Surface potential of phosphatidylserine monolayers. I. Divalent ion binding effect

Surface potentials of phosphatidylserine monolayers have been measured in the presence of different divalent ion concentrations in order to determine the way in which divalent ions bind to the membrane surface. The association constants for divalent ions (Mg²⁺, Ca²⁺ and Mn²⁺) with the phosphatidylserine membrane have been obtained from the experimental data and simple ion binding theory. The order of divalent ion binding to the membrane is Mn²⁺ > Ca²⁺ > Mg²⁺. However, none of the divalent ions used completely neutralized the negative charge of phosphatidylserine even at relatively high concentrations. The amounts of the divalent ions bound depended upon the concentration of the monovalent ions present in the subphase. It is suggested that the amounts of bound ions obtained from the use of radioisotope tracer methods may include a considerable contribution from the excess free ions in the double layer region of the phosphatidylserine membrane.     

Domaine de la biologie théorique

Summary In order to delimit the field of theoretical biology, the author distinguishes in empirical biology a substructure and a superstructure. Empirical biology cannot be constituted without a minimum of reference to philosophical ideas such as the principle of identity (substructure); having regard to its objective, which is explanation by means of physico-chemical models, it does not easily avoid ontological aspirations. Further, experimental research makes great use of scientific theories. Finally, the elaboration of the empirical data of biology may find its fulfilment in an ontological science, philosophical biology, which is a branch of the philosophy of nature. Philosophical biology, superstructure of experimental biology, is a discipline intermediate between the special sciences of life, defined in an empirical sense, and metaphysics.

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Zusammenfassung Um den Bereich der theoretischen Biologie abzugrenzen, unterscheidet der Autor in der positiven Biologie eine “Infrastruktur” und eine “Suprastruktur”. Die positive Biologie kann nicht ohne ein Minimum von Beziehungen zu den philosophischen Begriffen aufgebaut werden, wie z.B. zu dem Prinzip der Identit?t (Infrastruktur); ihrem Ziele nach betrachtet, welches in der Erkl?rung durch physiko-chemische Grundbegriffe besteht, kann sie sich nicht gut den ontologischen Ansprüchen entziehen. Darüber hinaus bedient sich die experimentelle Forschung weitgehend der wissenschaftlichen Theorien. Zum Schluss kann die Bearbeitung der empirischen Erkenntnisse in der Biologie ihre Vollendung in einer ontologischen Wissenschaft, die philosophische Biologie, welche ein Zweig der Philosophie der Natur ist, finden. Die philosophische Biologie, die der experimentellen übergeordnet ist, steht als Disziplin zwischen den besonderen Wissenschaften vom Leben, verstanden im empirischen Sinne, und der Metaphysik.

     

Phenylalanyl-tRNA synthetase of baker's yeast. Modulation of adenosine triphosphate-pyrophosphate exchange by transfer ribonucleic acid

Native and modified phenylalanine transfer ribonucleic acid (tRNAPhe) can modulate phenylalanine-dependent adenosine triphosphate--inorganic [32P]pyrophosphate (ATP--[32P]PPi) exchange activity via inhibition of adenylate synthesis. Inhibition is visualized if concentrations of L-phenylalanine, ATP, and pyrophosphate are subsaturating. In the proposed mechanism, tRNAPhe is a noncompetitive inhibitor at conditions where only one of the two active sites per molecule of enzyme is occupied by L-phenylalanine, ATP, and pyrophosphate. At saturating concentrations of these reactants, both active sites are occupied and, according to the model, inhibition is eliminated. Occupation by these reactants is assumed to follow homotropic negative cooperativity. The type of effects depends on modification of tRNAPhe. Native tRNAPhe, tRNA2'-dAPhe, and tRNAoxi-redPhe are inhibitors, tRNAPhepCpC has no effect, and tRNAoxPhe is an activator. Kinetics of activation by tRNAoxPhe are slow, following the time course of Schiff base formation and subsequent reduction by added cyanoborohydride. Besides showing that a putative enzyme amino group is nonessential for substrate binding and adenylate synthesis, this result may suggest that an enzyme amino group could interact with the 3'-terminal adenyl group of cognate tRNA. In the case of asymmetrical occupation of the enzyme active sites by all of the small reactants ATP, L-phenylalanine, and pyrophosphate, the interaction with the amino group might trigger the observed noncompetitive inhibition of the pyrophosphate exchange by tRNAPhe.     

Using the beta-lactamase protein-fragment complementation assay to probe dynamic protein-protein interactions

We have developed a general experimental strategy that enables the quantitative detection of dynamic protein-protein interactions in intact living cells, based on protein-fragment complementation assays (PCAs). In this method, protein interactions are coupled to refolding of enzymes from cognate fragments where reconstitution of enzyme activity acts as the detector of a protein interaction. We have described a number of assays with different reporter readouts, but of particular value to studies of protein interaction dynamics are assays based on enzyme reporters that catalyze the creation of products, thus taking advantage of the amplification of signal afforded. Here we describe protocols for one such PCA based on the enzyme TEM beta-lactamase as a reporter in mammalian cells. The beta-lactamase PCA consists of fusing complementary fragments of beta-lactamase to two proteins of interest. If the proteins interact, the fragments are brought together and fold into active beta-lactamase. Here we describe a protocol for this PCA that can be completed in a few hours, using two different substrates that are converted to fluorescent or colored products by beta-lactamase.     

Discovery of Drug Synergies in Gastric Cancer Cells Predicted by Logical Modeling

Discovery of efficient anti-cancer drug combinations is a major challenge, since experimental testing of all possible combinations is clearly impossible. Recent efforts to computationally predict drug combination responses retain this experimental search space, as model definitions typically rely on extensive drug perturbation data. We developed a dynamical model representing a cell fate decision network in the AGS gastric cancer cell line, relying on background knowledge extracted from literature and databases. We defined a set of logical equations recapitulating AGS data observed in cells in their baseline proliferative state. Using the modeling software GINsim, model reduction and simulation compression techniques were applied to cope with the vast state space of large logical models and enable simulations of pairwise applications of specific signaling inhibitory chemical substances. Our simulations predicted synergistic growth inhibitory action of five combinations from a total of 21 possible pairs. Four of the predicted synergies were confirmed in AGS cell growth real-time assays, including known effects of combined MEK-AKT or MEK-PI3K inhibitions, along with novel synergistic effects of combined TAK1-AKT or TAK1-PI3K inhibitions. Our strategy reduces the dependence on a priori drug perturbation experimentation for well-characterized signaling networks, by demonstrating that a model predictive of combinatorial drug effects can be inferred from background knowledge on unperturbed and proliferating cancer cells. Our modeling approach can thus contribute to preclinical discovery of efficient anticancer drug combinations, and thereby to development of strategies to tailor treatment to individual cancer patients.     

Alternative interactions define gyrase specificity in the CcdB family

Toxin-antitoxin (TA) modules are small operons associated with stress response of bacteria. F-plasmid CcdB(F) was the first TA toxin for which its target, gyrase, was identified. Plasmidic and chromosomal CcdBs belong to distinct families. Conserved residues crucial for gyrase poisoning activity of plasmidic CcdBs are not conserved among these families. Here we show that the chromosomal CcdB(Vfi) from Vibrio fischeri is an active gyrase poison that interacts with its target via an alternative energetic mechanism. Changes in the GyrA14-binding surface of the Vibrio and F-plasmid CcdB family members illustrate neutral drift where alternative interactions can be used to achieve the same functionality. Differences in affinity between V. fischeri and F-plasmid CcdB for gyrase and their corresponding CcdA antitoxin possibly reflect distinct roles for TA modules located on plasmids and chromosomes.     

Hyrtioreticulins A-E, indole alkaloids inhibiting the ubiquitin-activating enzyme, from the marine sponge Hyrtios reticulatus

Hyrtioreticulins A-E (1-5) were isolated from the marine sponge Hyrtios reticulatus, along with a known alkaloid, hyrtioerectine B (6). Structural elucidation on the basis of spectral data showed that 1, 2, and 5 are new tetrahydro-β-carboline alkaloids, while 3 and 4 are new azepinoindole-type alkaloids. Hyrtioreticulins A and B (1 and 2) inhibited ubiquitin-activating enzyme (E1) with IC(50) values of 0.75 and 11μg/mL, respectively, measured by their inhibitory abilities against the formation of an E1-ubiquitin intermediate. So far, only five E1 inhibitors, panapophenanthrine, himeic acid A, largazole, and hyrtioreticulins A and B (1 and 2), have been isolated from natural sources and, among them, 1 is the most potent E1 inhibitor.