Physiological State Of Cells Research Articles

Redox sensing: orthogonal control in cell cycle and apoptosis signalling

Living systems have three major types of cell signalling systems that are dependent upon high-energy chemicals, redox environment and transmembranal ion-gating mechanisms. Development of integrated systems biology descriptions of cell signalling require conceptual models incorporating all three. Recent advances in redox biology show that thiol-disulphide redox systems are regulated under dynamic, nonequilibrium conditions, progressively oxidized with the life cycle of cells and distinct in terms of redox potentials amongst subcellular compartments. This article uses these observations as a basis to distinguish 'redox-sensing' mechanisms, which are more global biologic redox control mechanisms, from 'redox signalling', which involves conveyance of discrete activating or inactivating signals. Both redox sensing and redox signalling use sulphur switches, especially cysteine (Cys) residues in proteins which are sensitive to reversible oxidation, nitrosylation, glutathionylation, acylation, sulfhydration or metal binding. Unlike specific signalling mechanisms, the redox-sensing mechanisms provide means to globally affect the rates and activities of the high-energy, ion-gating and redox-signalling systems by controlling sensitivity, distribution, macromolecular interactions and mobility of signalling proteins. Effects mediated through Cys residues not directly involved in signalling means redox-sensing control can be orthogonal to the signalling mechanisms. This provides a capability to integrate signals according to cell cycle and physiologic state without fundamentally altering the signalling mechanisms. Recent findings that thiol-disulphide pools in humans are oxidized with age, environmental exposures and disease risk suggest that redox-sensing thiols could provide a central mechanistic link in disease development and progression.

Proteins tightly bound to DNA: New data and old problems

Proteins tightly bound to DNA (TBP) comprise a group of proteins that remain bound to DNA after usual deproteinization procedures such as salting out and treatment with phenol or chloroform. TBP bind to DNA by covalent phosphotriester and noncovalent ionic and hydrogen bonds. Some TBP are conservative, and they are usually covalently bound to DNA. However, the TBP composition is very diverse and significantly different in different tissues and in different organisms. TBP include transcription factors, enzymes of the ubiquitin-proteasome system, phosphatases, protein kinases, serpins, and proteins of retrotransposons. Their distribution within the genome is nonrandom. However, the DNA primary structure or DNA curvatures do not define the affinity of TBP to DNA. But there are repetitive DNA sequences with which TBP interact more often. The TBP distribution within genes and chromosomes depends on a cell's physiological state, differentiation type, and stage of organism development. TBP do not interact with DNA in the sites of its association with nuclear matrix and most likely they are not components of the latter.

Plasma membrane microdomains in aging and disease

The plasma membrane of eukaryotic cells participates in signal transduction and many other cellular events to maintain the physiological state of cells. In recent decades, much attention has been paid to membrane microdomains, called lipid rafts or membrane rafts, as signaling platforms in the plasma membrane. Lipid rafts are lateral lipid clusters enriched in cholesterol and sphingolipids in which particular molecules are concentrated and participate in membrane-mediated signaling events. Recent studies have shown a close relationship between lipid rafts and the age-associated decline and dysregulation of cellular signaling pathways, such as T-cell receptor signaling and cellular senescence-related signaling. Lipid rafts have also been implicated in senile diseases and in lifestyle-related diseases whose incidences increase with age.

Inferring cluster-based networks from differently stimulated multiple time-course gene expression data

Motivation: Clustering and gene network inference often help to predict the biological functions of gene subsets. Recently, researchers have accumulated a large amount of time-course transcriptome data collected under different treatment conditions to understand the physiological states of cells in response to extracellular stimuli and to identify drug-responsive genes. Although a variety of statistical methods for clustering and inferring gene networks from expression profiles have been proposed, most of these are not tailored to simultaneously treat expression data collected under multiple stimulation conditions.Results: We propose a new statistical method for analyzing temporal profiles under multiple experimental conditions. Our method simultaneously performs clustering of temporal expression profiles and inference of regulatory relationships among gene clusters. We applied this method to MCF7 human breast cancer cells treated with epidermal growth factor and heregulin which induce cellular proliferation and differentiation, respectively. The results showed that the method is useful for extracting biologically relevant information.Availability: A MATLAB implementation of the method is available from http://csb.gsc.riken.jp/yshira/software/clusterNetwork.zipContact: yshira@riken.jpSupplementary information: Supplementary data are available at Bioinformatics online.

Monitoring Real-Time Metabolism of Living Cells by Fast Two-Dimensional NMR Spectroscopy

Living cell metabolism is often monitored by 1D NMR spectroscopy, but the spectral resolution and the short cell lifetime are certainly limiting aspects. 2D spectroscopy does yield higher resolution but is time-consuming since acquisition of the second dimension requires several minutes. However, after only few minutes, oxygen starvation changes cell metabolism, and long acquisition times may yield spectra that do not represent the cell physiological state. Accordingly, metabolic studies of cells require fast NMR data acquisition. Here, we have applied band-selective optimized flip-angle short-transient (SOFAST)-HMQC techniques to (15)N-labeled cells, showing for the first time that it is possible to obtain 2D (1)H-(15)N correlation spectra of small metabolites directly in living cells, in a few seconds and with a high S/N ratio. SOFAST-HMQC spectra of (15)N-labeled Thalassiosira rotula diatoms cells can be acquired in 10-15 s, and, as an application, we have detected a progressive variation of the amino acid content when diatoms are exposed to UV-B radiation, with no need of long analytical procedures to quantify the metabolic changes. We believe that fast acquisition techniques can easily be extended to other cell systems, foreseeing a wide application in the emerging fields of metabolomics and metabonomics, being able to picture the "instantaneous" in-cell metabolism.

Alterations in core histone variant ratios during maize root differentiation, callus formation and in response to plant hormone treatment.

Although several histone variants have been studied in both animal and plant organisms, little is known about their distribution during processes that involve alterations in chromatin function, such as differentiation, dedifferentiation and hormone treatment. In this study we evaluated the ratio of each histone variant in each of the four core histone classes in the three developmental zones of maize (Zea mays L.) root and in callus cultures derived from them, in order to define possible alterations either during plant cell differentiation or dedifferentiation. We also evaluated core histone variant ratios in the developmental zones of roots treated with auxin and gibberellin in order to examine the effects of exogenously applied plant hormones to histone variant distribution. Finally, immunohistochemical detection was used to identify the root tissues containing modified forms of core histones and correlates them with the physiological status of the plant cells. According to the results presented in this study, histone variant ratios are altered in all the cases examined, i.e. in the developmental zones of maize root, in callus cultures derived from them and in the developmental zones of roots treated either with auxin or gibberellin. We propose that the alterations in linker histone variant ratios are correlated with plant cell differentiation and physiological status in each case.

Phage adsorption to Lactobacillus plantarum: Influence of physiological and environmental factors

Bacteriophage infection of lactic acid bacteria (LAB) constitutes one of the major problems in the dairy industry, causing economic losses and a constant risk of low quality and/or unsafe foods. The first step in the phage biology is the adsorption on the host cell surface. In a previous study, a remarkable thermal, chemical and photocatalytic resistance was demonstrated by four phages of Lactobacillus plantarum (ATCC 8014-B1, ATCC 8014-B2, FAGK1 and FAGK2) . In the present work, these phages were used to characterize the adsorption process on L. plantarum ATCC 8014. Clearly, the characterization of this process could increase the possibilities of design useful strategies in order to prevent phage infections. The influence of Ca 2+, temperature, pH and physiological cell state on phage adsorption was investigated. Burst sizes of phages ATCC 8014-B1 and ATCC 8014-B2 were 60 and 83 PFU/infective centre, respectively. The four phages exhibited a high infectivity even at pH 4 and pH 11. Calcium or magnesium ions were not indispensable for cell lysis and plaque formation, and more than 99% of phage particles were adsorbed either in the presence or absence of Ca 2+, after 15 min at 37 °C. Phage adsorption was only partially affected at 50 °C, while reached its maximum between 30 and 42 °C. The highest adsorption values (99.9%) were observed from pH 5 to 7, after 30 min at 37 °C. Adsorption rates decreased after the thermal inactivation of cells, though, when 20 μg/ml of chloramphenicol was used, adsorption values were similar on treated and untreated cells. All these results showed that the adsorption process was only partially affected by a few conditions: thermally killed host cells, an incubation temperature of 50 °C and pH values of 9 and 10. Nevertheless, and unfortunately, those conditions are not commonly applied during fermented food manufacturing, thus restricting highly the application of strategies currently available to reduce phage infections in industrial environments. This work also contributes to increase the currently knowledge on the biological aspects of L. plantarum bacteriophages.

Influence of cell geometry and number of replicas in the reproducibility of whole cell FTIR analysis

Fourier Transform InfraRed (FTIR) spectroscopy is an increasingly used technique in biology, especially for whole cell metabolomic fingerprint. The reproducibility of this technique is influenced by a large number of factors such as the physiological state of cells, sample manipulation and growth conditions. Evidence exists suggesting that the cell shape and dimension can be further elements to consider in whole cell FTIR analysis. In this study we aimed to address the effect of cell geometry on the FTIR spectra and to define the extent of variability occurring between machine and biological replicas with a standardized protocol. The yeast species Saccharomyces cerevisiae (large oval-shaped cells) and Debaryomyces hansenii (small round shaped cells) were employed for their different morphology. Thirty machine replicas of each were analyzed separately and after averaging in groups of three, showing a three to four-fold reduction of the variability. Similarly, a two-fold reduction of variability was observed when thirty biological replicas of the two yeast species were analyzed. The optimal number of replicas to average was then estimated with a bootstrap-like procedure in which biological and machine replicas were randomly resampled 2000 times and averaged in groups spanning from 2 to 12 replicas. This simulation has shown that little if any advantage can be obtained by increasing the number of replicas over five and that the variability exhibited by the small regular cells of D. hansenii was always roughly half of that displayed by the large S. cerevisiae cells, confirming the results obtained with standard non-bootstrapped averages.

Morphology, physiology, molecular phylogeny and sexual compatibility of the cryptic Pseudo-nitzschia delicatissima complex (Bacillariophyta), including the description of P. arenysensis sp. nov.

Quijano-Scheggia S., Garcés E., Lundholm N., Moestrup Ø., Andree K. and Camp J. 2009. Morphology, physiology, molecular phylogeny and sexual compatibility of the cryptic Pseudo-nitzschia delicatissima complex (Bacillariophyta), including the description of P. arenysensis sp. nov. Phycologia 48: 492–509. DOI: 10.2216/08-21.1Several strains of Pseudo-nitzschia delicatissima were isolated from the northwestern Mediterranean Sea and compared using light and electron microscopy, phylogenetic analyses of internal transcribed spacer rDNA, together with studies of their mating system, estimations of growth rates and reduction in cell size with time. Morphological and phylogenetic analyses identified all strains as Pseudo-nitzschia delicatissima, but in the phylogenetic analyses they fell into two genetically distinct clades. Studies of fine structural morphological characteristics by electron microscopy did not allow discrimination of the two clades. Mating experiments showed successful sexualisation among strains of clade B/1; whereas, mating attempts in clade A/2 and between clade A/2 and clade B/1 were unsuccessful under the examined laboratory conditions. The morphological characteristics of the daughter generations F1 and F2 were analyzed and abnormalities in the F2 generation described. We describe for the first time the rapid size reduction in the F2 generation. Culture studies showed physiological differences between the two clades; strains from clade A/2 showed a tendency for higher growth rates and slower reduction in cell size than strains from clade B/1. Morphological and phylogenetic studies comparing strains from different parts of the world showed P. delicatissima as a cryptic species complex comprising at least two different genotypes, both apparently cosmopolitan. The two genotypes were shown to occur sympatrically in the Mediterranean Sea, and we hypothesize that the genotypes were most likely separated by reproductive barriers associated with sexualisation occurring at different temperatures and hence different seasons. The molecular and physiological differences allow us to describe one of the genotypes, clade B/1, as a new species, Pseudo-nitzschia arenysensis sp. nov. Differences in growth capabilities of the cultures were observed in relation to cell size and physiological status and showed a nonlinear relationship between cell size and growth. Possibly, such capability plays a key role in the occurrence and dynamics of phytoplankton algae.

Affinity Purification Strategy to Capture Human Endogenous Proteasome Complexes Diversity and to Identify Proteasome-interacting Proteins

An affinity purification strategy was developed to characterize human proteasome complexes diversity as well as endogenous proteasome-interacting proteins (PIPs). This single step procedure, initially used for 20 S proteasome purification, was adapted to purify all existing physiological proteasome complexes associated to their various regulatory complexes and to their interacting partners. The method was applied to the purification of proteasome complexes and their PIPs from human erythrocytes but can be used to purify proteasomes from any human sample as starting material. The benefit of in vivo formaldehyde cross-linking as a stabilizer of protein-protein interactions was studied by comparing the status of purified proteasomes and the identified proteins in both protocols (with or without formaldehyde cross-linking). Subsequent proteomics analyses identified all proteasomal subunits, known regulators, and recently assigned partners. Moreover other proteins implicated at different levels of the ubiquitin-proteasome system were also identified for the first time as PIPs. One of them, the ubiquitin-specific protease USP7, also known as HAUSP, is an important player in the p53-HDM2 pathway. The specificity of the interaction was further confirmed using a complementary approach that consisted of the reverse immunoprecipitation with HAUSP as a bait. Altogether we provide a valuable tool that should contribute, through the identification of partners likely to affect proteasomal function, to a better understanding of this complex proteolytic machinery in any living human cell and/or organ/tissue and in different cell physiological states.

Mitochondria-Targeted Vitamin E Antioxidant: An Agent for Cardiovascular Protection

Mitochondria play an important role in controlling the bioenergetic status of cells in physiological conditions. Consequently, mitochondrial dysfunction leads to a range of human vascular diseases in humans. Although the molecular mechanisms responsible for mitochondria-mediated disease processes are not altogether clear, oxidative stress appears to be closely linked to the subsequent increase in the amount of free radicals. Accordingly, strategies for the targeted deliv- ery of antioxidants to mitochondria are being developed. In this review, we shall briefly discuss the metabolism of cellular reactive oxygen species and its role in pathophysiology, currently available antioxidants and possible reasons for their in- effectiveness in ameliorating oxidative stress-mediated diseases, and recent developments in mitochondria-targeted anti- oxidants (in particular mitochondria-targeted vitamin E) and their potential for the treatment of cardiovascular disease.

Effect ofEscherichia coliSTb toxin on NIH-3T3 cells

Previous studies have shown that STb causes microscopic histological alterations in animal intestinal models. Disrupted intestinal epithelium at the villous tips could be the result of an altered physiological cell state induced by the toxin. As a cellular model we used NIH-3T3 cells, a mouse fibroblast cell line, previously shown to be capable of internalizing the STb toxin. Using various probes specific for the cellular physiological state or cell organelles, we investigated STb activity using flow cytometry and confocal microscopy. In NIH-3T3 cells, labelled with propidium iodide and carboxyfluorescein diacetate, STb permeabilized the plasma membrane but the cellular esterases remained active. Confocal microscopy showed that fluorescein isothiocyanate (FITC)-labelled STb toxin molecules were internalized and were found scattered in the cytoplasm. Moreover, important clusters of FITC-STb were observed inside the cells after 6 h and these clusters matched with mitochondria labelling. After cell treatment with STb, using a fluorescent mitochondrial potential sensor, we observed mitochondria hyperpolarization, as an early event of intoxication. This phenomenon increased linearly with the dose of STb. The cell population treated with STb showed histological alterations such as membrane budding, granular cytoplasm and enlarged nucleus. Altogether, these results provide new information, at the cellular level, on the effect of the STb toxin.

A non-catalytic disulphide bond regulating redox flux in the ER oxidative folding pathway

A non-catalytic disulphide bond regulating redox flux in the ER oxidative folding pathway

Dielectric characterization of complete mononuclear and polymorphonuclear blood cell subpopulations for label-free discrimination

Dielectric spectroscopy is a powerful technique for the elucidation of a number of important cell biophysical properties, and it can provide information about cell morphology, physiological state, viability and identity. A high-impact application for dielectric cell analysis would be microfluidic flow-through impedance sensing to perform what is perhaps the most routinely ordered medical diagnostic assay, a complete blood count with white blood cell differential enumeration. To assess the biophysical feasibility of such an analysis, we obtained reference dielectric measurements of the complete complement of purified leukocyte subpopulations using the dielectrophoretic crossover frequency method. The sensitivity of this method can detect subtle changes in cell morphology and physiology, so we developed a leukocyte isolation protocol based on a suite of negative selection techniques to yield cell subpopulations that were minimally processed and in an as near native state as possible. This is the first reported study of the dielectric properties of all the major leukocyte subpopulations that includes separate analysis of the polymorphonuclear neutrophil, basophil and eosinophil cell types. We show that T-lymphocytes, B-lymphocytes, monocytes and granulocytes possess distinct membrane dielectric properties and that the morphologically similar granulocyte subpopulations can be identified via their dielectric and size properties. Finally, we discuss the application of our findings to label-free systems for the analysis of leukocytes.

Alterations in core histone variant ratios during maize root differentiation, callus formation and in response to plant hormone treatment

Although several histone variants have been studied in both animal and plant organisms, little is known about their distribution during processes that involve alterations in chromatin function, such as differentiation, dedifferentiation and hormone treatment. In this study we evaluated the ratio of each histone variant in each of the four core histone classes in the three developmental zones of maize (Zea mays L.) root and in callus cultures derived from them, in order to define possible alterations either during plant cell differentiation or dedifferentiation. We also evaluated core histone variant ratios in the developmental zones of roots treated with auxin and gibberellin in order to examine the effects of exogenously applied plant hormones to histone variant distribution. Finally, immunohistochemical detection was used to identify the root tissues containing modified forms of core histones and correlates them with the physiological status of the plant cells. According to the results presented in this study, histone variant ratios are altered in all the cases examined, i.e. in the developmental zones of maize root, in callus cultures derived from them and in the developmental zones of roots treated either with auxin or gibberellin. We propose that the alterations in linker histone variant ratios are correlated with plant cell differentiation and physiological status in each case.

Maximum entropy decomposition of flux distribution at steady state to elementary modes

Enzyme Control Flux (ECF) is a method of correlating enzyme activity and flux distribution. The advantage of ECF is that the measurement integrates proteome data with metabolic flux analysis through Elementary Modes (EMs). But there are a few methods of effectively determining the Elementary Mode Coefficient (EMC) in cases where no objective biological function is available. Therefore, we proposed a new algorithm implementing the maximum entropy principle (MEP) as an objective function for estimating the EMC. To demonstrate the feasibility of using the MEP in this way, we compared it with Linear Programming and Quadratic Programming for modeling the metabolic networks of Chinese Hamster Ovary, Escherichia coli, and Saccharomyces cerevisiae cells. The use of the MEP presents the most plausible distribution of EMCs in the absence of any biological hypotheses describing the physiological state of cells, thereby enhancing the prediction accuracy of the flux distribution in various mutants.

Nuclear Phosphatidylcholine and Sphingomyelin Metabolism of Thyroid Cells Changes during Stratospheric Balloon Flight

Nuclear sphingomyelin and phosphatidylcholine metabolism is involved in the response to ultraviolet radiation treatment in different ways related to the physiological state of cells. To evaluate the effects of low levels of radiation from the stratosphere on thyroid cells, proliferating and quiescent FRTL-5 cells were flown in a stratospheric balloon (BIRBA mission). After recovery, the activity of neutral sphingomyelinase, phosphatidylcholine-specific phospholipase C, sphingomyelin synthase, and reverse sphingomyelin synthase was assayed in purified nuclei and the nuclei-free fraction. In proliferating FRTL-5, space radiation stimulate nuclear neutral sphingomyelinase and reverse sphingomyelin synthase activity, whereas phosphatidylcholine-specific phospholipase C and sphingomyelin synthase were inhibited, thus inducing sphingomyelin degradation and phosphatidylcholine synthesis. This effect was lower in quiescent cells. The possible role of nuclear lipid metabolism in the thyroid damage induced by space radiations is discussed.

Studies related to antibody fragment (Fab) production in Escherichia coli W3110 fed‐batch fermentation processes using multiparameter flow cytometry

Microbiology is important to industry therefore rapid and statistically representative measurements of cell physiological state, proliferation, and viability are essential if informed decisions about fermentation bioprocess optimization or control are to be made, because process performance will depend largely on the number of metabolically active viable cells. Samples of recombinant Escherichia coli W3110, containing the gene for the D1.3 anti-lysozyme Fab fragment under the control of the lac-based expression system, were taken at various stages from fed-batch fermentation processes and stained with a mixture of bis-(1,3-dibutylbarbituric acid) trimethine oxonol and propidium iodide (PI/BOX). Where appropriate, measurements of dissolved oxygen tension (DOT), OD600nm and Fab concentration were made. Depending on time of induction the maximum amount of Fab accumulating in the supernatant varied quite markedly from 1 to 4 microg ml(-1) as did subsequent cell physiological state with respect to PI/BOX staining with a concomitant drop in maximum biomass concentration. Depending on point of induction a fourfold increase in Fab production could be achieved accompanied by a approximately 50% drop in maximum biomass concentration but with a higher proportion of viable cells as measured by multiparameter flow cytometry.

The Expanding Role of APRIL in Cancer and Immunity

Proteins of the tumour necrosis factor (TNF) family are implicated in the regulation of essential cell processes such as proliferation, differentiation, survival and cell death. Altered expression of TNF family members is often associated with pathological conditions such as autoimmune disease and cancer. The TNF-like ligand APRIL (A PRoliferation Inducing Ligand), first described in 1998, was named for its capacity to stimulate tumour cell proliferation in vitro. APRIL expression was initially reported in haematopoietic cells in physiological conditions, and it is overexpressed in certain tumour tissues. APRIL is now known to be involved in activation and immune responses of B cells, as well as in B cell malignancies. This review focuses on recent advances in understanding APRIL and its receptors in physiology and tumour pathology, including the accumulating evidence that specific Toll-like receptor ligands can trigger APRIL-mediated responses, and the identification of new sources of APRIL such as epithelial cells and tumour-infiltrating neutrophils.

Physiological heterogeneity of suspended microbial aggregates

Suspended microbial aggregates, which are always in dynamic equilibrium with suspended cells and cells attached to surface, are very common structures in natural and engineering environmental systems. To study and design physiologically diverse suspended microbial aggregates the physiological classification of chemotrophic prokaryotes in 12 groups formed by four evolutionary periods (fermenting, anaerobic respiring, microaerophilic and facultative aerobic, aerobic prokaryotes) and three parallel lines (Gram-negative, Gram-positive Eubacteria, and Archaea) could be used. This type of physiological heterogeneity has been studied in microbial granules using fluorescence in situ hybridization, identification of 16S rRNA genes, and conferring the physiological properties from the description of the species. In spherical granules with diameter of 2.4 mm cells of aerobes were spread to the depth 0.55 mm below surface (85% of granule volume), facultative anaerobes dominated between the depths 0.55 mm and 0.85 mm (13% of granule volume), and anaerobes were concentrated at the depths from 0.85 to 1.0 mm (2% of granule volume). Percentages of aerobic, facultative anaerobic, and anaerobic species in granules, identified by 16S rRNA gene sequencing, were 69%, 9%, and 2% of total number of bacterial clones, respectively. Another type of physiological heterogeneity on the cellular level was due to the changes of cell physiological status during cell cycle. This type of heterogeneity has been studied in the populations of Escherichia coli, Bacillus megaterium, Saccharomyces cerevisiae, and Candida tropicalis. A significant proportion of cells from the exponential phase were killed after 10 min treatment with 1% solution of allyl alcohol, which specifically kills cells with high activity of alcohol dehydrogenase (ADH). However, there was no such effect in starved cell population. Percentage of cells with high activity ADH in microbial population can be used to monitor its physiological status. Physiological diversity of ecosystem may be due to mechanical mixing of cells from the different inflows. An example of such system is an ecosystem of aeration tank in municipal wastewater treatment plant. This ecosystem contains a mechanical mixture of dead anaerobic and live aerobic bacteria as well as attached and suspended cell aggregates supplied from anaerobic digester, raw sewage, and settling tank.