ABSTRACT
Elevated blood pressure (BP) is rising in children. Significant proportions of children have reactive hypertension or masked hypertension, making ambulatory BP monitoring (ABPM) a valuable tool, although with potential economic implications. In youth referred for elevated BP, we sought clinic BP combinations that obviated the need for ABPM and to specify the economic role of ABPM. In a retrospective pediatric referral cohort (N = 170), we examine clinic systolic BP (SBP) predictors of components of ABPM hypertension and their combination. In economic analyses, we compared effectiveness and charges of three diagnostic pathways: (1) clinic BP alone; (2) abnormal clinic BP prompting ABPM; or (3) universal ABPM. ABPM hypertension occurred in 55 (32.4%) and reactive hypertension in 37 (21.8%), average automated (ß = 0.208; 95% confidence interval, 0.027, 0.389; P = .03) and maximum auscultatory clinic SBP (ß = 0.160; 95% confidence interval 0.022, 0.299; P = .02) were associated with ABPM SBP mean, but none predicted SBP load. No clinic SBP combination was associated with ABPM hypertension. Universal ABPM accrued the lowest average charge per hypertensive youth identified ($10,948). We did not identify a clinic SBP combination that predicted ABPM hypertension in youth referred for elevated BP. Universal ABPM, in this context, may be the most economically and clinically efficient diagnostic strategy.
Subject(s)
Blood Pressure Monitoring, Ambulatory/methods , Blood Pressure/physiology , Hypertension/physiopathology , Referral and Consultation , Adolescent , Blood Pressure Monitoring, Ambulatory/economics , Cost-Benefit Analysis , Female , Humans , Male , Predictive Value of Tests , Retrospective StudiesABSTRACT
BAX is a proapoptotic BCL-2 family member that lies dormant in the cytosol until converted into a killer protein in response to cellular stress. Having recently identified the elusive trigger site for direct BAX activation, we now delineate by NMR and biochemical methods the essential allosteric conformational changes that transform ligand-triggered BAX into a fully activated monomer capable of propagating its own activation. Upon BAX engagement by a triggering BH3 helix, the unstructured loop between α helices 1 and 2 is displaced, the carboxy-terminal helix 9 is mobilized for membrane translocation, and the exposed BAX BH3 domain propagates the death signal through an autoactivating interaction with the trigger site of inactive BAX monomers. Our structure-activity analysis of this seminal apoptotic process reveals pharmacologic opportunities to modulate cell death by interceding at key steps of the BAX activation pathway.
Subject(s)
Apoptosis , bcl-2-Associated X Protein/chemistry , bcl-2-Associated X Protein/metabolism , Amino Acid Sequence , Binding Sites , Humans , Hydrophobic and Hydrophilic Interactions , Ligands , Mitochondria/metabolism , Models, Biological , Models, Molecular , Molecular Sequence Data , Protein Binding , Protein Structure, Secondary , Protein Transport , Structure-Activity RelationshipABSTRACT
The antineoplastic prodrug Cloretazine exerts its cytotoxicity via a synergism between 2-chloroethylating and carbamoylating activities that are cogenerated upon activation in situ. Cloretazine is reported here to inhibit the nucleotidyl-transferase activity of purified human DNA polymerase beta (Pol beta), a principal enzyme of DNA base excision repair (BER). The 2-chloroethylating activity of Cloretazine alkylates DNA at the O(6) position of guanine bases resulting in 2-chloroethoxyguanine monoadducts, which further react to form cytotoxic interstrand DNA crosslinks. Alkylated DNA is often repaired via BER in vivo. Inhibition of the polymerase activity of Pol beta may account for some of the synergism between Cloretazine's two reactive subspecies in cytotoxicity assays. This inhibition was only observed using agents with carbamoylating activity. Furthermore, while therapeutically relevant concentrations of Cloretazine inhibited the polymerase activity of Pol beta, the enzyme's lyase activity, which may also participate in BER, was not significantly inhibited.
Subject(s)
Antineoplastic Agents/pharmacology , DNA Polymerase beta/antagonists & inhibitors , Hydrazines/pharmacology , Prodrugs/pharmacology , Sulfonamides/pharmacology , DNA Polymerase I/antagonists & inhibitors , DNA Repair/drug effects , Humans , Inhibitory Concentration 50ABSTRACT
BAX is a pro-apoptotic protein of the BCL-2 family that is stationed in the cytosol until activated by a diversity of stress stimuli to induce cell death. Anti-apoptotic proteins such as BCL-2 counteract BAX-mediated cell death. Although an interaction site that confers survival functionality has been defined for anti-apoptotic proteins, an activation site has not been identified for BAX, rendering its explicit trigger mechanism unknown. We previously developed stabilized alpha-helix of BCL-2 domains (SAHBs) that directly initiate BAX-mediated mitochondrial apoptosis. Here we demonstrate by NMR analysis that BIM SAHB binds BAX at an interaction site that is distinct from the canonical binding groove characterized for anti-apoptotic proteins. The specificity of the human BIM-SAHB-BAX interaction is highlighted by point mutagenesis that disrupts functional activity, confirming that BAX activation is initiated at this novel structural location. Thus, we have now defined a BAX interaction site for direct activation, establishing a new target for therapeutic modulation of apoptosis.
