Bacteriophage T7 DNA ligase. Overexpression, purification, crystallization, and characterization

The bacteriophage T7 DNA ligase gene was amplified using polymerase chain reaction-based methods and cloned into a T7 promoter-based expression vector. The protein was overexpressed to greater than 15% of total soluble protein and purified to homogeneity, yielding 60-70 mg of protein per liter of bacterial culture. An initial physical and biochemical characterization of the enzyme reveals that it exists as a monomer and can ligate nicked, cohesive, and blunt-ended DNA fragments. Inhibition of the enzyme activity by a nonhydrolyzable ATP analogue was also investigated. The enzyme has been crystallized from methoxypolyethylene glycol. The crystals are of the orthorhombic space group P2(1)2(1)2 and diffract to 2.6 A. The unit cell dimensions are a = 66.1 A, b = 87.6 A, and c = 78.6 A, with one monomer in the asymmetric unit (Vm = 2.77 A3/Da). This is the first member of the DNA ligase family of enzymes to be crystallized.     

Rationale for en bloc vein resection in the treatment of pancreatic adenocarcinoma adherent to the superior mesenteric-portal vein confluence. Pancreatic Tumor Study Group

The enzymatic activity of protein kinase C (PKC) was measured in the cytosol and particulate fraction of parabrachial nucleus, the presumed site of conditioned taste aversion (CTA) engrams. At various time intervals after acquisition of the task (pairing saccharin consumption with subsequent LiCl poisoning) the nucleus was dissected from the frozen coronal sections. An increase (+40%) in the cytosol PKC activity was found 48 h after that pairing in comparison with controls (saline injection instead of LiCl). Particulate enzyme activity virtual did not change (-5%). Thus the total PKC activity increased significantly (21%). Qualitatively similar but less markedly expressed PKC shifts (+18% in cytosol) ere found 24 h following CTA. Twelve hours and 5 days after CTA acquisition the activity and distribution of PKC was similar to that seen in normal rats. The control experiments revealed that 6 h after LiCl injection alone (without previous saccharin consumption) translocation of PKC from the cytosol to the membrane fraction (found previously 1 h after LiCl injection alone) still persisted but did not differ from that found 6 h after its pairing with saccharin drinking (CTA). It is concluded that acquisition of conditioned taste aversion may be followed by synthesis of PKC rather than by its translocation or downregulation.     

RAPD analysis reveals low genetic variability in the endangered light-footed clapper rail

After 4 hours of treatment with TNF, newly synthesized endothelial leukocyte adhesion molecule 1, vascular cell adhesion molecule 1, and intercellular adhesion molecule 1 molecules are diffusely expressed on the apical surface of cultured umbilical vein endothelial cells. Such cells maintain the epithelioid, cobblestone appearance of untreated endothelial cells and display cytoskeletal actin largely arranged in dense peripheral bands. After 24 to 72 hours of treatment with TNF, cells become elongated and rearrange their actin filaments into longitudinal stress fibers. At this time, intercellular adhesion molecule 1 and vascular cell adhesion molecule 1 remain elevated but redistribute to the cell junctions. Intercellular adhesion molecule 2, beta 1 integrins, and beta 3 integrins also redistribute to cell junctions in TNF-treated cultures. IFN-gamma produces morphologic changes similar to those induced by TNF but does not cause surface protein redistribution. Cells treated with TNF plus IFN-gamma become even more elongated and display TNF-like redistributions. We conclude that TNF activates a program of membrane protein redistribution, and we speculate that this dynamic redistribution of adhesion molecules to cell junctions may contribute to the recruitment of leukocytes to sites of inflammation.     

An analysis of the effect of venous resistance on the performance of gravity-fed intravenous infusion systems

OBJECTIVE: Our objective was to assess the effect of venous resistance in a clinically relevant range on flow rates through intravenous (i.v.) cannulae. METHODS: Since resistances in series are additive, the sum of the resistance of the i.v. cannula and the vein equates to the total resistance to flow. Using resistance data from earlier work, the total resistance for various combinations of cannula and vein size was calculated, allowing a prediction to be made of the comparative flow rates between these combinations for a given driving pressure. Next, the clinical situation was simulated by measuring flow rates through i.v. cannulae connected in series to a variety of infusion devices with resistances known to be within the range of clinically relevant venous resistance. RESULTS: The effect of venous resistance was greatest on large-bore cannulae, with significant reductions in flow occurring when even low levels of venous resistance were added. Throughout much of the range of venous resistances encountered clinically, total flows through two small-gauge cannulae exceeded that through a single large-gauge cannula. CONCLUSION: To achieve maximum benefit from a large-bore cannula, a suitably large vein must be chosen. Further, where venous access is difficult and high flow potential from an infusion system is required, two separate infusions through small cannulae may be a preferable option to a single large bore.     

Use of a prognostic treadmill score in identifying diagnostic coronary disease subgroups

BACKGROUND: Exercise testing is useful in the assessment of symptomatic patients for diagnosis of significant or extensive coronary disease and to predict their future risk of cardiac events. The Duke treadmill score (DTS) is a composite index that was designed to provide survival estimates based on results from the exercise test, including ST-segment depression, chest pain, and exercise duration. However, its usefulness for providing diagnostic estimates has yet to be determined. METHODS AND RESULTS: A logistic regression model was used to predict significant (>/=75% stenosis) and severe (3-vessel or left main) coronary artery disease, and a Cox regression analysis was used to predict cardiac survival. After adjustment for baseline clinical risk, the DTS was effectively diagnostic for significant (P<0.0001) and severe (P<0.0001) coronary artery disease. For low-risk patients (score >/=+5), 60% had no coronary stenosis >/=75% and 16% had single-vessel >/=75% stenosis. By comparison, 74% of high-risk patients (score <-11) had 3-vessel or left main coronary disease. Five-year mortality was 3%, 10%, and 35% for low-, moderate-, and high-risk DTS groups (P<0.0001). CONCLUSIONS: The composite DTS provides accurate diagnostic and prognostic information for the evaluation of symptomatic patients evaluated for clinically suspected ischemic heart disease.     

Degradation kinetics of DMP 777, an elastase inhibitor

PURPOSE: The objective was to evaluate the degradation profile of the elastase inhibitor DMP 777 and lay the foundation for formulation development. METHODS: The pKa was determined by potentiometric titration in mixed-aqueous solvents. The degradation kinetics were studied as a function of pH, buffer concentration, ionic strength, methanol concentration and temperature using a stability-indicating HPLC assay. The degradation products were identified by LC-MS, NMR, and by comparison with authentic samples. RESULTS: The pKa for the protonated piperazine nitrogen was estimated to be 7.04. The pH-rate profile is described by specific acid-, water-, and specific base-catalyzed pathways. The pH of maximum stability is in the range of 4 to 4.5 where water is the principal catalyst in the reaction. Buffer catalysis, primary salt effects and medium effects were observed. The proposed mechanism for acid catalyzed degradation is the rarely observed AAL1 which involves alkyl-nitrogen heterolysis. The driving force for the reaction appears to lie in the stability of the benzylic carbocation. The proposed mechanism for base catalyzed degradation is BAC2 which involves beta-lactam ring opening. The beta-lactam ring of DMP 777, a monolactam, appears to be as reactive as that in benzylpenicillin in the KOH controlled region where a similar mechanism of hydrolysis should be operative. A contributing factor to this increased reactivity may lie in the reduced basicity of the beta-lactam nitrogen making it a good leaving group. CONCLUSIONS: The degradation profile indicates that development of a solution dosage form of DMP 777 with adequate shelf-life stability at room temperature is feasible.     

Markov chain Monte Carlo methods in biostatistics

Appropriate models in biostatistics are often quite complicated. Such models are typically most easily fit using Bayesian methods, which can often be implemented using simulation techniques. Markov chain Monte Carlo (MCMC) methods are an important set of tools for such simulations. We give an overview and references of this rapidly emerging technology along with a relatively simple example. MCMC techniques can be viewed as extensions of iterative maximization techniques, but with random jumps rather than maximizations at each step. Special care is needed when implementing iterative maximization procedures rather than closed-form methods, and even more care is needed with iterative simulation procedures: it is substantially more difficult to monitor convergence to a distribution than to a point. The most reliable implementations of MCMC build upon results from simpler models fit using combinations of maximization algorithms and noniterative simulations, so that the user has a rough idea of the location and scale of the posterior distribution of the quantities of interest under the more complicated model. These concerns with implementation, however, should not deter the biostatistician from using MCMC methods, but rather help to ensure wise use of these powerful techniques.