Determination of the cell lytic properties of amphiphilic inhibitors of the cytosolic phospholipase A2 against human platelets by measuring the liberation of serotonin with high-performance liquid chromatography and fluorescence detection

A procedure for the determination of the influence of detergents on the cell integrity of human platelets by measuring the release of serotonin with high-performance liquid chromatography and fluorescence detection is presented. After exposure of the platelets to the test compounds the cells and cell fragments, respectively, are centrifuged off. In the supernatants the liberated serotonin is determined directly without a further sample clean up. The amphiphilic inhibitors of cytosolic phospholipase A2 (cPLA2), arachidonyltrifluoromethyl ketone (AACOCF3), palmityltrifluoromethyl ketone (PACOCF3) and methyl arachidonylfluorophosphonate (MAFP), and the polyoxyethylene detergent Brij 58 were investigated for their cell lytic properties with this method. All compounds lysed the platelets liberating serotonin at a concentration of 33 microM. AACOCF, and Brij 58 even caused cell lysis at lower concentrations.     

Do Committees Ru(i)n the Bio‐Political Culture? On the Democratic Legitimacy of Bioethics Committees

Bioethical and bio-political questions are increasingly tackled by committees, councils, and other advisory boards that work on different and often interrelated levels. Research ethics committees work on an institutional or clinical level; local advisory boards deal with biomedical topics on the level of particular political regions; national and international political advisory boards try to answer questions about morally problematic political decisions in medical research and practice. In accordance with the increasing number and importance of committees, the quality of their work and their functional status are being subjected to more and more scrutiny. Besides overall criticism regarding the quality of their work, particular committees giving political advice are often suspected of being incompatible with democratic values, such as respect for affected parties, representation of diverse values and transparency in the decision-making processes. Based on the example of the German National Ethics Council, whose inauguration caused a still ongoing debate on the aims and scopes of committees in general, this paper discusses: (1) the requirements of modern democratic societies in dealing with complex scientific-technical problems; (2) the composition and organisation of committees working as political advisory boards; and (3) the appointment procedures and roles of laymen and experts, and here in particular of ethicists, who may legitimately be taken on by a committee. I will argue that bioethics committees do not necessarily endanger democratic values, but can considerably improve their realisation in democratic decision-making procedures--if, and only if, they do not act as substitutes for parliamentarian processes, but help prepare parliamentarian processes to be organised as rationally as possible.     

Proteomic analysis of the lake trout (Salvelinus namaycush) heart and blood: The beginning of a comprehensive lake trout protein database

Lake trout (Salvelinus namaycush) are a top-predator species in the Laurentian Great Lakes that are often used as bioindicators of chemical stressors in the ecosystem. Although many studies are done using these fish to determine concentrations of stressors like legacy persistent, bioaccumulative and toxic chemicals, there are currently no proteomic studies on the biological effects these stressors have on the ecosystem. This lack of proteomic studies on Great Lakes lake trout is because there is currently no complete, comprehensive protein database for this species. Here, we employed proteomics approaches to develop a lake trout protein database that could aid in future research on this fish, in particular exposomics and adductomics. The current study utilized heart tissue and blood from two lake trout. Our previous work using lake trout liver revealed 4194 potential protein hits in the NCBI databases and 3811 potential protein hits in the UniProtKB databases. In the current study, using the NCBI databases we identified 838 proteins for the heart and 580 proteins for the blood tissues in the biological replicate 1 (BR1) and 1180 potential protein hits for the heart and 561 potential protein hits for the blood in BR2. Similar results were obtained using the UniProtKB databases. This study builds on our previous work by continuing to build the first comprehensive lake trout protein database and provides insight into protein homology through evolutionary relationships. This data is available via the PRIDE partner repository with the dataset identifier PXD023970.     

Utilization of Splicing Elements and Polyadenylation Signal Elements in the Coupling of Polyadenylation and Last-Intron Removal

Polyadenylation (PA) is the process by which the 3' ends of most mammalian mRNAs are formed. In nature, PA is highly coordinated, or coupled, with splicing. In mammalian systems, the most compelling mechanistic model for coupling arises from data supporting exon definition (2, 34, 37). We have examined the roles of individual functional components of splicing and PA signals in the coupling process by using an in vitro splicing and PA reaction with a synthetic pre-mRNA substrate containing an adenovirus splicing cassette and the simian virus 40 late PA signal. The effects of individually mutating splicing elements and PA elements in this substrate were determined. We found that mutation of the polypyrimidine tract and the 3' splice site significantly reduced PA efficiency and that mutation of the AAUAAA and the downstream elements of the PA signal decreased splicing efficiency, suggesting that these elements are the most significant for the coupling of splicing and PA. Although mutation of the upstream elements (USEs) of the PA signal dramatically decreased PA, splicing was only modestly affected, suggesting that USEs modestly affect coupling. Mutation of the 5' splice site in the presence of a viable polypyrimidine tract and the 3' splice site had no effect on PA, suggesting no effect of this element on coupling. However, our data also suggest that a site for U1 snRNP binding (e.g., a 5' splice site) within the last exon can negatively effect both PA and splicing; hence, a 5' splice site-like sequence in this position appears to be a modulator of coupling. In addition, we show that the RNA-protein complex formed to define an exon may inhibit processing if the definition of an adjacent exon fails. This finding indicates a mechanism for monitoring the appropriate definition of exons and for allowing only pre-mRNAs with successfully defined exons to be processed.     

The distribution of the Rocky Mountain tailed frog (Ascaphus montanus) in relation to the fluvial system: implications for management and conservation

The mating, egg-laying, and larval development of tailed frogs occur in dynamic mountain streams. During the lengthy (up to 5 years) aquatic residency these species are vulnerable to channel disturbances that can be exacerbated by land uses. Researchers have highlighted specific tailed frog habitat associations but never in the context of fluvial system processes. Based on an extensive regional study with a watershed-wide sampling strategy, we demonstrate that the Rocky Mountain tailed frog (Ascaphus montanus) is limited to contributing basins of roughly 0.3–100 km² in size, with peak numbers in basins up to 35 km². We conclude that the primary determinant of tailed frog distribution patterns in a watershed is basin area, a proximate variable for channel process domain and regional stream discharge: tailed frogs are adapted to cascade and step-pool channel morphologies that characterize these small basins, presumably because they afford more bedform stability and pore-space refugia than do smaller, colluvial headwaters, or larger, floodplain-forming plane bed and pool-riffle bedforms of mainstem rivers. Secondarily, climate and physiography interact to influence occurrence and abundance at the watershed level by controlling such variables as runoff, water temperature, and sedimentation regime. This point has important management implications because it forces us to recognize that in complex ecosystems, wildlife habitat associations are contingent on site-specific interactions amongst fluvial system control variables: significance levels of any one variable to tailed frog distribution will not necessarily be consistent among basins. The study clearly shows that case studies can produce conflicting results when they lack a process-based understanding of ecological response.     

Activation of the SV40 late promoter: direct effects of T antigen in the absence of viral DNA replication

We have examined the activation of the SV40 late promoter by inserting the late promoter and the viral origin of replication into chloramphenicol acetyltransferase (CAT) transient expression vectors. Very little late promoter activity was detected in CV-1 cells, compared with high activity in COS cells, in which replication occurs due to endogenous T antigen. Nonreplicative counterparts of these plasmids, containing a mutated origin of replication, produced significantly more late promoter activity in COS cells than any of the plasmids in CV-1 cells. When plasmids were cotransfected into CV-1 cells with a plasmid that supplies T antigen, the nonreplicative plasmid displayed 30% of the activity of the replicative plasmid. Using mutant T antigens unable to replicate viral DNA, late promoter activation occurred only with mutant T antigens that retain DNA binding activity. These results demonstrate that T antigen can substantially stimulate late promoter activity directly and independent of viral DNA replication.     

Plasma coenzyme Q10 concentrations are not decreased in male patients with coronary atherosclerosis

Coenzyme Q₁₀ (CoQ₁₀) is an important mitochondrial electron transfer component and has been postulated to function as a powerful antioxidant protecting LDL from oxidative damage. It could thus reduce the risk of cardiovascular disease. Thus far, beneficial effects of supplementation with CoQ₁₀ have been reported. To study the relation between unsupplemented concentrations of plasma CoQ₁₀ and coronary atherosclerosis, we performed a case-control study among 71 male cases with angiographically documented severe coronary atherosclerosis and 69 healthy male controls free from symptomatic cardiovascular disease and without atherosclerotic plaques in the carotid artery.

Plasma CoQ₁₀ concentrations (mean ± SE) were 0.86 ± 0.04 vs. 0.83 ± 0.04 μmol/l for cases and controls, respectively. The CoQ₁₀/LDL-cholesterol ratio (μmol/mmol) was slightly lower in cases than in controls (0.22 ± 0.01 vs. 0.26 ± 0.03). Differences in CoQ₁₀ concentrations and CoQ₁₀/LDL-cholesterol ratio did not reach significance. The odds ratios (95% confidence interval) for the risk of coronary atherosclerosis calculated per μmol/l increase of CoQ₁₀ was 1.12 (0.28–4.43) after adjustment for age, smoking habits, total cholesterol and diastolic blood pressure.

We conclude that an unsupplemented plasma CoQ₁₀ concentration is not related to risk of coronary atherosclerosis.     

PKR-Like Endoplasmic Reticulum Kinase Is Necessary for Lipogenic Activation during HCMV Infection

PKR-like endoplasmic reticulum (ER) kinase (PERK) is an ER-associated stress sensor protein which phosphorylates eukaryotic initiation factor 2α (eIF2α) to induce translation attenuation in response to ER stress. PERK is also a regulator of lipogenesis during adipocyte differentiation through activation of the cleavage of sterol regulatory element binding protein 1 (SREBP1), resulting in the upregulation of lipogenic enzymes. Our recent studies have shown that human cytomegalovirus (HCMV) infection in human fibroblasts (HF) induces adipocyte-like lipogenesis through the activation of SREBP1. Here, we report that PERK expression is highly increased in HCMV-infected cells and is necessary for HCMV growth. Depletion of PERK, using short hairpin RNA (shRNA), resulted in attenuation of HCMV growth, inhibition of lipid synthesis and reduction of lipogenic gene expression. Examination of the cleavage of SREBP proteins showed PERK depletion inhibited the cleavage of SREBP1, but not SREBP2, in HCMV-infected cells, suggesting different cleavage regulatory mechanisms for SREBP1 and 2. Further studies showed that the depletion of SREBP1, but not SREBP2, reduced lipid synthesis in HCMV infection, suggesting that activation of SREBP1 is sufficient to induce lipogenesis in HCMV infection. The reduction of lipid synthesis by PERK depletion can be partially restored by expressing a Flag-tagged nuclear form of SREBP1a. Our studies also suggest that the induction of PERK in HCMV-infected cells stimulates SREBP1 cleavage by reducing levels of Insig1 (Insulin inducible gene 1) protein; this occurs independent of the phosphorylation of eIF2α. Introduction of an exogenous Insig1-Myc into HCMV infected cells significantly reduced HCMV growth and lipid synthesis. Our data demonstrate that the induction of PERK during HCMV infection is necessary for full activation of lipogenesis; this effect appears to be mediated by limiting the levels of Insig1 thus freeing SREBP1-SCAP complexes for SREBP1 processing.