Efficient parallel synthesis of macrocyclic peptidomimetics

A new method for solid phase parallel synthesis of chemically and conformationally diverse macrocyclic peptidomimetics is reported. A key feature of the method is access to broad chemical and conformational diversity. Synthesis and mechanistic studies on the macrocyclization step are reported.     

Mitochondrial bovine ADP/ATP carrier in detergent is predominantly monomeric but also forms multimeric species

ADP/ATP carriers (AACs) are major and essential constituents of the inner mitochondrial membrane. They drive the import of ADP and the export of newly synthesized ATP. They were described as functional dimers from the 1980s until the structures of the AAC shed doubt on this consensus. We aimed to ascertain the published biophysical data claiming that AACs are dimers and to characterize the oligomeric state of the protein before crystallization. Analytical ultracentrifugation sedimentation velocity experiments clearly show that the bovine AAC is a monomer in 3-laurylamido-N,N'-dimethylpropylaminoxide (LAPAO), whereas in Triton X-100 and reduced Triton X-100, higher molecular mass species can also be identified. Neutron scattering data for monomeric bovine AAC in LAPAO does not give definite conclusions on the association state, because the large amount of detergent and lipids is imperfectly matched by contrast methods. We discuss a possible way to integrate previously published biochemical evidence in favor of assemblies, the lack of well-defined multimers that we observe, and the information from the high-resolution structures, considering supramolecular organizations of AACs within the mitochondrial membrane.     

Bax-induced cell death in yeast depends on mitochondrial lipid oxidation.

The oxidant function of pro-apoptotic protein Bax was investigated through heterologous expression in yeast. Direct measurements of fatty acid content show that Bax-expression induces oxidation of mitochondrial lipids. This effect is prevented by the coexpression of Bcl-xL. The oxidation actually could be followed on isolated mitochondria as respiration-induced peroxidation of polyunsaturated cis-parinaric acid and on whole cells as the increase in the amount of thiobarbituric acid-reactive products. Treatments that increase the unsaturation ratio of lipids, making them more sensitive to oxidation, increase kinetics of Bax-induced death. Conversely, inhibitors of lipid oxidation and treatments that decrease the unsaturation ratio of fatty acids decrease kinetics of Bax-induced death. Taken together, these results show that Bax-induced mitochondrial lipid oxidation is relevant to Bax-induced cell death. Conversely, lipid oxidation is poorly related to the massive Bax-induced superoxide and hydrogen peroxide accumulation, which occurs at the same time, as chemical or enzymatic scavenging of ROS does not prevent lipid oxidation nor has any effects on kinetics of Bax-induced cell death. Whatever the origin of mitochondrial lipid oxidation, these data show that it represents a major step in the cascade of events leading to Bax-induced cell death. These results are discussed in the light of the role of lipid oxidation both in mammalian apoptosis and in other forms of cell death in other organisms.     

A novel, high conductance channel of mitochondria linked to apoptosis in mammalian cells and Bax expression in yeast.

During apoptosis, proapoptotic factors are released from mitochondria by as yet undefined mechanisms. Patch-clamping of mitochondria and proteoliposomes formed from mitochondrial outer membranes of mammalian (FL5.12) cells has uncovered a novel ion channel whose activity correlates with onset of apoptosis. The pore diameter inferred from the largest conductance state of this channel is approximately 4 nm, sufficient to allow diffusion of cytochrome c and even larger proteins. The activity of the channel is affected by Bcl-2 family proteins in a manner consistent with their pro- or antiapoptotic properties. Thus, the channel activity correlates with presence of proapoptotic Bax in the mitochondrial outer membrane and is absent in mitochondria from cells overexpressing antiapoptotic Bcl-2. Also, a similar channel activity is found in mitochondrial outer membranes of yeast expressing human Bax. These findings implicate this channel, named mitochondrial apoptosis-induced channel, as a candidate for the outer-membrane pore through which cytochrome c and possibly other factors exit mitochondria during apoptosis.     

Evaluation of tissue and cellular biomarkers to assess 2,4,6-trinitrotoluene (TNT) exposure in earthworms: effects-based assessment in laboratory studies using Eisenia andrei

The lysosomal neutral red retention time (NRRT) assay, a biomarker for lysosomal membrane stability, and the total immune activity (TIA) assay, a measure of non-specific immune system activity, were used in laboratory studies to assess the toxic effects of 2,4,6-trinitrotoluene (TNT) on earthworms (Eisenia andrei) in vivo. The results were compared with the concentration of TNT and its metabolites in earthworm tissue, as well as standard sublethal toxicity endpoints including growth (i.e. weight change) and reproduction effects from previously published studies. Filter paper experiments indicated a significant decrease in NRRT at ≥1.8 µg TNT cm^-2, whereas sublethal (weight loss) and lethal effects to earthworms were detected at ≥3.5 and 7.1 µg TNT cm^-2, respectively. Experiments in artificial soil showed that NRRT effects could be detected at lower TNT concentrations ( ≥55 mg TNT kg^-1 soil dry weight) compared with other sublethal endpoints (effects on growth and reproduction). The TIA biomarker did not significantly respond to TNT. Copper (as CuSO₄, filter paper contact tests) and 2-chloroacetamide (soil tests), which were used as reference toxicants, also decreased the NRRT. The use of the NRRT assay linked with tissue concentrations of TNT metabolites in earthworms was identified as a potentially appropriate biomarker approach for TNT exposure assessment under laboratory conditions and a novel tool for effects-based risk assessment.     

The role of zinc in caspase activation and apoptotic cell death

In addition to its diverse role in many physiological systems, zinc (Zn) has now been shown to be an important regulator of apoptosis. The purpose of this review is to integrate previously published knowledge on Zn and apoptosis with current attempts to elucidate the mechanisms of action of this biometal. This paper begins with an introduction to apoptosis and then briefly reviews the evidence relating Zn to apoptosis. The major focus of this review is the mechanistic actions of Zn and its candidate intracellular targets. In particular, we examine the cytoprotective functions of Zn which suppress major pathways leading to apoptosis, as well as the more direct influence of Zn on the apoptotic regulators, especially the caspase family of enzymes. These two mechanisms are closely related since a decline in intracellular Zn below a critical threshold level may not only trigger pathways leading to caspase activation but may also facilitate the process by which the caspases are activated. Studies by our laboratory in airway epithelial cells show that Zn is co-localized with the precursor form of caspase-3, mitochondria and microtubules, suggesting this Zn is critically placed to control apoptosis. Further understanding the different pools of Zn and how they interact with apoptotic pathways should have importance in human disease.     

Life in a fragment: Evolution of foraging strategies of translocated collared brown lemurs,Eulemur collaris,over an 18-year period

While the drivers of primate persistence in forest fragments have been often considered at the population level, the strategies to persist in these habitats have been little investigated at the individual or group level. Considering the rapid variation of fragment characteristics over time, longitudinal data on primates living in fragmented habitats are necessary to understand the key elements for their persistence. Since translocated animals have to cope with unfamiliar areas and face unknown fluctuations in food abundance, they offer the opportunity to study the factors contributing to successful migration between fragments. Here, we illustrated the evolution of the foraging strategies of translocated collared brown lemurs (Eulemur collaris) over an 18-year period in the Mandena Conservation Zone, south-east Madagascar. Our aim was to explore the ability of these frugivorous lemurs to adjust to recently colonized fragmented forests. Although the lemurs remained mainly frugivorous throughout the study period, over the years we identified a reduction in the consumption of leaves and exotic/pioneer plant species. These adjustments were expected in frugivorous primates living in a degraded area, but we hypothesize that they may also reflect the initial need to cope with an unfamiliar environment after the translocation. Since fragmentation is often associated with the loss of large trees and native vegetation, we suggest that the availability of exotic and/or pioneer plant species can provide an easy-to-access, nonseasonal food resource and be a key factor for persistence during the initial stage of the recolonization.     

Prostatic Alpha-Linolenic Acid (ALA) Is Positively Associated with Aggressive Prostate Cancer: A Relationship Which May Depend on Genetic Variation in ALA Metabolism

Previous observational studies have reported associations between prostate cancer and alpha-linolenic acid (ALA). However, few investigations have been able to study this relationship prospectively and in well-controlled settings. Moreover, no studies have determined whether single nucleotide polymorphisms (SNPs) that influence ALA metabolism are associated with this common cancer. The purpose of this study was to explore associations between prostatic levels of ALA, SNPs and prostate cancer-specific biomarkers in samples collected from a previous randomized clinical trial conducted using a presurgical model and which tested the effects of flaxseed supplementation, a rich source of ALA, prior to prostatectomy (n = 134). Serum prostate-specific antigen (PSA) was determined and immunohistochemistry was used to assess tumor proliferation rate (Ki67). Prostatic ALA was determined with gas chromatography. Seven previously identified SNPs associated with delta-6 desaturase activity (rs99780, rs174537, rs174545, rs174572, rs498793, rs3834458 and rs968567) were tested for associations with prostatic ALA, PSA and Ki67. Despite consuming seven times more ALA per day, men in the flaxseed arm had similar amounts of prostatic ALA relative to men not consuming flaxseed. In unadjusted analysis, there were significant positive associations between prostatic ALA and PSA (ρ = 0.191, p = 0.028) and Ki67 (ρ = 0.186, p = 0.037). After adjusting for covariates (flaxseed, age, race, BMI and statin-use) the association between ALA and PSA remained (p = 0.004) but was slightly attenuated for Ki67 (p = 0.051). We did not observe associations between any of the SNPs studied and prostatic ALA; however, in models for PSA there was a significant interaction between rs498793 and ALA and for Ki67 there were significant interactions with ALA and rs99780 and rs174545. Independent and inverse associations were observed between rs174572 and Ki67. This study provides evidence that prostatic ALA, independent of the amount of ALA consumed, is positively associated with biomarkers of aggressive prostate cancer and that genetic variation may modify this relationship.     

Apolipoprotein A-V is a potential target for treating coronary artery disease: evidence from genetic and metabolomic analyses

Triglyceride (TG)-lowering LPL variants in combination with genetic LDL-C-lowering variants are associated with reduced risk of coronary artery disease (CAD). Genetic variation in the APOA5 gene encoding apolipoprotein A-V also strongly affects TG levels, but the potential clinical impact and underlying mechanisms are yet to be resolved. Here, we aimed to study the effects of APOA5 genetic variation on CAD risk and plasma lipoproteins through factorial genetic association analyses. Using data from 309,780 European-ancestry participants from the UK Biobank, we evaluated the effects of lower TG levels as a result of genetic variation in APOA5 and/or LPL on CAD risk with or without a background of reduced LDL-C. Next, we compared lower TG levels via APOA5 and LPL variation with over 100 lipoprotein measurements in a combined sample from the Netherlands Epidemiology of Obesity study (N = 4,838) and the Oxford Biobank (N = 6,999). We found that lower TG levels due to combined APOA5 and LPL variation and genetically-influenced lower LDL-C levels afforded the largest reduction in CAD risk (odds ratio: 0.78 (0.73–0.82)). Compared to patients with genetically-influenced lower TG via LPL, genetically-influenced lower TG via APOA5 had similar and independent, but notably larger, effects on the lipoprotein profile. Our results suggest that lower TG levels as a result of APOA5 variation have strong beneficial effects on CAD risk and the lipoprotein profile, which suggest apo A-V may be a potential novel therapeutic target for CAD prevention.