Significant contribution of the 3'→5' exonuclease activity to the high fidelity of nucleotide incorporation catalyzed by human DNA polymerase ϵ.

Most eukaryotic DNA replication is performed by A- and B-family DNA polymerases which possess a faithful polymerase activity that preferentially incorporates correct over incorrect nucleotides. Additionally, many replicative polymerases have an efficient 3'→5' exonuclease activity that excises misincorporated nucleotides. Together, these activities contribute to overall low polymerase error frequency (one error per 10(6)-10(8) incorporations) and support faithful eukaryotic genome replication. Eukaryotic DNA polymerase ϵ (Polϵ) is one of three main replicative DNA polymerases for nuclear genomic replication and is responsible for leading strand synthesis. Here, we employed pre-steady-state kinetic methods and determined the overall fidelity of human Polϵ (hPolϵ) by measuring the individual contributions of its polymerase and 3'→5' exonuclease activities. The polymerase activity of hPolϵ has a high base substitution fidelity (10(-4)-10(-7)) resulting from large decreases in both nucleotide incorporation rate constants and ground-state binding affinities for incorrect relative to correct nucleotides. The 3'→5' exonuclease activity of hPolϵ further enhances polymerization fidelity by an unprecedented 3.5 × 10(2) to 1.2 × 10(4)-fold. The resulting overall fidelity of hPolϵ (10(-6)-10(-11)) justifies hPolϵ to be a primary enzyme to replicate human nuclear genome (0.1-1.0 error per round). Consistently, somatic mutations in hPolϵ, which decrease its exonuclease activity, are connected with mutator phenotypes and cancer formation.

Effect of hyperinsulinaemia-hyperaminoacidaemia on leg muscle protein synthesis and breakdown: reassessment of the two-pool arterio-venous balance model.

Accurate measurement of muscle protein turnover is critical for understanding the physiological processes underlying muscle atrophy and hypertrophy. Several mathematical approaches, used in conjunction with a tracer amino acid infusion, have been described to derive protein synthesis and breakdown rates from a two-pool (artery-vein) model. Despite apparently common underlying principles, these approaches differ significantly (some seem to not take into account arterio-venous shunting of amino acids, which comprises ∼80-90% of amino acids appearing in the vein) and most do not specify how tracer enrichment (i.e. mole percent excess (MPE) or tracer-to-tracee ratio (TTR)) and amino acid concentration (i.e. unlabelled only or total labelled plus unlabelled) should be expressed, which could have a significant impact on the outcome when using stable isotope labelled tracers. We developed equations that avoid these uncertainties and used them to calculate leg phenylalanine (Phe) kinetics in subjects who received a [(2) H5 ]Phe tracer infusion during postabsorptive conditions and during a hyperinsulinaemic-euglycaemic clamp with concomitant protein ingestion. These results were compared with those obtained by analysing the same data with previously reported equations. Only some of them computed the results correctly when used with MPE as the enrichment measure and total (tracer+tracee) Phe concentrations; errors up to several-fold in magnitude were noted when the same approaches were used in conjunction with TTR and/or unlabelled concentration only, or when using the other approaches (irrespective of how concentration and enrichment are expressed). Our newly developed equations should facilitate accurate calculation of protein synthesis and breakdown rates.

Kinetic mechanism of DNA polymerization catalyzed by human DNA polymerase ε.

Eukaryotes require highly accurate and processive DNA polymerases to ensure faithful and efficient replication of their genomes. DNA polymerase ε (Polε) has been shown to catalyze leading-strand DNA synthesis during replication in vivo, but little is known about the kinetic mechanism of polymerization catalyzed by this replicative enzyme. To elucidate this mechanism, we have generated a truncated, exonuclease-deficient mutant of the catalytic subunit of human Polε (Polε exo-) and carried out pre-steady-state kinetic analysis of this enzyme. Our results show that Polε exo-, as other DNA polymerases, follows an induced-fit mechanism when catalyzing correct nucleotide incorporation. Polε exo- binds DNA with a Kd(DNA) of 79 nM and dissociates from the E·DNA binary complex with a rate constant of 0.021 s(-1). Although Polε exo- binds a correct incoming nucleotide weakly with a Kd(dTTP) of 31 µM, it catalyzes correct nucleotide incorporation at a fast rate constant of 248 s(-1) at 20 °C. Both a large reaction amplitude difference (42%) between pulse-chase and pulse-quench assays and a small elemental effect (0.9) for correct dTTP incorporation suggest that a slow conformational change preceding the chemistry step limits the rate of correct nucleotide incorporation. In addition, our kinetic analysis shows that Polε exo- exhibits low processivity during polymerization. To catalyze leading-strand synthesis in vivo, Polε likely interacts with its three smaller subunits and additional replication factors in order to assemble a replication complex and significantly enhance its polymerization processivity.

Prospective determination of candidates for thrombolysis in patients with acute proximal deep vein thrombosis.

PURPOSE: To prospectively determine the distribution, extent, and age of venous thrombosis in patients presenting with acute signs and symptoms of venous thromboembolism and identify candidates for thrombolysis. MATERIALS AND METHODS: Five hundred seventy-six consecutive patients (281 male, 295 female; mean age 58) referred for lower extremity deep vein thrombosis (DVT) assessment between November 2007 and April 2008 were included in the study. Documented cases of DVT were categorized by age (acute, chronic, and acute on chronic), anatomic location, and extent. Patients with iliofemoral and femoropopliteal DVT were evaluated for thrombolysis using standard criteria. RESULTS: DVT was found in 19% of patients (112/576). Of these, 31 patients (27.7%, 31/112) had isolated calf DVT, 61 patients (54.5%, 61/112) had proximal vein thrombosis extending into the femoropopliteal venous segments, and 20 patients (17.9%, 20/112) presented with iliofemoral DVT. Using standard criteria, 12 patients were selected as potential candidates for pharmacomechanical thrombolysis (PhMT). This equated to an incidence of 2% (12/576) in the population studied, 11% of patients (12/112) with DVT, 26.1% of patients (12/46) presenting with acute proximal DVT, and 20% of patients (4/20) with iliofemoral DVT. CONCLUSION: The incidence of potential candidates for thrombolysis is low. These data should be considered when recruiting centers to participate in ongoing clinical trials assessing the efficacy of these techniques.

Protein Ingestion Induces Muscle Insulin Resistance Independent of Leucine-Mediated mTOR Activation.

Increased plasma branched-chain amino acid concentrations are associated with insulin resistance, and intravenous amino acid infusion blunts insulin-mediated glucose disposal. We tested the hypothesis that protein ingestion impairs insulin-mediated glucose disposal by leucine-mediated mTOR signaling, which can inhibit AKT. We measured glucose disposal and muscle p-mTOR(Ser2448), p-AKT(Ser473), and p-AKT(Thr308) in 22 women during a hyperinsulinemic-euglycemic clamp procedure with and without concomitant ingestion of whey protein (0.6 g/kg fat-free mass; n = 11) or leucine that matched the amount given with whey protein (n = 11). Both whey protein and leucine ingestion raised plasma leucine concentration by approximately twofold and muscle p-mTOR(Ser2448) by ∼30% above the values observed in the control (no amino acid ingestion) studies; p-AKT(Ser473) and p-AKT(Thr308) were not affected by whey protein or leucine ingestion. Whey protein ingestion decreased insulin-mediated glucose disposal (median 38.8 [quartiles 30.8, 61.8] vs. 51.9 [41.0, 77.3] µmol glucose/µU insulin · mL(-1) · min(-1); P < 0.01), whereas ingestion of leucine did not (52.3 [43.3, 65.4] vs. 52.3 [43.9, 73.2]). These results indicate that 1) protein ingestion causes insulin resistance and could be an important regulator of postprandial glucose homeostasis and 2) the insulin-desensitizing effect of protein ingestion is not due to inhibition of AKT by leucine-mediated mTOR signaling.

Pancreaticoportal fistula and disseminated fat necrosis after revision of a transjugular intrahepatic portosystemic shunt.

A 59-year old man with alcohol related cirrhosis and portal hypertension was referred for transjugular intrahepatic portosystemic shunt (TIPS) to treat his refractory ascites. Ten years later, two sequential TIPS revisions were performed for shunt stenosis and recurrent ascites. After these revisions, he returned with increased serum pancreatic enzyme levels and disseminated superficial fat necrosis; an iatrogenic pancreaticoportal vein fistula caused by disruption of the pancreatic duct was suspected. The bare area of the TIPS was subsequently lined with a covered stent-graft, and serum enzyme levels returned to baseline. In the interval follow-up period, the patient has clinically improved.

Acute deep vein thrombosis cases in the real world.

Practicing interventional radiologists (IRs) are routinely faced with challenging decisions that pertain to the management of patients with acute deep vein thrombosis (DVT). In this article, we describe five questions that are commonly posed by interventionalists and discuss both the indirect published evidence as well as our own experience in dealing with these issues. Our aim is to address procedural and, perhaps more importantly, nonprocedural-related knowledge domains with which the IR physician is less familiar and are often not directly addressed by published data or evidence-based clinical practice guidelines. This discussion is meant to facilitate a stronger understanding of the published literature as it pertains to the justifiable indications for endovascular thrombolytic therapy, the optimal use of anticoagulant therapy, and the reasonable use of adjuncts such as inferior vena cava filters and elastic compression stockings. Our goal is to provide a framework for practicing IRs to help them make the best clinical decisions for their individual patients and, ultimately, achieve optimal DVT treatment outcomes.