DNA buckling in bacteriophage cavities as a mechanism to aid virus assembly.

While relatively simple biologically, bacteriophages are sophisticated biochemical machines that execute a precise sequence of events during virus assembly, DNA packaging, and ejection. These stages of the viral life cycle require intricate coordination of viral components whose structures are being revealed by single molecule experiments and high resolution (cryo-electron microscopy) reconstructions. For example, during packaging, bacteriophages employ some of the strongest known molecular motors to package DNA against increasing pressure within the viral capsid shell. Located upstream of the motor is an elaborate portal system through which DNA is threaded. A high resolution reconstruction of the portal system for bacteriophage ϕ29 reveals that DNA buckles inside a small cavity under large compressive forces. In this study, we demonstrate that DNA can also buckle in other bacteriophages including T7 and P22. Using a computational rod model for DNA, we demonstrate that a DNA buckle can initiate and grow within the small confines of a cavity under biologically-attainable force levels. The forces of DNA-cavity contact and DNA-DNA electrostatic repulsion ultimately limit cavity filling. Despite conforming to very different cavity geometries, the buckled DNA within T7 and P22 exhibits near equal volumetric energy density (∼1kT/nm(3)) and energetic cost of packaging (∼22kT). We hypothesize that a DNA buckle creates large forces on the cavity interior to signal the conformational changes to end packaging. In addition, a DNA buckle may help retain the genome prior to tail assembly through significantly increased contact area with the portal.

Airway drug pharmacokinetics via analysis of exhaled breath condensate.

Although the airway surface is the anatomic target for many lung disease therapies, measuring drug concentrations and activities on these surfaces poses considerable challenges. We tested whether mass spectrometric analysis of exhaled breath condensate (EBC) could be utilized to non-invasively measure airway drug pharmacokinetics and predicted pharmacological activities. Mass spectrometric methods were developed to detect a novel epithelial sodium channel blocker (GS-9411/P-680), two metabolites, a chemically related internal standard, plus naturally occurring solutes including urea as a dilution marker. These methods were then applied to EBC and serum collected from four (Floridian) sheep before, during and after inhalation of nebulized GS-9411/P-680. Electrolyte content of EBC and serum was also assessed as a potential pharmacodynamic marker of drug activity. Airway surface concentrations of drug, metabolites, and electrolytes were calculated from EBC measures using EBC:serum urea based dilution factors. GS-9411/P-680 and its metabolites were quantifiable in the sheep EBC, with peak airway concentrations between 1.9 and 3.4 µM measured 1 h after inhalation. In serum, only Metabolite #1 was quantifiable, with peak concentrations ∼60-fold lower than those in the airway (45 nM at 1 h). EBC electrolyte concentrations suggested a pharmacological effect; but this effect was not statistical significant. Analysis of EBC collected during an inhalation drug study provided a method for quantification of airway drug and metabolites via mass spectrometry. Application of this methodology could provide an important tool in development and testing of drugs for airways diseases.

Structural ensemble and dynamics of toroidal-like DNA shapes in bacteriophage ϕ29 exit cavity.

In the bacteriophage ϕ29, DNA is packed into a preassembled capsid from which it ejects under high pressure. A recent cryo-EM reconstruction of ϕ29 revealed a compact toroidal DNA structure (30-40 basepairs) lodged within the exit cavity formed by the connector-lower collar protein complex. Using multiscale models, we compute a detailed structural ensemble of intriguing DNA toroids of various lengths, all highly compatible with experimental observations. In particular, coarse-grained (elastic rod) and atomistic (molecular dynamics) models predict the formation of DNA toroids under significant compression, a largely unexplored state of DNA. Model predictions confirm that a biologically attainable compressive force of 25 pN sustains the toroid and yields DNA electron density maps highly consistent with the experimental reconstruction. The subsequent simulation of dynamic toroid ejection reveals large reactions on the connector that may signal genome release.

DNA modeling reveals an extended lac repressor conformation in classic in vitro binding assays.

Protein-mediated DNA looping, such as that induced by the lactose repressor (LacI) of Escherichia coli, is a well-known gene regulation mechanism. Although researchers have given considerable attention to DNA looping by LacI, many unanswered questions about this mechanism, including the role of protein flexibility, remain. Recent single-molecule observations suggest that the two DNA-binding domains of LacI are capable of splaying open about the tetramerization domain into an extended conformation. We hypothesized that if recent experiments were able to reveal the extended conformation, it is possible that such structures occurred in previous studies as well. In this study, we tested our hypothesis by reevaluating two classic in vitro binding assays using a computational rod model of DNA. The experiments and computations evaluate the looping of both linear DNA and supercoiled DNA minicircles over a broad range of DNA interoperator lengths. The computed energetic minima align well with the experimentally observed interoperator length for optimal loop stability. Of equal importance, the model reveals that the most stable loops for linear DNA occur when LacI adopts the extended conformation. In contrast, for DNA minicircles, optimal stability may arise from either the closed or the extended protein conformation depending on the degree of supercoiling and the interoperator length.

Real-World Evidence of Prostatic Urethral Lift Confirms Pivotal Clinical Study Results: 2-Year Outcomes of a Retrospective Multicenter Study.

Introduction: This study expands results from recent prostatic urethral lift (PUL) clinical trials by examining outcomes within a large unconstrained multicenter data set. Methods: Retrospective chart review and analysis of 1413 consecutive patients who received PUL in North America and Australia was performed. International Prostate Symptom Score (IPSS), quality of life (QoL), and maximum urinary flow rate (Qmax) were evaluated at 1, 3, 6, 12, and 24 months post-procedure for all nonurinary retention subjects (Group A) and retention subjects (Group B). Within Group A outcomes were further analyzed using paired t-tests and 95% mean confidence intervals under the following parameters: IPSS baseline ≥13, age, prostate size, site of service, prostate cancer treatment, and diabetic status. Adverse events, surgical interventions, and catheterization rates were summarized in detail. Results: Compared with the randomized controlled prosatic urethral lift (L.I.F.T.) study, subjects in this retrospective study were older and less symptomatic. After PUL, mean IPSS for Group A improved significantly from baseline by at least 8.1 points throughout follow-up. No significant differences were observed between Group A and B follow-up symptom scores. Within Group A, subjects with an IPSS baseline ≥13 behaved similarly to L.I.F.T. subjects. Age, prostate volume, site of service, prior cancer treatment, and diabetic status did not significantly affect PUL outcomes. When completed in a clinic office, PUL resulted in less side effects and catheter placement compared to other sites of service. Previous prostate cancer treatment did not elevate adverse events of high concern such as incontinence and infection. Conclusion: PUL performs well in a real-world setting in terms of symptom relief, morbidity, and patient experience for all studied patient cohorts.

A2B adenosine receptors regulate the mucus clearance component of the lung's innate defense system.

Adenosine (ADO) signaling is altered in both asthma and chronic obstructive pulmonary disease, and the A(2B) adenosine receptor (A(2B)-R) may drive pulmonary inflammation. Accordingly, it has been proposed that specific inhibition of the A(2B)-R could treat inflammatory lung diseases. However, stimulation of the cystic fibrosis transmembrane conductance regulator (CFTR) by ADO may be crucial in permitting the superficial epithelium to maintain airway surface liquid (ASL) volume, which is required to ensure hydrated and clearable mucus. Our goal was to determine which ADO receptor (ADO-R) underlies ASL volume regulation in bronchial epithelia. We used PCR techniques to determine ADO-R expression in bronchial epithelia and used nasal potential difference measurements, Ussing chambers studies, and XZ-confocal microscopy to look at Cl- secretion and ASL volume regulation. The A(2B)-R was the most highly expressed ADO-R in donor specimens of human bronchial epithelia, and inhibition of ADO-R in vivo prevented activation of CFTR. A(2B)-R was the only ADO-R detected in cultured human bronchial epithelial cells and inhibition of this receptor with specific A(2B)-R antagonists resulted in ASL height collapse and a failure to effect ASL height homeostasis. Removal of ADO with ADO deaminase and replacement with 5'N-ethylcarboxamide adenosine resulted in dose-dependent changes in ASL height, and suggested that the cell surface (ADO) may be in excess of 1 microM, which is sufficient to activate A(2B)-R. A(2B)-R are required for ASL volume homeostasis in human airways, and therapies directed at inhibiting A(2B)-R may lead to a cystic fibrosis-like phenotype with depleted ASL volume and mucus stasis.

Pharmacological properties of N-(3,5-diamino-6-chloropyrazine-2-carbonyl)-N'-4-[4-(2,3-dihydroxypropoxy)phenyl]butyl-guanidine methanesulfonate (552-02), a novel epithelial sodium channel blocker with potential clinical efficacy for cystic fibrosis lung disease.

Amiloride improves mucociliary clearance (MC) by blocking airway epithelial sodium channels (ENaC) and expanding airway surface liquid (ASL). However, the low potency and rapid absorption of amiloride by airway epithelia translated into a short duration of efficacy as an aerosolized therapy for cystic fibrosis (CF) patients. To improve ENaC blocker CF pharmacotherapy, a more potent and durable ENaC blocker tailored for aerosol delivery was synthesized. Parion compound N-(3,5-diamino-6-chloropyrazine-2-carbonyl)-N'-4-[4-(2,3-dihydroxypropoxy)phenyl]butyl-guanidine methanesulfonate (552-02) was tested for potency and reversibility of ENaC block, epithelial absorption and biotransformation, selectivity, durability of ASL expansion under isotonic and hypertonic conditions in canine and human CF bronchial epithelial cells, and drug dissociation on ENaC in Xenopus oocytes. Short-circuit current assessed compound potency and reversibility, patch-clamp recordings of ENaC current assessed drug off-rate (k(off)), a gravimetric method and confocal microscopy measured mucosal water retention and ASL height, and drug absorption and biotransformation were assessed using liquid chromatography-mass spectrometry. Amiloride and 552-02 were tested in vivo for MC activity in sheep immediately and 4 to 6 h after aerosol dosing. Compared with amiloride, compound 552-02 was 60 to 100-fold more potent, it was 2 to 5-fold less reversible, it was slower at crossing the epithelium, and it exhibited a 170-fold slower k(off) value. 552-02 exhibited greater ASL expansion over 8 h in vitro, and it was more effective than amiloride at increasing MC immediately and 4 to 6 h after dosing. When combining hypertonic saline and 552-02, a synergistic effect on ASL expansion was measured in canine or CF bronchial epithelia. In summary, the preclinical data support the clinical use of 552-02 +/- hypertonic saline for CF lung disease.

Design, synthesis, and structure-activity relationships of novel 2-substituted pyrazinoylguanidine epithelial sodium channel blockers: drugs for cystic fibrosis and chronic bronchitis.

Amiloride (1), the prototypical epithelial sodium channel (ENaC) blocker, has been administered with limited success as aerosol therapy for improving pulmonary function in patients with the genetic disorder cystic fibrosis. This study was conducted to synthesize and identify more potent, less reversible ENaC blockers, targeted for aerosol therapy and possessing minimal systemic renal activity. A series of novel 2-substituted acylguanidine analogues of amiloride were synthesized and evaluated for potency and reversibility on bronchial ENaC. All compounds tested were more potent and less reversible at blocking sodium-dependent short-circuit current than amiloride. Compounds 30-34 showed the greatest potency on ENaC with IC(50) values below 10 nM. A regioselective difference in potency was found (compounds 30, 39, and 40), whereas no stereospecific (compounds 33, 34) difference in potency on ENaC was displayed. Lead compound 32 was 102-fold more potent and 5-fold less reversible than amiloride and displayed the lowest IC(50) value ever reported for an ENaC blocker.

Altering airway surface liquid volume: inhalation therapy with amiloride and hyperosmotic agents.

The thin layer of liquid lining the entire respiratory tract is the first line of defense against the continuous insult of inhaled bacteria and noxious chemicals. Many chronic obstructive diseases of the airway may reflect decreased airway surface liquid, which results from imbalances in ion transport and mucus production. Reduction in the thickness of airway surface liquid leads to reduced mucociliary clearance rates, causing mucus accumulation and infection in the airway. In this chapter, two inhalation therapies to replenish airway surface liquid and enhance mucociliary clearance are discussed: (1) aerosolized hyperosmotic agents; and (2) aerosolized sodium channel blockers. The advantages and disadvantages of each therapy are discussed, as well as future directions for improving airway surface liquid volume by inhalation pharmacotherapy.

Role of microscopic flexibility in tightly curved DNA.

The genetic material in living cells is organized into complex structures in which DNA is subjected to substantial contortions. Here we investigate the difference in structure, dynamics, and flexibility between two topological states of a short (107 base pair) DNA sequence in a linear form and a covalently closed, tightly curved circular DNA form. By employing a combination of all-atom molecular dynamics (MD) simulations and elastic rod modeling of DNA, which allows capturing microscopic details while monitoring the global dynamics, we demonstrate that in the highly curved regime the microscopic flexibility of the DNA drastically increases due to the local mobility of the duplex. By analyzing vibrational entropy and Lipari-Szabo NMR order parameters from the simulation data, we propose a novel model for the thermodynamic stability of high-curvature DNA states based on vibrational untightening of the duplex. This novel view of DNA bending provides a fundamental explanation that bridges the gap between classical models of DNA and experimental studies on DNA cyclization, which so far have been in substantial disagreement.

Effect of topically applied epithelial sodium channel inhibitors on tear production in normal mice and in mice with induced aqueous tear deficiency.

PURPOSE: Dry eye syndromes affect a significant proportion of the population worldwide with reported prevalence ranging from 6% to more than 34%. Patients with dry eye can experience intense pain due to eye irritation, gritty/scratchy feeling in the eyes, blurry vision, and light sensitivity. Available treatments for dry eye syndromes remain mainly palliative. The purpose of the present study was to test the hypothesis that inhibiting sodium absorption via the epithelial sodium channel (ENaC) will increase ocular hydration in both normal as well as in animals with experimentally induced dry eye. METHODS: ENaC inhibitors were dissolved in an aqueous buffer that mimics the composition of tears and were applied topically to the ocular surface of isoflurane-anesthetized mice. The effect of ENaC inhibitors was compared with that of the secretagogue uridine triphosphate (UTP; 1%), a purinergic receptor agonist which was shown to increase tear volume in animals. Tear production was measured for 10 s using phenol red-impregnated cotton threads. Fluorescein staining that assesses ocular surface damage was performed at baseline and then at days 1, 2, and 3 after the induction of dry eye in mice. RESULTS: Our data show that the inhibition of ENaC led to a time- and concentration-dependent increase in tear volume in normal mice. The effect of ENaC inhibition after a single application outperformed UTP, as it was long-lasting with tear volume still above baseline values 8 h postdosing. ENaC inhibition, which led to increased tear production, improved fluorescein scores in our dry eye model, when compared with nontreated or animals treated with buffer or UTP. CONCLUSION: We conclude that the inhibition of ENaC provides long-lasting increases in ocular surface hydration and that ENaC blockers could provide an effective new therapy for chronic dry eye.

Induction of stable drug resistance in human breast cancer cells using a combinatorial zinc finger transcription factor library.

Combinatorial libraries of artificial zinc-finger transcription factors (ZF-TFs) provide a robust tool for inducing and understanding various functional components of the cancer phenotype. Herein, we utilized combinatorial ZF-TF library technology to better understand how breast cancer cells acquire resistance to fulvestrant, a clinically important anti-endocrine therapeutic agent. From a diverse collection of nearly 400,000 different ZF-TFs, we isolated six ZF-TF library members capable of inducing stable, long-term anti-endocrine drug-resistance in two independent estrogen receptor-positive breast cancer cell lines. Comparative gene expression profile analysis of the six different ZF-TF-transduced breast cancer cell lines revealed five distinct clusters of differentially expressed genes. One cluster was shared among all 6 ZF-TF-transduced cell lines and therefore constituted a common fulvestrant-resistant gene expression signature. Pathway enrichment-analysis of this common fulvestrant resistant signature also revealed significant overlap with gene sets associated with an estrogen receptor-negative-like state and with gene sets associated with drug resistance to different classes of breast cancer anti-endocrine therapeutic agents. Enrichment-analysis of the four remaining unique gene clusters revealed overlap with myb-regulated genes. Finally, we also demonstrated that the common fulvestrant-resistant signature is associated with poor prognosis by interrogating five independent, publicly available human breast cancer gene expression datasets. Our results demonstrate that artificial ZF-TF libraries can be used successfully to induce stable drug-resistance in human cancer cell lines and to identify a gene expression signature that is associated with a clinically relevant drug-resistance phenotype.

Adenosine deaminase 1 and concentrative nucleoside transporters 2 and 3 regulate adenosine on the apical surface of human airway epithelia: implications for inflammatory lung diseases.

Adenosine is a multifaceted signaling molecule mediating key aspects of innate and immune lung defenses. However, abnormally high airway adenosine levels exacerbate inflammatory lung diseases. This study identifies the mechanisms regulating adenosine elimination from the apical surface of human airway epithelia. Experiments conducted on polarized primary cultures of nasal and bronchial epithelial cells showed that extracellular adenosine is eliminated by surface metabolism and cellular uptake. The conversion of adenosine to inosine was completely inhibited by the adenosine deaminase 1 (ADA1) inhibitor erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA). The reaction exhibited Km and Vmax values of 24 microM and 0.14 nmol x min(-1) x cm(-2). ADA1 (not ADA2) mRNA was detected in human airway epithelia. The adenosine/mannitol permeability coefficient ratio (18/1) indicated a minor contribution of paracellular absorption. Adenosine uptake was Na+-dependent and was inhibited by the concentrative nucleoside transporter (CNT) blocker phloridzin but not by the equilibrative nucleoside transporter (ENT) blocker dipyridamole. Apparent Km and Vmax values were 17 microM and 7.2 nmol x min(-1) x cm(-2), and transport selectivity was adenosine = inosine = uridine > guanosine = cytidine > thymidine. CNT3 mRNA was detected throughout the airways, while CNT2 was restricted to nasal epithelia. Inhibition of adenosine elimination by EHNA or phloridzin raised apical adenosine levels by >3-fold and stimulated IL-13 and MCP-1 secretion by 6-fold. These responses were reproduced by the adenosine receptor agonist 5'-(N-ethylcarboxamido)adenosine (NECA) and blocked by the adenosine receptor antagonist, 8-(p-sulfophenyl) theophylline (8-SPT). This study shows that adenosine elimination on human airway epithelia is mediated by ADA1, CNT2, and CNT3, which constitute important regulators of adenosine-mediated inflammation.

The role of endogenous kallikrein inhibition in perioperative transfusion and adverse outcome in cardiac surgical patients.

OBJECTIVE: The goal of this study was to explore the relationship among endogenous plasma kallikrein inhibition (KI), perioperative bleeding, and adverse outcomes in cardiac surgery. DESIGN: A prospective, observational study. SETTING: University teaching hospitals. PARTICIPANTS: Cardiac surgical patients. INTERVENTIONS: Endogenous plasma KI levels (%) and kallikrein-like activity (KKA) were measured preoperatively, 30 minutes into cardiopulmonary bypass, and at the end of surgery. Patients were divided into quartiles of preoperative KI. Data including risk factors, blood loss, transfusion requirements, and postoperative outcomes were collected. MEASUREMENTS AND MAIN RESULTS: Preoperative endogenous KI ranged from 40% to 175%, where 100% represents the activity of pooled healthy volunteer plasma. The quartiles of KI levels were as follows: quartile 1, KI = 40% to 83.8% (n = 40); quartile 2, KI = 84% to 101.5% (n = 40); quartile 3, KI = 102% to 120% (n = 42); and quartile 4, KI = 121% to 175% (n = 38). The hematocrits on admission to the intensive care unit were as follows: quartile 1, 28% +/- 4%; quartile 2, 26% +/- 4%; quartile 3, 26% +/- 4%; and quartile 4, 24% +/- 4% (p = 0.009). Blood product use was similar among quartiles in the operating room. Quartiles 3 and 4 received more blood (p = 0.003) and platelet (p = 0.04) transfusions than quartiles 1 and 2 in the first 24 hours after surgery. More patients in quartile 4 were ventilated for more than 24 hours after surgery (p < 0.05). Hospital length of stay was longest in quartile 4 (p = 0.002). CONCLUSION: Contrary to expectation, higher endogenous KI levels were associated with more blood product transfusion, longer postoperative mechanical ventilation, and hospital length of stay. These findings raise questions as to the role of KI in postoperative outcomes.

Standardized reagents and protocols for engineering zinc finger nucleases by modular assembly.

Engineered zinc finger nucleases can stimulate gene targeting at specific genomic loci in insect, plant and human cells. Although several platforms for constructing artificial zinc finger arrays using "modular assembly" have been described, standardized reagents and protocols that permit rapid, cross-platform "mixing-and-matching" of the various zinc finger modules are not available. Here we describe a comprehensive, publicly available archive of plasmids encoding more than 140 well-characterized zinc finger modules together with complementary web-based software (termed ZiFiT) for identifying potential zinc finger target sites in a gene of interest. Our reagents have been standardized on a single platform, enabling facile mixing-and-matching of modules and transfer of assembled arrays to expression vectors without the need for specialized knowledge of zinc finger sequences or complicated oligonucleotide design. We also describe a bacterial cell-based reporter assay for rapidly screening the DNA-binding activities of assembled multi-finger arrays. This protocol can be completed in approximately 24-26 d.

Thromboelastography maximum amplitude predicts postoperative thrombotic complications including myocardial infarction.

Postoperative thrombotic complications increase hospital length of stay and health care costs. Given the potential for thrombotic complications to result from hypercoagulable states, we sought to determine whether postoperative blood analysis using thromboelastography could predict the occurrence of thrombotic complications, including myocardial infarction (MI). We prospectively enrolled 240 patients undergoing a wide variety of surgical procedures. A cardiac risk score was assigned to each patient using the established revised Goldman risk index. Thromboelastography was performed immediately after surgery and maximum amplitude (MA), representing clot strength, was determined. Postoperative thrombotic complications requiring confirmation by a diagnostic test were assessed by a blinded observer. Ten patients (4.2%) suffered a total of 12 postoperative thrombotic complications. The incidence of thrombotic complications with increased MA (8 of 95 = 8.4%) was significantly (P = 0.0157) more frequent than that of patients with MA < or =68 (2 of 145 = 1.4%). Furthermore, the percentage suffering postoperative MI in the increased MA group (6 of 95 = 6.3%) was significantly larger than that in the MA < or =68 group (0 of 145 = 0%) (P = 0.0035). In a multivariate analysis, increased MA (P = 0.013; odds ratio, 1.16; 95% confidence interval, 1.03-1.20) and Goldman risk score (P = 0.046; odds ratio, 2.39; 95% confidence interval, 1.02-5.61) both independently predicted postoperative MI. A postoperative hypercoagulable state as determined by thromboelastography is associated with postoperative thrombotic complications, including MI, in a diverse group of surgical patients.

Ecto 5'-nucleotidase and nonspecific alkaline phosphatase. Two AMP-hydrolyzing ectoenzymes with distinct roles in human airways.

In human airways, extracellular adenosine regulates epithelial functions supporting mucociliary clearance, an important airway defense mechanism against bacterial infection. Thus, defining the mechanisms of adenosine generation is critical for elucidating the role of this nucleoside in airway homeostasis. In this study, we identified the source of adenosine on the mucosal surface of human airway epithelia. Polarized primary cultures of human nasal or bronchial epithelial cells were assayed for transepithelial transport, cytosolic and cell surface adenosine production. Ussing chamber experiments indicated that serosal 1 microM [(3)H]adenosine was not transported to the mucosal compartment. Messenger RNA for the cytosolic AMP-specific 5'-nucleotidase (CN-I) was not detected in human bronchial epithelial cells, suggesting that mucosal adenosine did not originate from intracellular pools. In contrast, extracellular 0.1 mm ATP was rapidly dephosphorylated into adenosine on the mucosal epithelial surface. We identified two ectonucleotidases that mediated the conversion of AMP to adenosine: ecto 5'-nucleotidase (ecto 5'-NT, CD73) and alkaline phosphatase (AP). Both mucosal and serosal epithelial surfaces displayed ecto 5'-NT activity (K(m) = 14 microM, V(max) = 0.5 nmol x min(-1) x cm(-2)), whereas AP activity was restricted to the mucosal surface (K(m,)(high) = 36 microM, V(max) = 1.2 nmol x min(-1) x cm(-2); K(m,)(low) = 717 microM, V(max) = 2.8 nmol x min(-1) x cm(-2)). In bronchial cultures and tissues, ecto 5'-NT accounted for >80% of total activity toward 0.01 mm AMP, compared with <15% for 5 mm AMP. The proximal airway AP isoform was identified as nonspecific AP (NS AP) by levamisole sensitivity and mRNA expression. The two ectoenzymes presented opposite airway distributions, ecto 5'-NT and NS AP mRNA dominating in higher and lower airways, respectively. Collectively, these experiments support a major role for extracellular nucleotide catalysis and for ecto 5'-NT and NS AP in the regulation of adenosine concentrations on airway surfaces.

Differences in arterial and venous thromboelastography parameters: potential roles of shear stress and oxygen content.

OBJECTIVE: To investigate potential mechanisms for the differences in thromboelastography variables observed between arterial blood samples and venous blood samples. DESIGN: Prospective cohort study. SETTING: University hospital. PARTICIPANTS: Patients undergoing cardiac surgery (n = 33). INTERVENTIONS: After the withdrawal of 10 mL of discarded blood (>3 deadspace volumes), 3 blood samples were withdrawn simultaneously from the central venous port of the pulmonary artery catheter (CVP), the radial arterial catheter (ART), and the side port of the 9F sheath introducer (SI). MEASUREMENTS AND MAIN RESULTS: Thromboelastography was done simultaneously on each sample. All thromboelastography analyses were performed with 1% celite and heparinase according to the manufacturer's guidelines. A total of 80 ART, SI, and CVP comparisons were obtained. Mean hematocrit values were not different between sampling sites (27 +/- 4 v 27 +/- 4 v 27 +/- 3). Thromboelastography R time values (mean +/- SD) were CVP, 8 +/- 3; ART, 10 +/- 3; and SI, 13 +/- 5 (p = 0.004). Thromboelastography maximal amplitude (MA) values (mean +/- SD) were CVP, 60.4 +/- 11.7; ART, 56.2 +/- 11.4; and SI, 50.5 +/- 13.2 (p = 0.008). Calculated maximal shear stresses were CVP, 48 dyne/cm(2); ART, 36 dyne/cm(2); and SI, 0.3 dyne/cm(2). Blood samples obtained from the CVP (highest shear stress) resulted in faster (shorter R) and stronger (larger MA) coagulation compared with the arterial site (intermediate shear stress) and sheath introducer (lowest shear stress). CONCLUSION: These data show that differences exist in thromboelastography values between arterial and venous blood samples and, more importantly, show that the differences observed are not related to differences in oxygen content. These differences seem to be related to differences in catheter lumen diameter and, presumably, shear forces.

Highly specific zinc finger proteins obtained by directed domain shuffling and cell-based selection.

Engineered Cys2His2 zinc finger proteins (ZFPs) can mediate regulation of endogenous gene expression in mammalian cells. Ideally, all zinc fingers in an engineered multifinger protein should be optimized concurrently because cooperative and context-dependent contacts can affect DNA recognition. However, the simultaneous selection of key contacts in even three fingers from fully randomized libraries would require the consideration of >10(24) possible combinations. To address this challenge, we have developed a novel strategy that utilizes directed domain shuffling and rapid cell-based selections. Unlike previously described methods, our strategy is amenable to scale-up and does not sacrifice combinatorial diversity. Using this approach, we have successfully isolated multifinger proteins with improved in vitro and in vivo function. Our results demonstrate that both DNA binding affinity and specificity are important for cellular function and also provide a general approach for optimizing multidomain proteins.

Regulation of the epithelial sodium channel by serine proteases in human airways.

The epithelial sodium channel (ENaC) constitutes the rate-limiting step for sodium absorption across airway epithelia, which in turn regulates airway surface liquid (ASL) volume and the efficiency of mucociliary clearance. This role in ASL volume regulation suggests that ENaC activity is influenced by local factors rather than systemic signals indicative of total body volume homeostasis. Based on reports that ENaC may be regulated by extracellular serine protease activity in Xenopus and mouse renal epithelia, we sought to identify proteases that serve similar functions in human airway epithelia. Homology screening of a human airway epithelial cDNA library identified two trypsin-like serine proteases (prostasin and TMPRSS2) that, as revealed by in situ hybridization, are expressed in airway epithelia. Functional studies in the Xenopus oocyte expression system demonstrated that prostasin increased ENaC currents 60--80%, whereas TMPRSS2 markedly decreased ENaC currents and protein levels. Studies of primary nasal epithelial cultures in Ussing chambers revealed that inhibition of endogenous serine protease activity with aprotinin markedly decreased ENaC-mediated currents and sensitized the epithelia to subsequent channel activation by exogenous trypsin. These data, therefore, suggest that protease-mediated regulation of sodium absorption is a function of human airway epithelia, and prostasin is a likely candidate for this activity.