Protein tyrosine nitration in cellular signal transduction pathways

How specificity and reversibility in tyrosine nitration are defined biologically in cellular systems is poorly understood. As more investigations identify proteins involved in cell regulatory pathways in which only a small fraction of that protein pool is modified by nitration to affect cell function, the mechanisms of biological specificity and reversal should come into focus. In this review experimental evidence has been summarized to suggest that tyrosine nitration is a highly selective modification and under certain physiological conditions fulfills the criteria of a physiologically relevant signal. It can be specific, reversible, occurs on a physiological time scale, and, depending on a target, can result in either activation or inhibition.     

Reversible and noisy progression towards a commitment point enables adaptable and reliable cellular decision-making

Cells must make reliable decisions under fluctuating extracellular conditions, but also be flexible enough to adapt to such changes. How cells reconcile these seemingly contradictory requirements through the dynamics of cellular decision-making is poorly understood. To study this issue we quantitatively measured gene expression and protein localization in single cells of the model organism Bacillus subtilis during the progression to spore formation. We found that sporulation proceeded through noisy and reversible steps towards an irreversible, all-or-none commitment point. Specifically, we observed cell-autonomous and spontaneous bursts of gene expression and transient protein localization events during sporulation. Based on these measurements we developed mathematical population models to investigate how the degree of reversibility affects cellular decision-making. In particular, we evaluated the effect of reversibility on the 1) reliability in the progression to sporulation, and 2) adaptability under changing extracellular stress conditions. Results show that reversible progression allows cells to remain responsive to long-term environmental fluctuations. In contrast, the irreversible commitment point supports reliable execution of cell fate choice that is robust against short-term reductions in stress. This combination of opposite dynamic behaviors (reversible and irreversible) thus maximizes both adaptable and reliable decision-making over a broad range of changes in environmental conditions. These results suggest that decision-making systems might employ a general hybrid strategy to cope with unpredictably fluctuating environmental conditions.     

Abstract biological systems as sequential machines: Behavioral reversibility

Rosen’s identification of abstract biological systems, called (M,R)-systems, with sequential machines is formally characterized. It is then shown that the determination of environmental alterations of (M,R)-systems from a knowledge of the response sequence and the structure of the system, which we call behavioral reversibility, can be interpreted as information-losslessness of sequential machines. Applying this relationship, necessary conditions for behavioral reversibility are derived. It is further shown that, similar to Rosen’s work on structural reversibility, (M,R)-systems are behaviorally reversible only if the number of physically realizable mappings are restricted.     

Multispectral/hyperspectral image enhancement for biological cell analysis.

BACKGROUND: Microscopes form projected images from illuminated objects, such as cellular tissue, which are recorded at a distance through the optical system's field of view. A telescope on a satellite or airplane also forms images with a similar optical projection of objects on the ground. Typical visible illuminations form a displayed set of three-color channels (Red Green Blue [RGB]) that are combined from three image sensor arrays (e.g., focal plane arrays) into a single pixel coding for each color present in the image. Analysis of these RGB color images develops a qualitative image representation of the objects. METHODS: Independent component analysis (ICA) is used for analysis and enhancement of multispectral images, and compared with the similar and widely used principal component analysis. RESULTS: The data examples indicate that the ICA enhancement, and the resulting RGB image combination display, can be useful in processing datacubes of cellular data where isolation of unknown subtle image elements representing objects is desired. CONCLUSIONS: ICA image enhancement can aid processing of datacubes of cellular data by clarifying subtle image elements. These parallelizable algorithms can be implemented for real-time, online analysis.     

Oxidant Sensing by Reversible Disulfide Bond Formation

Maintenance of the cellular redox balance is crucial for cell survival. An increase in reactive oxygen, nitrogen, or chlorine species can lead to oxidative stress conditions, potentially damaging DNA, lipids, and proteins. Proteins are very sensitive to oxidative modifications, particularly methionine and cysteine residues. The reversibility of some of these oxidative protein modifications makes them ideally suited to take on regulatory roles in protein function. This is especially true for disulfide bond formation, which has the potential to mediate extensive yet fully reversible structural and functional changes, rapidly adjusting the protein''s activity to the prevailing oxidant levels.     

Studies on rapid reversible and non-reversible inactivation of fructose-1,6-bisphosphatase and malate dehydrogenase in wild-type and glycolytic block mutants of Saccharomyces cerevisiae

Experimental conditions have been elaborated to test for reversibility of the malate dehydrogenase inactivation (E.C.1.1.1.37) after addition of glucose to derepressed yeast cells. Malate dehydrogenase inactivation was shown to be irreversible at all stages of inactivation. In contrast fructose-1,6-bisphosphatase inactivation (E.C.3.1.11) remained reversible for at least 30 min after addition of glucose. Rapid reversible inactivation of fructose-1,6-bisphosphatase and irreversible inactivation of malate dehydrogenase were additionally investigated in glycolytic block mutants. Normal inactivation kinetics were observed in mutants without catalytic activity of phosphoglucose isomerase (E.C.5.3.1.9), phosphofructokinase (E.C.2.7.1.11), triosephosphate isomerase (E.C.5.3.1.1) and phosphoglycerate kinase (E.C.2.7.2.3). Hence, neither type of inactivation depended on the accumulation of any glucose metabolite beyond glucose-6-phosphate. Under anaerobic conditions irreversible inactivation was completely abolished in glycolytic block mutants. In contrast rapid reversible inactivation was independent of energy provided by respiration or fermentation. Reversibility of fructose-1,6-bisphosphatase inactivation was tested under conditions which prevented irreversible malate dehydrogenase inactivation. In these experiments, fructose-1,6-bisphosphatase inactivation remained reversible for at least 120 min, whereas reversibility was normally restricted to about 30 min. This indicated a common mechanism between the irreversible part of fructose-1,6-bisphosphatase inactivation and irreversible malate dehydrogenase inactivation.     

HEMETβ: improvement of hepatocyte metabolism mathematical model

This article describes hepatocyte metabolism mathematical model (HEMETβ), which is an improved version of HEMET, an effective and versatile virtual cell model based on hepatic cell metabolism. HEMET is based on a set of non-linear differential equations, implemented in Simulink?, which describes the biochemical reactions and energetic cell state, and completely mimics the principal metabolic pathways in hepatic cells. The cell energy function and modular structure are the core of this model. HEMETβ as HEMET model describes hepatic cellular metabolism in standard conditions (cell culture in a plastic multi-well placed in an incubator at 37° C with 5% of CO2) and with excess substrates concentration. The main improvements in HEMETβ are the introductions of Michaelis-Menten models for reversible reactions and enzymatic inhibition. In addition, we eliminated hard non-linearities and modelled cell proliferation and every single aminoacid degradation pathway. All these innovations, combined with a user-friendly aspect, allow researchers to create new cell types and validate new experimental protocols just varying 'peripheral' pathways or model inputs.     

HEMETβ: improvement of hepatocyte metabolism mathematical model

This article describes hepatocyte metabolism mathematical model (HEMETβ), which is an improved version of HEMET, an effective and versatile virtual cell model based on hepatic cell metabolism. HEMET is based on a set of non-linear differential equations, implemented in Simulink®, which describes the biochemical reactions and energetic cell state, and completely mimics the principal metabolic pathways in hepatic cells. The cell energy function and modular structure are the core of this model. HEMETβ as HEMET model describes hepatic cellular metabolism in standard conditions (cell culture in a plastic multi-well placed in an incubator at 37°C with 5% of CO₂) and with excess substrates concentration. The main improvements in HEMETβ are the introductions of Michaelis–Menten models for reversible reactions and enzymatic inhibition. In addition, we eliminated hard non-linearities and modelled cell proliferation and every single aminoacid degradation pathway. All these innovations, combined with a user-friendly aspect, allow researchers to create new cell types and validate new experimental protocols just varying ‘peripheral’ pathways or model inputs.     

Reversibility of monensin inhibition of oligosaccharide processing of human fibronectin

Monensin impairs oligosaccharide processing in fibronectin primarily by inhibiting the conversion of oligosaccharides from the high mannose type to the complex type. The separate effects of monensin and cations on alpha-mannosidase activity in fibroblasts were examined using an in vitro assay system. The results indicated that monensin did not directly inhibit alpha-mannosidase activity in vitro, although prior treatment of fibroblasts with monensin caused an irreversible suppression of enzyme activity. The reversibility of monensin action on oligosaccharide processing was also examined. Analyses using concanavalin A (ConA) Sepharose affinity chromatography showed that the inhibitory action of monensin on oligosaccharide processing was biologically reversible. A progressive return to complex type oligosaccharides began about 11 h after the removal of the monensin. These composite results indicate that the reversibility of monensin action on oligosaccharide processing in fibronectin may be attributed to the restoration of enzyme activity, although the mechanism by which restoration occurs remains to be deciphered.     

Effect of decimation on the classification rate of non-linear analysis methods applied to uterine EMG signals

Recently, much attention has been paid to the use of non-linear analysis techniques for the characterization of biological signals. Several measures have been proposed to detect non-linear characteristics in time series. The effect of sampling frequency on the performance of these non-linear methods has rarely been evaluated. In this paper, we present a preliminary study of this effect for four methods that are widely used in non-linearity detection: Time reversibility, Sample Entropy, Lyapunov Exponents and Delay Vector Variance. These methods have been applied to real uterine EMG signals with the aim of distinguishing between pregnancy and labor contractions. The signals were used to classify contractions before and after decimating them by a factor 10. The results show that decimation improves the performance for sample entropy. It reduces it considerably for Delay Vector Variance and only slightly for Time reversibility and Lyapunov exponents. Time reversibility still gives the highest classification rate. The methods were much less computationally expensive after down sampling.     

A Possible Role for Taurine in Osmoregulation Within the Brain

Intracranial microdialysis was used to measure changes in extracellular amino acids within the rat brain during local osmotic alteration of the extracellular microenvironment or during systemic water intoxication. Increased cellular hydration produced by either of these methods was accompanied by a marked increase in extracellular taurine levels without affecting the other amino acids measured. With local osmotic alteration, this increase was osmolarity dependent and reversible. The specificity, sensitivity, and reversibility of the increase in extracellular taurine strongly suggest a functional role in osmoregulation in the brain under normal as well as pathological conditions.     

Reversibility and efficiency in coding protein information

Why the genetic code has a fixed length? Protein information is transferred by coding each amino acid using codons whose length equals 3 for all amino acids. Hence the most probable and the least probable amino acid get a codeword with an equal length. Moreover, the distributions of amino acids found in nature are not uniform and therefore the efficiency of such codes is sub-optimal. The origins of these apparently non-efficient codes are yet unclear. In this paper we propose an a priori argument for the energy efficiency of such codes resulting from their reversibility, in contrast to their time inefficiency. Such codes are reversible in the sense that a primitive processor, reading three letters in each step, can always reverse its operation, undoing its process.We examine the codes for the distributions of amino acids that exist in nature and show that they could not be both time efficient and reversible. We investigate a family of Zipf-type distributions and present their efficient (non-fixed length) prefix code, their graphs, and the condition for their reversibility. We prove that for a large family of such distributions, if the code is time efficient, it could not be reversible. In other words, if pre-biotic processes demand reversibility, the protein code could not be time efficient. The benefits of reversibility are clear: reversible processes are adiabatic, namely, they dissipate a very small amount of energy. Such processes must be done slowly enough; therefore time efficiency is non-important. It is reasonable to assume that early biochemical complexes were more prone towards energy efficiency, where forward and backward processes were almost symmetrical.     

Dephosphorylation of the hepatic insulin receptor: absence of intrinsic phosphatase activity in purified receptors

We have compared here the reversibility of phosphorylation of insulin receptors either partially purified by lectin chromatography, or highly purified by specific immunoprecipitation with anti-receptor antibodies. We found that the beta subunit of partially purified insulin receptors was rapidly dephosphorylated (t 1/2 = 15 min). In contrast, the level of phosphorylation of immunoprecipitated receptors remained unchanged for up to 4 hours at 37 degrees C. However, cytosolic phosphatases, which are inhibited by vanadate, were able to induce a complete dephosphorylation of immunoprecipitated receptors. These results show that 1. phosphorylation of insulin receptors is reversible; and 2. no phosphatase activity is contained in the insulin receptor structure itself.     

Immobilization of histidine-tagged proteins on gold surfaces using chelator thioalkanes

The reversible, oriented immobilization of proteins on solid surfaces is a prerequisite for the investigation of molecular interactions and the controlled formation of supramolecular assemblies. This paper describes a generally applicable method using a synthetic chelator thioalkane that can be self-assembled on gold surfaces. The reversible binding of an anti-lysozyme F(ab) fragment modified with a C-terminal hexahistidine extension was monitored and the apparent dissociation constants determined using surface plasmon resonance. Infra-red spectroscopy demonstrated that the secondary structure of the protein was unaffected by the immobilization process. The retention of functionality of the immobilized protein was also successfully demonstrated. Given the mild reaction conditions and reversibility, this method of oriented immobilization of proteins opens possibilities for the development of biosensors.     

Correlation of structure and active transport in the teleost nephron.

In the present study we have extended our investigations concerning the correlation between ultrastructure and active transport in the isolated flounder nephron. The composition of the fish nephron is defined in ultrastructural terms and its behavior when incubated in vitro under short term and long term culture conditions is described. Using the in vitro system originally described by Forster, a variety of inhibitors and conditions which modify cell structure and function were tested. Ultrastructure was correlated with chlorphenol red dye transport. In general, conditions altering active transport also markedly altered cellular ultrastructure. The principal alterations consisted of membrane changes involving various organelles--most importantly the plasma membrane and the mitochondria. Conditions associated with irreversible cell injury could be rapidly produced by interference either with mitochondrial ATP synthesis or with the integrity of the plasma membrane. Both of these rapidly lead to irreversible events which are preceded by reversible structural changes. Organelle changes progress in a rather well-defined sequence of reversible and irreversible stages which are defined. One difference between the two types of interactions is the presence of intramitochondrial calcification which does not occur with direct modification of the mitochondrial electron transport system. The concept of utilizing long term explant organ cultures of fish nephrons for environmental studies is introduced.     

Small molecule inhibition of protein depalmitoylation as a new approach towards downregulation of oncogenic Ras signalling

The H- and N-Ras GTPases are prominent examples of proteins, whose localizations and signalling capacities are regulated by reversible palmitoylations and depalmitoylations. Recently, the novel small molecule inhibitor palmostatin B has been described to inhibit Ras depalmitoylation and to revert the phenotype of oncogenic HRasG12V transformed cells. This demonstrates that palmostatin B is a tool to investigate the biochemical effects of the inhibition of cellular Ras depalmitoylation on Ras signalling, which is relevant for oncology. Furthermore, it is to be expected that many proteins, of which the signalling capacities depend on reversible palmitoylation, will be discovered in the near future. This stresses the urgent need for further development of small molecule inhibitors of palmitoylation and depalmitoylation in order to study their functions in cellular signalling.     

Reversibility of the thermal transitions of chloroplast thylakoid membranes

Thylakoid membranes from cucumbers and peas have been examined by high-sensitivity differential scanning calorimetry. Data was collected during both heating and subsequent cooling scans in order to observe reversibility. Cucumber thylakoids exhibited almost no reversibility; a very small reversible exothermic peak was observed at approximately 12 degrees C in cooling scans. However, thylakoids from peas had reversible transitions at 50 and 68 degrees C, as well as other transitions which were visible as shoulders in a second heating scan. When pea grana thylakoids were unstacked, the high temperature transitions were sharpened and their reversibility was enhanced. This is the first report of chloroplast thylakoid membranes exhibiting reversible high temperature transitions. The results indicate that considerable variation can occur in the calorimetric profiles of thylakoids from different plants.     

Pseudoirreversible inhibition of prostate-specific membrane antigen by phosphoramidate peptidomimetics

The mode of inhibition for phosphoramidate peptidomimetic inhibitors of prostate-specific membrane antigen was determined by inhibition reversibility experiments. The results revealed that these inhibitors can be classified into three types: pseudoirreversible (compounds 1-3), moderately reversible (compounds 4-9), and rapidly reversible inhibitors (compounds 10 and 11). Representative compounds from each class were further evaluated for their ability to induce cellular internalization of PSMA. Results from these experiments revealed that the pseudoirreversible inhibitor 1 induced the greatest PSMA internalization. The discovery of pseudoirreversible PSMA inhibitors is expected to provide a new avenue of investigation and therapeutic applications for prostate cancer and neurological disorders.     

Reversibility of Acid-Inactivation of Bacillus subtilis’ α-Amylase

The inactivation of Bacillus subtilis’ α-amylase by acid was shown to be reversible. In the experiment, two different Bac. subtilis’ α-amylases, saccharifying and liquefying types, were used and the reversibility was investigated deviding into two processes of inactivation and reactivation. Both amylases showed the reversibility in a similar degree and in general the inactivated enzymes by acid were reactivated only by adjusting the pH to slightly alkaline values followed by incubation under certain conditions. However, the reversibility, especially, the reactivation was greatly influenced by several chemicals, the effect of certain chemicals being different according to the type of the bacterial amylase. Contrary to liquefying amylase, saccharifying amylase was insensitive to metal chelators but, nevertheless, the reactivation of the amylase was prevented by metal chelators. Also the reactivation of saccharifying amylase was inhibited by sulfhydryl reagents, although the native enzyme was quite insensitive to the chemicals. In the acid-inactivation and reactivation process, a reversible change in the ultraviolet absorption spectra of the enzymes was observed, and some discussion of the implication was presented.