Membrane binding by tBid initiates an ordered series of events culminating in membrane permeabilization by Bax.

In normal circumstances, the Bcl-2 family dutifully governs when cells die. However, the rules of engagement between the pro- and antiapoptotic family members are still contested, and how Bax is transformed from a cytosolic monomer to an outer mitochondrial membrane-permeabilizing oligomer is unclear. With fluorescence techniques and an in vitro system, the combination of tBid and Bax produced dramatic membrane permeabilization. The membrane is not a passive partner in this process beause membranes are required for the protein-protein interactions to occur. Simultaneous measurements of these interactions revealed an ordered series of steps required for outer membrane permeabilization: (1) tBid rapidly binds to membranes, where (2) tBid interacts with Bax, causing (3) Bax insertion into membranes and (4) oligomerization, culminating in (5) membrane permeabilization. Bcl-XL prevents membrane-bound tBid from binding Bax. Bad releases tBid from Bcl-XL, restoring both tBid binding to Bax and membrane permeabilization.

OBSERVE-5: Comparison of Etanercept-Treated Psoriasis Patients From Canada and the United States.

BACKGROUND: OBSERVE-5 surveillance registry results evaluating etanercept safety and effectiveness in patients with moderate to severe psoriasis from Canada and the United States have been reported from data collected between May 2006 and December 2012. Although both countries have an identical indicated starting dose, the maintenance dose can differ and thus affect management strategies and outcomes. OBJECTIVE: To compare the long-term safety and effectiveness outcomes of etanercept in the Canadian and US cohorts. METHODS: Primary end points included exposure-adjusted event incidence rates of serious adverse events and serious infectious events. Secondary end points included exposure-adjusted event incidence rates of events of medical interest and efficacy outcomes. RESULTS: Over 5 years, Canadian patients received a higher maintenance dose of etanercept (50 mg twice/week) more frequently than those from the United States. Safety outcome comparisons revealed that Canadian patients had a significantly lower occurrence of serious adverse events than patients from the United States, with an overall exposure-adjusted event incidence rate per 100 patient-years of 4.46 (95% confidence interval [CI], 3.05-6.29) vs 7.76 (95% CI 7.04-8.54), respectively. Serious infectious event rates were not significantly different between the 2 countries. Secondary outcomes of events of medical interest and effectiveness also did not reveal significant differences between the 2 cohorts. CONCLUSION: After 5 years of etanercept use, safety and effectiveness outcomes were similar between patients from Canada and the United States, with the exception of a significantly lower rate of serious adverse events in the Canadian population.

Bcl-XL inhibits membrane permeabilization by competing with Bax.

Although Bcl-XL and Bax are structurally similar, activated Bax forms large oligomers that permeabilize the outer mitochondrial membrane, thereby committing cells to apoptosis, whereas Bcl-XL inhibits this process. Two different models of Bcl-XL function have been proposed. In one, Bcl-XL binds to an activator, thereby preventing Bax activation. In the other, Bcl-XL binds directly to activated Bax. It has been difficult to sort out which interaction is important in cells, as all three proteins are present simultaneously. We examined the mechanism of Bax activation by tBid and its inhibition by Bcl-XL using full-length recombinant proteins and measuring permeabilization of liposomes and mitochondria in vitro. Our results demonstrate that Bcl-XL and Bax are functionally similar. Neither protein bound to membranes alone. However, the addition of tBid recruited molar excesses of either protein to membranes, indicating that tBid activates both pro- and antiapoptotic members of the Bcl-2 family. Bcl-XL competes with Bax for the activation of soluble, monomeric Bax through interaction with membranes, tBid, or t-Bid-activated Bax, thereby inhibiting Bax binding to membranes, oligomerization, and membrane permeabilization. Experiments in which individual interactions were abolished by mutagenesis indicate that both Bcl-XL-tBid and Bcl-XL-Bax binding contribute to the antiapoptotic function of Bcl-XL. By out-competing Bax for the interactions leading to membrane permeabilization, Bcl-XL ties up both tBid and Bax in nonproductive interactions and inhibits Bax binding to membranes. We propose that because Bcl-XL does not oligomerize it functions like a dominant-negative Bax in the membrane permeabilization process.

Sequence diversity, metal specificity, and catalytic proficiency of metal-dependent phosphorylating DNA enzymes.

Although DNA has not been found responsible for biological catalysis, many artificial DNA enzymes have been created by "in vitro selection." Here we describe a new selection approach to assess the influence of four common divalent metal ions (Ca(2+), Cu(2+), Mg(2+), and Mn(2+)) on sequence diversity, metal specificity, and catalytic proficiency of self-phosphorylating deoxyribozymes. Numerous autocatalytic DNA sequences were isolated, a majority of which were selected using Cu(2+) or Mn(2+) as the divalent metal cofactor. We found that Cu(2+)- and Mn(2+)-derived deoxyribozymes were strictly metal specific, while those selected by Ca(2+) and Mg(2+) were less specific. Further optimization by in vitro evolution resulted in a Mn(2+)-dependent deoxyribozyme with a k(cat) of 2.8 min(-1). Our findings suggest that DNA has sufficient structural diversity to facilitate efficient catalysis using a broad scope of metal cofactor utilizing mechanisms.

Cell-free analysis of tail-anchor protein targeting to membranes.

We describe cell-free strategies for analysis of the biogenesis of membrane proteins. Special emphasis is placed on the unique challenges presented by tail-anchor proteins for the analysis of: targeting specificity, binding to membranes such as endoplasmic reticulum, mitochondria and pure lipid membranes, protein topology in the membrane and the dependence of various steps on additional proteins. Tail-anchor proteins such as cytochrome b(5), Bcl-2 and Bcl-X(L) are used to address the suitability of both cell-free techniques based on in vitro transcription translation systems and the use of purified recombinant protein and pure lipid membranes. Together these cell-free systems permit detailed characterization of membrane protein biogenesis.

Bcl-2 changes conformation to inhibit Bax oligomerization.

Bcl-2 inhibits apoptosis by regulating the release of cytochrome c and other proteins from mitochondria. Oligomerization of Bax promotes cell death by permeabilizing the outer mitochondrial membrane. In transfected cells and isolated mitochondria, Bcl-2, but not the inactive point mutants Bcl-2-G145A and Bcl-2-V159D, undergoes a conformation change in the mitochondrial membrane in response to apoptotic agonists such as tBid and Bax. A mutant Bcl-2 with two cysteines introduced at positions predicted to result in a disulfide bond that would inhibit the mobility of alpha5-alpha6 helices (Bcl-2-S105C/E152C) was only active in a reducing environment. Thus, Bcl-2 must change the conformation to inhibit tBid-induced oligomerization of integral membrane Bax monomers and small oligomers. The conformationally changed Bcl-2 sequesters the integral membrane form of Bax. If Bax is in excess, apoptosis resumes as Bcl-2 is consumed by the conformational change and in complexes with Bax. Thus, Bcl-2 functions as an inhibitor of mitochondrial permeabilization by changing conformation in the mitochondrial membrane to bind membrane-inserted Bax monomers and prevent productive oligomerization of Bax.

Secondary-structure characterization of two proficient kinase deoxyribozymes.

Dk1 and Dk2 are two catalytically proficient, manganese-dependent, guanine-rich deoxyribozymes previously isolated for DNA phosphorylation. In this study, we carried out a series of experiments that aimed to understand the structural properties of Dk1 and Dk2 and compare the structural similarities or differences of these two distinct deoxyribozymes that carry out similar catalytic functions. First, we performed reselections from two partially randomized DNA libraries on the basis of the original Dk1 and Dk2 sequences to isolate catalytically active sequence variants and identify nucleotides that are invariable, well-conserved, or highly mutagenized. Sequence analysis of these variants assisted by secondary-structure predictions led to the identification of possible Watson-Crick base-pairing regions within each deoxyribozyme. Sequence truncation and base-pair partner exchange experiments were conducted to confirm, or rule out, the existence of the predicted secondary-structure elements. Finally, methylation interference experiments were applied to identify nucleotides that are potentially important for the tertiary structure folding of the deoxyribozymes. Our data suggest that Dk1 and Dk2, despite the differences in their primary sequences and NTP requirements, use an analogous stem-loop element to anchor a structural domain of substantial tertiary interactions to execute their catalytic functions.

Synthesis and characterization of topologically linked single-stranded DNA rings.

We investigated the synthesis of linked-ring DNAs by two DNA-ligation-based methods. In the first method, two DNA oligonucleotides were associated through a duplex segment of more than a full helical turn. Circularization of the entwined oligonucleotides by T4 DNA ligase resulted in two linked-ring DNAs with a total yield of approximately 40%. In the second method, a DNA oligonucleotide was circularized over a circular DNA template, resulting in the formation of approximately 10% linked-ring product. The circular nature of linked-ring DNAs was verified with exonuclease digestion and the existence of topological linkages was demonstrated by analyzing the electrophoretic mobility pattern of DNA products obtained from the digestion of each linked-ring DNA using specific restriction endonucleases. A linked-ring DNA library in which one of the two rings contained random-sequence nucleotides was also constructed and tested for compatibility with in vitro selection.