On the dynamics of the adenylate energy system: homeorhesis vs homeostasis.

Biochemical energy is the fundamental element that maintains both the adequate turnover of the biomolecular structures and the functional metabolic viability of unicellular organisms. The levels of ATP, ADP and AMP reflect roughly the energetic status of the cell, and a precise ratio relating them was proposed by Atkinson as the adenylate energy charge (AEC). Under growth-phase conditions, cells maintain the AEC within narrow physiological values, despite extremely large fluctuations in the adenine nucleotides concentration. Intensive experimental studies have shown that these AEC values are preserved in a wide variety of organisms, both eukaryotes and prokaryotes. Here, to understand some of the functional elements involved in the cellular energy status, we present a computational model conformed by some key essential parts of the adenylate energy system. Specifically, we have considered (I) the main synthesis process of ATP from ADP, (II) the main catalyzed phosphotransfer reaction for interconversion of ATP, ADP and AMP, (III) the enzymatic hydrolysis of ATP yielding ADP, and (IV) the enzymatic hydrolysis of ATP providing AMP. This leads to a dynamic metabolic model (with the form of a delayed differential system) in which the enzymatic rate equations and all the physiological kinetic parameters have been explicitly considered and experimentally tested in vitro. Our central hypothesis is that cells are characterized by changing energy dynamics (homeorhesis). The results show that the AEC presents stable transitions between steady states and periodic oscillations and, in agreement with experimental data these oscillations range within the narrow AEC window. Furthermore, the model shows sustained oscillations in the Gibbs free energy and in the total nucleotide pool. The present study provides a step forward towards the understanding of the fundamental principles and quantitative laws governing the adenylate energy system, which is a fundamental element for unveiling the dynamics of cellular life

Photosystem II and cellular membrane stability evaluation in hexaploid wheat seedlings under salt stress conditions

Salinity limits crop production in large areas of the world. The application of in vitro Photosystem II (PS-II) activity measurements to screen hexaploid wheat (Triticum aestivum, L.) genotypes for NaCl tolerance has been investigated by comparing their responses under stress and control (no added NaCl) conditions. One of the four cultivars used in the study was 'Kharchia' known for its high salt tolerance. Wheat seedlings were grown hydroponically in environmental chambers and treated with a range of NaCl concentrations (0.034 M, 0.17 M, 0.68 M, or 3.42 M) over a 1, 3, and 5-day period. The salt treatments were started in the appropriate time so that they were all ten-day-old during harvest. Cellular membrane stability (CMS) as measured by a conductivity method and PS-LT. activity values were affected adversely by NaCl concentration and duration of treatment. Both methods clearly distinguish between salt-sensitive and salt-tolerant genotypes. Statistical analysis showed that PS-II activity and CMS measurements are well correlated (r=0.7589) suggesting that PS-II activity would be used as an additional screening method besides CMS to evaluate salt tolerance of wheat

2-DIMENSIONAL ELECTROPHORESIS OF PROTEINS WITH A DIFFERENT APPROACH TO ISOELECTRIC-FOCUSING

A simple method for the two-dimensional analysis of proteins was developed based on direct extraction of a protein sample from dried/precipitated material into isoelectric focusing (IEF) gel solution. This sample solution is equally useful for sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis and two-dimensional electrophoresis. For the former method this sample solution is applied directly to the gel. For the tatter, sample solution containing a desired amount of proteins is mixed with IEF gel solution followed by polymerization. The method thus allows solubilization in volumes as large as the total IEF gel volume, uses no salt and SDS in sample solublization and needs no boiling. It is thus simple and can be used for the analysis of samples containing very small amounts of proteins and/or with low radioactive incorporation without involving concentration procedures that result in further losses of proteins. The presence of proteins within the polymerized gel prior to the run removes any doubt regarding their entry into the gel, and an extended pH gradient results in perfect resolution

Detection of extracellular ATP in the tumor microenvironment, using the pmeLUC biosensor

ATP is one of the main components of the tumor microenvironment, where it affects cell growth, tumor progression and antitumor immune response. The development of the pmeLUC probe, a luciferase engineered to be expressed on the outer facet of the plasma membrane, allowed real-time measurement of extracellular ATP in vitro and in vivo systems, among which the tumor microenvironment. Here we describe the experimental procedures to measure extracellular ATP levels in the tumor microenvironment of three different cancer models generated by the implant of pmeLUC-expressing tumor cells into the appropriate mice strain: ACN human neuroblastoma (nude/nude mice host), WEHI-3B murine leukemia (BALB/c host), and B16F10 murine melanoma (C57Bl/6 host). The procedure to obtain stable expression of pmeLUC in different cell types and methods for the measurement of extracellular ATP with pmeLUC in vitro are also described