusions

These new cellular models, which are the first to spontaneously reproduce all the biochemical phenotypes associated with FRDA, are important tools to gain new insights into the in vivo consequences of pathological missense mutations as well as for large-scale pharmacological screening aimed at compensating frataxin deficiency.     

Targeted deletion of the genes encoding NTH1 and NEIL1 DNA N-glycosylases reveals the existence of novel carcinogenic oxidative damage to DNA

We have generated a strain of mice lacking two DNA N-glycosylases of base excision repair (BER), NTH1 and NEIL1, homologs of bacterial Nth (endonuclease three) and Nei (endonuclease eight). Although these enzymes remove several oxidized bases from DNA, they do not remove the well-known carcinogenic oxidation product of guanine: 7,8-dihydro-8-oxoguanine (8-OH-Gua), which is removed by another DNA N-glycosylase, OGG1. The Nth1^?/?Neil1^?/? mice developed pulmonary and hepatocellular tumors in much higher incidence than either of the single knockouts, Nth1^?/? and Neil1^?/?. The pulmonary tumors contained, exclusively, activating GGT → GAT transitions in codon 12 of K-ras of their DNA. Such transitions contrast sharply with the activating GGT → GTT transversions in codon 12 of K-ras of the pathologically similar pulmonary tumors, which arose in mice lacking OGG1 and a second DNA N-glycosylase, MUTY. To characterize the biochemical phenotype of the knockout mice, the content of oxidative DNA base damage was analyzed from three tissues isolated from control, single and double knockout mice. The content of 8-OH-Gua was indistinguishable among all genotypes. In contrast, the content of 4,6-diamino-5-formamidopyrimidine (FapyAde) and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua) derived from adenine and guanine, respectively, were increased in some but not all tissues of Neil1^?/? and Neil1^?/?Nth1^?/? mice. The high incidence of tumors in our Nth1^?/?Neil1^?/? mice together with the nature of the activating mutation in the K-ras gene of their pulmonary tumors, reveal for the first time, the existence of mutagenic and carcinogenic oxidative damage to DNA which is not 8-OH-Gua.     

Single Vector System for Efficient N-myristoylation of Recombinant Proteins in E. coli

Background

N-myristoylation is a crucial covalent modification of numerous eukaryotic and viral proteins that is catalyzed by N-myristoyltransferase (NMT). Prokaryotes are lacking endogeneous NMT activity. Recombinant production of N-myristoylated proteins in E. coli cells can be achieved by coexpression of heterologous NMT with the target protein. In the past, dual plasmid systems were used for this purpose.

Methodology/Principal Findings

Here we describe a single vector system for efficient coexpression of substrate and enzyme suitable for production of co- or posttranslationally modified proteins. The approach was validated using the HIV-1 Nef protein as an example. A simple and efficient protocol for production of highly pure and completely N-myristoylated Nef is presented. The yield is about 20 mg myristoylated Nef per liter growth medium.

Conclusions/Significance

The single vector strategy allows diverse modifications of target proteins recombinantly coexpressed in E. coli with heterologous enzymes. The method is generally applicable and provides large amounts of quantitatively processed target protein that are sufficient for comprehensive biophysical and structural studies.     

Effects of landscape features on gene flow of valley oaks (<Emphasis Type="Italic">Quercus lobata</Emphasis>)

Landscape features affect habitat connectivity and patterns of gene flow and hence influence genetic structure among populations. We studied valley oak (Quercus lobata), a threatened species of California (USA) savannas and oak woodlands, with a distribution forming a ring around the Central Valley grasslands. Our main goal was to determine the role of topography and land cover on patterns of gene flow and to test whether elevation or land cover forms stronger barriers to gene flow among valley oak populations. We sampled valley oaks in 12 populations across the range of this species, genotyped each tree at eight nuclear microsatellite loci, and created a series of resistance surfaces by assigning different resistance values to land cover type and elevation. We also estimated recent migration rates and evaluated them with regard to landscape features. There was a significant but weak relationship between Euclidian distance and genetic distance. There was no relationship between genetic distances and land cover, but a significant relationship between genetic distances and elevation resistance. We conclude that gene flow is restricted by high elevations in the northern part of the valley oak range and by high elevations and the Central Valley further south. Migration rate analysis indicated some gene flow occurring east–west but we suggest that the high connectivity in the northern Central Valley is facilitating the formation of these links. We predict that southern populations may become more differentiated in the future through genetic isolation and local adaptation taking place in the face of climate change.     

Pileated Gibbon Density in Relation to Habitat Characteristics and Post‐logging Forest Recovery

ABSTRACT Although it is known that forest loss and degradation negatively impact most forest‐dwelling primates, such relationships are difficult to quantify because many primates are difficult to survey over large areas. Furthermore, recovery times are also difficult to assess due to a lack of long‐term data. Here, we determined how forest characteristics and habitat disturbance correlate with the abundance of pileated gibbons, Hylobates pileatus. We studied a population in Khao Ang Rue Nai Wildlife Sanctuary in southeastern Thailand, assessed its density using an auditory method combined with distance sampling at 24 randomly placed sample sites. In addition, we determined how simple forest structural characteristics and habitat disturbance correlate with the gibbon abundance. Average gibbon density per site was 1.02 ± 0.16 (SE) groups/km² (range 0–2.74). Bivariate analyses indicated that densities depended on food tree biomass, level of disturbance, evergreen forest cover, time since protection, and distance to the sanctuary boundary. Multiple regression analysis suggested evergreen forest cover and distance to boundary were the most influential factors. Because evergreen forest cover, time since protection, and habitat disturbance are correlated, these results suggest a direct dependence of gibbon densities on mature, undisturbed evergreen forest. While gibbons can persist in disturbed areas if the forest is protected, it appears that recovery to previous densities may take decades. We suggest that this is due to the slow pace of forest regeneration and/or poor recovery potential of gibbons.