Doctors in the decent society: torture, ill-treatment and civic duty

How should physicians act when faced with corporal punishment, such as amputation, or torture? In most cases, the answer is clear: international law, UN resolutions and universal codes of medical ethics absolutely forbid physicians from countenancing torture and corporal punishment in any form. An acute problem arises, however, in decent societies, but not necessarily liberal states, that are, nonetheless, welcome in the world community. The decent society is often governed, in whole or in part, by religious laws, and while these states abridge various human rights they are peace loving, generally tolerant, and offer their citizens wide avenues for political participation. Under these circumstances the prohibition against corporal punishment and torture weakens, often compelling physicians to participate. This is true in two cases. In Rawls’ hypothetical nation of Kazanistan, Islamic law is the order of the day, and amputations and corporal punishment play an integral part in the execution of traditional Islamic justice. In Israel, torture is sometimes used to elicit the information needed to thwart impending terror attacks. In each case, a physician's participation is essential. In light of the near universal condemnation that accompanies torture and corporal punishment, physicians can only appeal to norms anchored in collective well‐being and concern for life that override respect for human dignity in these societies. Western societies have consistently rejected this reasoning, but it is part and parcel of life in the decent society.     

Developmental Regulation of the (1,3)-beta-Glucan (Callose) Synthase from Tomato : Possible Role of Endogenous Phospholipases

Activity levels of UDP-glucose: (1,3)-β-glucan (callose) synthase in microsomal membranes of pericarp tissue from tomato fruit (Lycoperisicon esculentum Mill, cv Rutgers) were determined during development and ripening. Addition of the phospholipase inhibitors O-phosphorylcholine and glycerol-1-phosphate to homogenization buffers was necessary to preserve enzyme activity during homogenization and membrane isolation. Enzyme activity declined 90% from the immature green to the red ripe stage. The polypeptide composition of the membranes did not change significantly during ripening. The enzyme from immature fruit was inactivated by exogenously added phospholipases A₂, C, and D. These results suggest that the decline in callose synthase activity during ontogeny may be a secondary effect of endogenous lipase action.     

Transcriptional Repressor HIC1 Contributes to Suppressive Function of Human Induced Regulatory T Cells

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Downregulation of Serine Protease HTRA1 Is Associated with Poor Survival in Breast Cancer

HTRA1 is a highly conserved serine protease which has been implicated in suppression of epithelial-to-mesenchymal-transition (EMT) and cell motility in breast cancer. Its prognostic relevance for breast cancer is unclear so far. Therefore, we evaluated the impact of HTRA1 mRNA expression on patient outcome using a cohort of 131 breast cancer patients as well as a validation cohort including 2809 publically available data sets. Additionally, we aimed at investigating for the presence of promoter hypermethylation as a mechanism for silencing the HTRA1 gene in breast tumors. HTRA1 downregulation was detected in more than 50% of the breast cancer specimens and was associated with higher tumor stage (p = 0.025). By applying Cox proportional hazard models, we observed favorable overall (OS) and disease-free survival (DFS) related to high HTRA1 expression (HR = 0.45 [CI 0.23–0.90], p = 0.023; HR = 0.55 [CI 0.32–0.94], p = 0.028, respectively), with even more pronounced impact in node-positive patients (HR = 0.21 [CI 0.07–0.63], p = 0.006; HR = 0.29 [CI 0.13–0.65], p = 0.002, respectively). Moreover, HTRA1 remained a statistically significant factor predicting DFS among established clinical parameters in the multivariable analysis. Its impact on patient outcome was independently confirmed in the validation set (for relapse-free survival (n = 2809): HR = 0.79 [CI 0.7–0.9], log-rank p = 0.0003; for OS (n = 971): HR = 0.63 [CI 0.48–0.83], log-rank p = 0.0009). In promoter analyses, we in fact detected methylation of HTRA1 in a small subset of breast cancer specimens (two out of a series of 12), and in MCF-7 breast cancer cells which exhibited 22-fold lower HTRA1 mRNA expression levels compared to unmethylated MDA-MB-231 cells. In conclusion, we show that downregulation of HTRA1 is associated with shorter patient survival, particularly in node-positive breast cancer. Since HTRA1 loss was demonstrated to induce EMT and cancer cell invasion, these patients might benefit from demethylating agents or histone deacetylase inhibitors previously reported to lead to HTRA1 upregulation, or from novel small-molecule inhibitors targeting EMT-related processes.     

Mutagenesis of the BH3 Domain of BAX Identifies Residues Critical for Dimerization and Killing

The BCL-2 family of proteins is comprised of proapoptotic as well as antiapoptotic members (S. N. Farrow and R. Brown, Curr. Opin. Genet. Dev. 6:45–49, 1996). A prominent death agonist, BAX, forms homodimers and heterodimerizes with multiple antiapoptotic members. Death agonists have an amphipathic α helix, called BH3; however, the initial assessment of BH3 in BAX has yielded conflicting results. Our BAX deletion constructs and minimal domain constructs indicated that the BH3 domain was required for BAX homodimerization and heterodimerization with BCL-2, BCL-X_L, and MCL-1. An extensive site-directed mutagenesis of BH3 revealed that substitutions along the hydrophobic face of BH3, especially charged substitutions, had the greatest affects on dimerization patterns and death agonist activity. Particularly instructive was the BAX mutant mIII-1 (L63A, G67A, L70A, and M74A), which replaced the hydrophobic face of BH3 with alanines, preserving its amphipathic nature. BAXmIII-1 failed to form heterodimers or homodimers by yeast two-hybrid or immunoprecipitation analysis yet retained proapoptotic activity. This suggests that BAX’s killing function reflects mechanisms beyond its binding to BCL-2 or BCL-X_L to inhibit them or simply displace other protein partners. Notably, BAXmIII-1 was found predominantly in mitochondrial membranes, where it was homodimerized as assessed by homobifunctional cross-linkers. This characteristic of BAXmIII-1 correlates with its capacity to induce mitochondrial dysfunction, caspase activation, and apoptosis. These data are consistent with a model in which BAX death agonist activity may require an intramembranous conformation of this molecule that is not assessed accurately by classic binding assays.

Programmed cell death and its morphologic equivalent, apoptosis, are orchestrated by a distinct genetic pathway that is apparently possessed by all multicellular organisms (22). Moreover, the biochemical details of how encoded proteins function are beginning to emerge. The BCL-2 family of proteins constitutes a central decisional point within the common portion of the apoptotic pathway. This family possesses both proapoptotic (BAX, BAK, BCL-X_S, BAD, BIK, BID, HRK, and BIM) and antiapoptotic (BCL-2, BCL-X_L, MCL-1, and A1) molecules (5, 11). The ratio of antiapoptotic to proapoptotic molecules such as BCL-2/BAX determines the response to a proximal apoptotic signal (14). A striking characteristic of many family members is their propensity to form homo- and heterodimers (16, 19). The BCL-2 family has homology clustered principally within four conserved domains called BH1, BH2, BH3, and BH4 (5, 11). The multidimensional nuclear magnetic resonance (NMR) and X-ray crystallographic structure of a BCL-X_L monomer indicates that the BH1-4 domains correspond to α helices 1 to 7. Notably, the BH1, -2, and -3 domains are in close proximity and create a hydrophobic pocket presumably involved in interactions with other BCL-2 family members (13). The NMR analysis of a BCL-X_L-BAK BH3 peptide complex revealed both hydrophobic and electrostatic interactions between the BCL-X_L pocket and a BH3 amphipathic α-helical peptide from BAK (17).Prior mutagenesis studies of BCL-2 and BCL-X_L revealed the importance of BH1 and BH2 domains for both their antiapoptotic function and the capacity to heterodimerize with proapoptotic molecules like BAX or BAK (2, 19, 26). In general, most mutations that disrupt heterodimerization with BAX also lose their death repressor function. However, exceptions do exist; some mutants of BCL-X_L fail to bind BAX or BAK but still repress cell death, suggesting that these functions can be separated for antiapoptotic molecules (2). Moreover, a genetic approach with Bcl-2-deficient and Bax-deficient mice also suggested that BCL-2 and BAX could function independently of one another (10).Deletion studies of the death agonist BAK first implicated the BH3 domain as having the capacity to bind BCL-X_L and promote apoptosis (3). However, the functional significance of BH3 in BAX is uncertain as indicated in the literature. Three deletion analyses indicated the necessity of the BH3 domain in BAX to promote cell death as well as to heterodimerize with BCL-2 (3, 9, 28). Yet, two recent studies reported that BAX functions as a death activator independent of its heterodimerization (21, 27). Moreover, substitution mutants within the BH3 domain showed conflicting specificities of heterodimerization (20, 21, 27).Our initial screen of yeast two-hybrid libraries with BCL-2 as bait yielded multiple clones that possess only the NH₂ terminus of BAX, bearing the BH3 but not the BH1 or the BH2 domains. A similar set of isolates was obtained when BCL-2 (G145A) was used as bait (15). We also noted by deletion analysis and assessment of minimal domains of BAX that the BH3 domain was required for both homodimerization and heterodimerization. Consequently, we undertook an extensive site-directed mutagenesis of the BH3 domain of BAX. These studies demonstrate the importance of the hydrophobic face of the amphipathic α helix of BH3 for the dimerization and cell death activities of BAX. Furthermore, analysis of a BAX mutant indicates that its retained conformation as a cross-linkable dimer at mitochondrial membranes correlates with its intact apoptotic function.     

Effective small interfering RNAs and phosphorothioate antisense DNAs have different preferences for target sites in the luciferase mRNAs

Antisense DNA target sites can be selected by the accessibility of the mRNA target. It remains unknown whether a mRNA site that is accessible to an antisense DNA is also a good candidate target site for a siRNA. Here, we reported a parallel analysis of 12 pairs of antisense DNAs and siRNA duplexes for their potency to inhibit reporter luciferase activity in mammalian cells, both of the antisense DNA and siRNA agents in a pair being directed to same site in the mRNA. Five siRNAs and two antisense DNAs turned out to be effective, but the sites targeted by those effective siRNAs and antisense DNAs did not overlap. Our results indicated that effective antisense DNAs and siRNAs have different preferences for target sites in the mRNA.     

Introgressive hybridization of Senecio hercynicus and S. ovatus (Compositae,Senecioneae) along an altitudinal gradient in Harz National Park (Germany)

Introgressive hybridization of Senecio hercynicus and S. ovatus (Compositae, Senecioneae) was studied in a hybrid zone on the southern slopes of Mt Brocken (Harz Mountains, Germany). A total of 415 plants representing 10 stands along an altitudinal gradient were investigated using multivariate statistical analyses of morphological characters and molecular markers (random amplified polymorphic DNA[RAPD]). Both types of traits detected pure S. hercynicus stands on the summit plateau, pure S. ovatus stands at the lowest elevations, and hybrid swarms at intermediate elevations. While morphological and molecular patterns coincided, some individuals in hybrid stands combined morphological patterns typical of S. ovatus with RAPD patterns typical of S. hercynicus, and vice versa. In general, introgression was symmetrical within stands, though one stand combined S. ovatus characters with the glandular hair typical for S. hercynicus, and two stands combined a S. hercynicus typical RAPD genotype with morphological characters shifted towards S. ovatus. Because pure stands of S. hercynicus occurred only on the summit plateau of Mt Brocken, and markers typical for S. ovatus were detectable in stands up to 1040 m a.s.l., future fusion or assimilation of the rare form, S. hercynicus, by the more widespread S. ovatus appears possible at Mt Brocken.     

The QRxGRxGRxxxG motif of the vaccinia virus DExH box RNA helicase NPH-II is required for ATP hydrolysis and RNA unwinding but not for RNA binding.

Vaccinia virus NPH-II is an essential nucleic acid-dependent nucleoside triphosphate that catalyzes unidirectional unwinding of duplex RNA containing a 3' tail. NPH-II is the prototypal RNA helicase of the DExH box protein family, which is defined by several shared sequence motifs. The contribution of the conserved QRKGRVGRVNPG region to enzyme activity was assessed by alanine-scanning mutagenesis. Ten mutated versions of NPH-II were expressed in vaccinia virus-infected BSC-40 cells and purified by nickel affinity chromatography and glycerol gradient sedimentation. The mutated proteins were characterized with respect to RNA helicase, nucleic acid-dependent ATPase, and RNA binding functions. Individual alanine substitutions at invariant residues Q-491, G-494, R-495, G-497, R-498, and G-502 caused severe defects in RNA unwinding that correlated with reduced rates of ATP hydrolysis. None of these mutations affected the binding of NPH-II to single-strand RNA or to the tailed duplex RNA used as a helicase substrate. Mutation of the strictly conserved position R-492 inhibited ATPase and helicase activities and also caused a modest decrement in RNA binding. Alanine mutations at the nonconserved position N-500 and the weakly conserved residue P-501 had no apparent effect on any activity associated with NPH-II, whereas a mutation at the weakly conserved position K-493 reduced helicase to one-third and ATPase to two-thirds of the activity of wild-type required for ATP hydrolysis and RNA unwinding but not for RNA binding. Because mutations in the HRxGRxxR motif of the prototypal DEAD box RNA helicase eIF-4A abolish or severely inhibit RNA binding, we surmise that the contribution of conserved helicase motifs to overall protein function is context dependent.     

Single and Multiple Dose Pharmacokinetics,Pharmacodynamics and Safety of the Novel Lipoprotein-Associated Phospholipase A2 Enzyme Inhibitor Darapladib in Healthy Chinese Subjects: An Open Label Phase-1 Clinical Trial

Background and Objectives

Darapladib is a lipoprotein-associated phospholipase A₂ (Lp-PLA₂) inhibitor. This study evaluated the pharmacokinetics, pharmacodynamics and safety of darapladib in healthy Chinese subjects.

Methods

Twenty-four subjects received darapladib 160 mg orally, approximately 1 hour after a standard breakfast, as a single dose and once daily for 28 days. Non-compartmental methods were used to determine the single and multiple dose pharmacokinetics of darapladib and its metabolite SB-553253. Repeat dose Lp-PLA₂ activity and safety were evaluated.

Results

Systemic exposure (AUC_(0-T), Cmax geometric mean (CVb%)) of darapladib was higher after multiple-dosing (519 ng.h/mL (33.3%), 34.4 ng/mL (49.9%)) compared to single-dose administration (153 ng.h/mL (69.0%), 17.9 ng/mL (55.2%). The steady-state accumulation ratio was less than unity (Rs = 0.80), indicating time-dependent pharmacokinetics of darapladib. Darapladib steady-state was reached by Day 14 of once daily dosing. Systemic exposure to SB-553253 was lower than darapladib with median (SB-553253: darapladib) ratios for AUC_(0-τ) of 0.0786 for single dose and 0.0532 for multiple dose administration. On Day 28, pre-dose and maximum inhibition of Lp-PLA₂ activity was approximately 70% and 75% relative to the baseline value, respectively and was dependent of darapladib concentration. The most common adverse events (≥ 21% subjects) were abnormal faeces, abnormal urine odour, diarrhoea and nasopharyngitis.

Conclusion

Darapladib 160 mg single and repeat doses were profiled in healthy Chinese subjects. Single dose systemic exposure to darapladib in healthy Chinese subjects was consistent with that observed previously in Western subjects whereas steady-state systemic exposure was approximately 65% higher in Chinese than Western subjects. The Lp-PLA₂ activity and adverse event profile were similar in healthy Chinese and previous reports in Western subjects. Ethnic-specific dose adjustment of darapladib is not considered necessary for the Chinese population.

Trial Registration

ClinicalTrials.gov NCT02000804