Isolation of impermeable inside-out vesicles from an enriched sarcolemma fraction of rat heart

Sarcolemmal vesicles isolated from relaxed rat cardiac ventricles were 120-fold enriched in (Na+ + K+)-ATPase and 5'-nucleotidase activities (final recoveries, 50%). The alpha and beta chains of the former enzyme were visualized by the immunological approach. Inside-out sarcolemmal vesicles were isolated by affinity chromatography on immobilized concanavalin A. The yield of membranes was 0.45 mg of protein/g of muscle. The orientation of the unbound vesicles was studied by the increased accessibility of sarcolemma outer face markers (ouabain- and K+-binding sites, 5'-nucleotidase, and sialic acids) with permeability-increasing treatments: freeze-thaw cycles, sodium dodecyl sulfate, methanol, and valinomycin. The total ATP hydrolysis remained constant with a conversion of ouabain-insensitive activity into an ouabain-sensitive one. These agents caused a parallel increase in the ouabain sensitivity, the number of [3H]ouabain-binding sites, the monovalent cation stimulation of ATPase, and the 5'-nucleotidase activity. Valinomycin revealed that most vesicles were sealed to sequestered and exogenous K+. Inside-out vesicles were 80% pure in sidedness and sealing. The affinity chromatography did not affect the (Na+ + K+)-ATPase activity (200 mumol of product/mg of protein/h). This model of sarcolemma vesicles offers a new tool for ion transport studies.     

Effect of phenylhydrazine on red blood cell metabolism

In addition to the well known effect of phenylhydrazine on red blood cells (methaemoglobin and Heinz body formation, autologous IgG binding, lipid peroxidation, etc.) an increased glucose utilization was observed. Measurement of 14CO2 formation from [1-14C]-glucose showed a maximum value at 2mM phenylhydrazine followed by a progressive inhibition on increasing the drug concentration to 16 mM. Concomitantly we found a reduction in the reduced glutathione concentration but not a corresponding increase in the level of oxidized glutathione. Phenylhydrazine also causes ATP depletion. The ATP is in part dephosphorylated to ADP and AMP and in part converted to inosine monophosphate and hypoxanthine. Measurement of the cell content of reduced and oxidized pyridine nucleotides was also performed and showed a progressive increase in the reduced forms of these coenzymes. Thus phenylhydrazine promotes cellular ATP depletion followed by adenine nucleotide catabolism that is not efficiently counteracted by an increase in glucose utilization. The relevance of these data to the mechanism of phenylhydrazine-induced anemia is discussed.     

Aphidicolin does not inhibit the repair synthesis of mitotic chromosomes

In order to determine the possible role of α-polymerase in repair synthesis, we measured the unscheduled DNA synthesis stimulated by UV-irradiation in mitotic HeLa cells in the presence of Aphidicolin, a specific inhibitor of this enzyme. The results do not show any inhibition of the unscheduled DNA synthesis,indicating that the function of α-polymerase is not essential for repair synthesis of mitotic cells.     

Lower inflection point and recruitment with PEEP in ventilated patients with acute respiratory failure.

The lower inflection point (LIP) on the total respiratory system pressure-volume (P-V) curve is widely used to set positive end-expiratory pressure (PEEP) in patients with acute respiratory failure (ARF) on the assumption that LIP represents alveolar recruitment. The aims of this work were to study the relationship between LIP and recruited volume (RV) and to propose a simple method to quantify the RV. In 23 patients with ARF, respiratory system P-V curves were obtained by means of both constant-flow and rapid occlusion technique at four different levels of PEEP and were superimposed on the same P-V plot. The RV was measured as the volume difference at a pressure of 20 cm H(2)O. A third measurement of the RV was done by comparing the exhaled volumes after the same distending pressure of 20 cm H(2)O was applied (equal pressure method). RV increased with PEEP (P < 0.0001); the equal pressure method compares favorably with the other methods (P = 0.0001 by correlation), although individual data cannot be superimposed. No significant difference was found when RV was compared with PEEP in the group of patients with a LIP < or =5 cm H(2)O and the group with a LIP >5 cm H(2)O (76.9 +/- 94.3 vs. 61.2 +/- 51.3, 267.7 +/- 109.9 vs. 209.6 +/- 73.9, and 428.2 +/- 216.3 vs. 375.8 +/- 145.3 ml with PEEP of 5, 10, and 15 cm H(2)O, respectively). A RV was found even when a LIP was not present. We conclude that the recruitment phenomenon is not closely related to the presence of a LIP and that a simple method can be used to measure RV.     

Graphical modeling for gene set analysis: A critical appraisal

Current demand for understanding the behavior of groups of related genes, combined with the greater availability of data, has led to an increased focus on statistical methods in gene set analysis. In this paper, we aim to perform a critical appraisal of the methodology based on graphical models developed in Massa et al. ( 2010 ) that uses pathway signaling networks as a starting point to develop statistically sound procedures for gene set analysis. We pay attention to the potential of the methodology with respect to the organizational aspects of dealing with such complex but highly informative starting structures, that is pathways. We focus on three themes: the translation of a biological pathway into a graph suitable for modeling, the role of shrinkage when more genes than samples are obtained, the evaluation of respondence of the statistical models to the biological expectations. To study the impact of shrinkage, two simulation studies will be run. To evaluate the biological expectation we will use data from a network with known behavior that offer the possibility of carrying out a realistic check of respondence of the model to changes in the experimental conditions.