Showing papers in "Methods in Enzymology in 2007"

Mechanisms of high salinity tolerance in plants.

Abstract: Among abiotic stresses, high salinity stress is the most severe environmental stress, which impairs crop production on at least 20% of irrigated land worldwide. In response to high salinity stress, various genes get upregulated, the products of which are involved either directly or indirectly in plant protection. Some of the genes encoding osmolytes, ion channels, receptors, components of calcium signaling, and some other regulatory signaling factors or enzymes are able to confer salinity-tolerant phenotypes when transferred to sensitive plants. Overall, the susceptibility or tolerance to high salinity stress in plants is a coordinated action of multiple stress responsive genes, which also cross talk with other components of stress signal transduction pathways. High salinity exerts its negative impact mainly by disrupting the ionic and osmotic equilibrium of the cell. In saline soils, high levels of sodium ions lead to plant growth inhibition and even death; therefore, mechanisms of salinity tolerance involve sequestration of Na(+) and Cl(-) in vacuoles of the cells, blocking of Na(+) entry into the cell, Na(+) exclusion from the transpiration stream, and some other mechanisms that help in salinity tolerance. Understanding these mechanisms of stress tolerance, along with a plethora of genes involved in the stress signaling network, is important to improve high salinity stress tolerance in crops plants. This chapter first describes the adverse effect of salinity stress and general pathway for the plant stress response, followed by roles of various ion pumps, calcium, SOS pathways, ABA, transcription factors, mitogen-activated protein kinases, glycine betaine, proline, reactive oxygen species, and DEAD-box helicases in salinity stress tolerance. The cross-tolerance between stresses is also mentioned.

Mammalian stress granules and processing bodies.

Abstract: The packaging of cytoplasmic mRNA into discrete RNA granules regulates gene expression by delaying the translation of specific transcripts. Specialized RNA granules found in germ cells direct the timing of maternal mRNA translation to promote germ cell development in the early embryo and establish the germ line for the next generation. Similarly, select neuronal mRNA transcripts are packaged into translationally inert RNA granules, transported to sites where their protein products are required, and only then activated and translated. Following translation, however, newly inactivated mRNAs released from polysomes can also be packaged into dynamic, transient structures known as stress granules (SGs) and processing bodies (PBs). Stress granules are composed largely of stalled preinitiation complexes, and contain mRNA, small ribosomal subunits, eIF3, eIF4E, eIF4G, and PABP, as their core components. PBs are associated with mRNA decay and contain the decapping enzymes DCP1/2, the 5' to 3' exonuclease Xrn1, the Lsm proteins (1-7), and the scaffolding proteins hedls/GE-1 and GW182. Both SGs and PBs contain mRNA, eIF4E, microRNAs and argonaute proteins, and various regulators of mRNA stability and translation (TTP, RCK/p54, and CPEB). Thus, SGs and PBs share some protein and mRNA components, but also contain a number of unique markers specific to each structure. We describe markers and staining procedures used to identify these distinct types of RNA granules, describe conditions that promote their assembly and disassembly, and establish YB-1 as a useful marker of SGs and PBs.

Tumor hypoxia in cancer therapy.

Abstract: Human solid tumors are invariably less well-oxygenated than the normal tissues from which they arose. This so-called tumor hypoxia leads to resistance to radiotherapy and anticancer chemotherapy as well as predisposing for increased tumor metastases. In this chapter, we examine the resistance of tumors to radiotherapy produced by hypoxia and, in particular, address the question of whether this resistance is the result of the physicochemical free radical mechanism that produces resistance to radiation killing of cells in vitro. We conclude that a major part of the resistance, though perhaps not all, is the result of the physicochemical free radical mechanism of the oxygen effect in sensitizing cells to ionizing radiation. However, in modeling studies used to evaluate the effect of fractionated irradiation on tumor response, it is essential to consider the fact that the tumor cells are at a wide range of oxygen concentrations, not just at the extremes of oxygenated and hypoxic. Prolonged hypoxia of the tumor tissue also leads to necrosis, and necrotic regions are also characteristic of solid tumors. These two characteristics--hypoxia and necrosis--represent clear differences between tumors and normal tissues and are potentially exploitable in cancer treatment. We discuss strategies for exploiting these differences. One such strategy is to use drugs that are toxic only under hypoxic conditions. The second strategy is to take advantage of the selective induction under hypoxia of the hypoxia-inducible factor (HIF)-1. Gene therapy strategies based on this strategy are in development. Finally, tumor hypoxia can be exploited using live obligate anaerobes that have been genetically engineered to express enzymes that can activate nontoxic prodrugs into toxic chemotherapeutic agents.

Chapter Two – Yeast Osmoregulation

Abstract: Osmoregulation is the active control of the cellular water balance and encompasses homeostatic mechanisms crucial for life. The osmoregulatory system in the yeast Saccharomyces cerevisiae is particularly well understood. Key to yeast osmoregulation is the production and accumulation of the compatible solute glycerol, which is partly controlled by the high osmolarity glycerol (HOG) signaling system. Genetic analyses combined with studies on protein–protein interactions have revealed the wiring scheme of the HOG signaling network, a branched mitogen‐activated protein (MAP) kinase (MAPK) pathway that eventually converges on the MAPK Hog1. Hog1 is activated following cell shrinking and controls posttranscriptional processes in the cytosol as well as gene expression in the nucleus. HOG pathway activity can easily and rapidly be controlled experimentally by extracellular stimuli, and signaling and adaptation can be separated by a system of forced adaptation. This makes yeast osmoregulation suitable for studies on system properties of signaling and cellular adaptation via mathematical modeling. Computational simulations and parallel quantitative time course experimentation on different levels of the regulatory system have provided a stepping stone toward a holistic understanding, revealing how the HOG pathway can combine rigorous feedback control with maintenance of signaling competence. The abundant tools make yeast a suitable model for an integrated analysis of cellular osmoregulation. Maintenance of the cellular water balance is fundamental for life. All cells, even those in multicellular organisms with an organism‐wide osmoregulation, have the ability to actively control their water balance. Osmoregulation encompasses homeostatic processes that maintain an appropriate intracellular environment for biochemical processes as well as turgor of cells and organism. In the laboratory, the osmoregulatory system is studied most conveniently as a response to osmotic shock, causing rapid and dramatic changes in the extracellular water activity. Those rapid changes mediate either water efflux (hyperosmotic shock), and hence cell shrinkage, or influx (hypoosmotic shock), causing cell swelling. The yeast S. cerevisiae, as a free‐living organism experiencing both slow and rapid changes in extracellular water activity, has proven a suitable and genetically tractable experimental system in studying the underlying signaling pathways and regulatory processes governing osmoregulation. Although far from complete, the present picture of yeast osmoregulation is both extensive and detailed (de Nadal et al., 2002; Hohmann, 2002; Klipp et al., 2005). Simulations using mathematical models combined with time course measurements of different molecular processes in signaling and adaptation have allowed elucidation of the first system properties on the yeast osmoregulatory network.

Recombineering: in vivo genetic engineering in E. coli, S. enterica, and beyond

Abstract: "Recombineering," in vivo genetic engineering with short DNA homologies, is changing how constructs are made. The methods are simple, precise, efficient, rapid, and inexpensive. Complicated genetic constructs that can be difficult or even impossible to make with in vitro genetic engineering can be created in days with recombineering. DNA molecules that are too large to manipulate with classical techniques are amenable to recombineering. This technology utilizes the phage lambda homologous recombination functions, proteins that can efficiently catalyze recombination between short homologies. Recombineering can be accomplished with linear PCR products or even single-stranded oligos. In this chapter we discuss methods of and ways to use recombineering.

Identification of modified residues in RNAs by reverse transcription-based methods.

Abstract: Naturally occurring modified residues derived from canonical RNA nucleotides are present in most cellular RNAs. Their detection in RNA represents a difficult task because of their great diversity and their irregular distribution within RNA molecules. Over the decades, multiple experimental techniques were developed for the identification and localization of RNA modifications. Most of them are quite laborious and require purification of individual RNA to a homogeneous state. An alternative to these techniques is the use of reverse transcription (RT)-based approaches. In these approaches, purification of RNA to homogeneity is not necessary, because the selection of the analyzed RNA species is done by specific annealing of oligonucleotide DNA primers. However, results from primer extension analysis are difficult to interpret because of the unpredictable nature of RT pauses. They depend not only on the properties of nucleotides but also on the RNA primary and secondary structure. In addition, the degradation of cellular RNA during extraction, even at a very low level, may complicate the analysis of the data. RT-based techniques for the identification of modified residues were considerably improved by the development of selected chemical reagents specifically reacting with a given modified nucleotide. The RT profile obtained after such chemical modifications generally allows unambiguous identification of the chemical nature of the modified residues and their exact location in the RNA sequence. Here, we provide experimental protocols for selective chemical modification and identification of several modified residues: pseudouridine, inosine, 5-methylcytosine, 2'-O-methylations, 7-methylguanosine, and dihydrouridine. Advice for an optimized use of these methods and for correct interpretation of the data is also given. We also provide some helpful information on the ability of other naturally occurring modified nucleotides to generate RT pauses.

Integrins in cell migration.

Abstract: Integrins are cell-surface adhesion receptors that play a central role in regulating cell migration by mediating interactions between the extracellular matrix and the actin cytoskeleton. Substantial progress has been made in understanding the mechanisms by which the formation and breakdown of adhesions are regulated. Here we describe general methods used to study integrin-mediated cell migration. Furthermore, we outline detailed procedures to examine focal adhesion assembly and disassembly using time-lapse fluorescent microscopy. Finally, we provide methods for the analysis of podosomes, which are highly dynamic adhesive structures that form in immune cells and invasive cancer cells.

Cell volume regulatory ion channels in cell proliferation and cell death.

Abstract: Alterations of cell volume are key events during both cell proliferation and apoptotic cell death. Cell proliferation eventually requires an increase of cell volume, and apoptosis is typically paralleled by cell shrinkage. Alterations of cell volume require the participation of ion transport across the cell membrane, including appropriate activity of Cl(-) and K(+) channels. Cl(-) channels modify cytosolic Cl(-) activity and mediate osmolyte flux, and thus influence cell volume. Most Cl(-) channels allow exit of HCO(3)(-), leading to cytosolic acidification, which in turn inhibits cell proliferation and favors apoptosis. K(+) exit through K(+) channels decreases cytosolic K(+) concentration, which may sensitize the cell for apoptotic cell death. K(+) channel activity further maintains the cell membrane potential, a critical determinant of Ca(2+) entry through Ca(2+) channels. Ca(2+) may, in addition, enter through Ca(2+)-permeable cation channels, which, in some cells, are activated by hyperosmotic shock. Increases of cytosolic Ca(2+) activity may trigger both mechanisms required for cell proliferation and mechanisms, leading to apoptosis. Thereby cell proliferation and apoptosis depend on magnitude and temporal organization of Ca(2+) entry, as well as activity of other signaling pathways. Accordingly, the same ion channels may participate in the stimulation of both cell proliferation and apoptosis. Specific ion channel blockers may thus abrogate both cellular mechanisms, depending on cell type and condition.

Quantification of F2-isoprostanes in biological fluids and tissues as a measure of oxidant stress.

Abstract: Oxidant stress has been implicated in a wide variety of disease processes. One method to quantify oxidative injury is to measure lipid peroxidation. Quantification of a group of prostaglandin F(2)-like compounds derived from the nonezymatic oxidation of arachidonic acid, termed the F(2)-isoprostanes (F(2)-IsoPs), provides an accurate assessment of oxidative stress both in vitro and in vivo. In fact, in a recent National Institutes of Health-sponsored independent study, F(2)-IsoPs were shown to be the most reliable index of in vivo oxidant stress when compared against other well-known biomarkers. This article summarizes current methodology used to quantify these molecules. Our laboratory's method to measure F(2)-IsoPs in biological fluids and tissues using gas chromatography-mass spectrometry is detailed herein. In addition, other mass spectrometric approaches, as well as immunological methods to measure these compounds, are discussed. Finally, the utility of these molecules as in vivo biomarkers of oxidative stress is summarized.

Glycerophospholipid identification and quantitation by electrospray ionization mass spectrometry.

Abstract: Glycerophospholipids are the structural building blocks of the cellular membrane. In addition to creating a protective barrier around the cell, lipids are precursors of intracellular signaling molecules that modulate membrane trafficking and are involved in transmembrane signal transduction. Phospholipids are also increasingly recognized as important participants in the regulation and control of cellular function and disease. Analysis and characterization of lipid species by mass spectrometry (MS) have evolved and advanced with improvements in instrumentation and technology. Key advances, including the development of “soft” ionization techniques for MS such as electrospray ionization (ESI), matrix‐assisted laser desorption/ionization (MALDI), and tandem mass spectrometry (MS/MS), have facilitated the analysis of complex lipid mixtures by overcoming the earlier limitations. ESI‐MS has become the technique of choice for the analysis of multi‐component mixtures of lipids from biological samples due to its exceptional sensitivity and capacity for high throughput. This chapter covers qualitative and quantitative MS methods used for the elucidation of glycerophospholipid identity and quantity in cell or tissue extracts. Sections are included on the extraction, MS analysis, and data analysis of glycerophospholipids and polyphosphoinositides.

Detection and quantitation of eicosanoids via high performance liquid chromatography-electrospray ionization-mass spectrometry.

Abstract: Eicosanoids constitute a large class of biologically active arachidonic acid (AA) metabolites that play important roles in numerous physiological processes. Eicosanoids are produced by several distinct routes, including the cyclooxygenase, lipoxygenase, and P450 enzymatic pathways, as well as by nonenzymatic processes. In order to completely understand the eicosanoid response of a cell or tissue to a given stimulus, measuring the complete profile of eicosanoids produced is important. Since the eicosanoids are products of a single species, AA, and represent, for the most part, the addition of various oxygen species, the hundreds of eicosanoids have very similar structures, chemistries, and physical properties. The identification and quantitation of all eicosanoids in a single biological sample are a challenging task, one that high-performance liquid chromatography-mass spectrometry (LC-MS) is well suited to handle. We have developed a LC-MS/MS procedure for isolating, identifying, and quantitating a broad spectrum of eicosanoids in a single biological sample. We currently can measure over 60 eicosanoids in a 16-min LC-MS/MS analysis. Our method employs stable isotope dilution internal standards to quantitate these specific eicosanoids. In the course of setting up the LC-MS system, we have established a library that includes relative chromatographic retention times and tandem mass spectrometry data for the most common eicosanoids. This library is available to the scientific community on the website www.lipidmaps.org.

Reconstitution of yeast translation initiation

Abstract: To facilitate the mechanistic dissection of eukaryotic translation initiation we have reconstituted the steps of this process using purified Saccharomyces cerevisiae components. This system provides a bridge between biochemical studies in vitro and powerful yeast genetic techniques, and complements existing reconstituted mammalian translation systems (Benne and Hershey, 1978; Pestova and Hellen, 2000; Pestova et al., 1998; Trachsel et al., 1977). The following describes methods for synthesizing and purifying the components of the yeast initiation system and assays useful for its characterization.

Measuring distances by pulsed dipolar ESR spectroscopy: spin-labeled histidine kinases.

Abstract: Applications of dipolar ESR spectroscopy to structural biology are rapidly expanding, and it has become a useful method that is aimed at resolving protein structure and functional mechanisms. The method of pulsed dipolar ESR spectroscopy (PDS) is outlined in the first half of the chapter, and it illustrates the simplicity and potential of this developing technology with applications to various biological systems. A more detailed description is presented of the implementation of PDS to reconstruct the ternary structure of a large dimeric protein complex from Thermotoga maritima , formed by the histidine kinase CheA and the coupling protein CheW. This protein complex is a building block of an extensive array composed of coupled supramolecular structures assembled from CheA/CheW proteins and transmembrane signaling chemoreceptors, which make up a sensor that is key to controlling the motility in bacterial chemotaxis. The reconstruction of the CheA/CheW complex has employed several techniques, including X‐ray crystallography and pulsed ESR. Emphasis is on the role of PDS, which is part of a larger effort to reconstruct the entire signaling complex, including chemoreceptor, by means of PDS structural mapping. In order to precisely establish the mode of coupling of CheW to CheA and to globally map the complex, approximately 70 distances have already been determined and processed into molecular coordinates by readily available methods of distance geometry constraints.

The lipid maps initiative in lipidomics.

Abstract: The Lipid Metabolites and Pathways Strategy (LIPID MAPS) initiative constitutes the first broad scale national exploration of lipidomics and is supported by a U.S. National Institute of General Medical Sciences Large Scale Collaborative "Glue" Grant. The emerging field of lipidomics faces many obstacles to become a true systems biology approach on par with the other "omics" disciplines. With a goal to overcome these hurdles, LIPID MAPS has been developing the necessary infrastructure and techniques to ensure success. This review introduces a few of the challenges and solutions implemented by LIPID MAPS. Among these solutions is the new comprehensive classification system for lipids, along with a recommended nomenclature and structural drawing representation. This classification system was developed by the International Lipids Classification and Nomenclature Committee (ILCNC) in collaboration with LIPID MAPS and representatives from Europe and Asia. The latest changes implemented by the committee are summarized. In addition, we discuss the adoption of mass spectrometry (MS) as the instrumental platform to investigate lipidomics. This platform has the versatility to quantify known individual lipid molecular species and search for novel lipids affecting biological systems.

Transient receptor potential channels in mechanosensing and cell volume regulation.

Abstract: Transient receptor potential (TRP) channels are unique cellular sensors responding to a wide variety of extra‐ and intracellular signals, including mechanical and osmotic stress. In recent years, TRP channels from multiple subfamilies have been added to the list of mechano‐ and/or osmosensitive channels, and it is becoming increasingly apparent that Ca2+ influx via TRP channels plays a crucial role in the response to mechanical and osmotic perturbations in a wide range of cell types. Although the events translating mechanical and osmotic stimuli into regulation of TRP channels are still incompletely understood, the specific mechanisms employed vary between different TRP isoforms, and probably include changes in the tension and/or curvature of the lipid bilayer, changes in the cortical cytoskeleton, and signaling events such as lipid metabolism and protein phosphorylation/dephosphorylation. This chapter describes candidate mechanosensitive channels from mammalian TRP subfamilies, discusses inherent and technical issues potentially confounding evaluation of mechano‐ and/or osmosensitivity, and presents methods relevant to the study of TRP channel regulation by mechanical and osmotic stimuli and involvement in cell volume regulation.

Phenotype of the taurine transporter knockout mouse.

Abstract: This chapter reports present knowledge on the properties of mice with disrupted gene coding for the taurine transporter (taut-/- mice). Study of those mice unraveled some of the roles of taurine and its membrane transport for the development and maintenance of normal organ functions and morphology. When compared with wild-type controls, taut-/- mice have decreased taurine levels in skeletal and heart muscle by about 98%, in brain, kidney, plasma, and retina by 80 to 90%, and in liver by about 70%. taut-/- mice exhibit a lower body mass as well as a strongly reduced exercise capacity compared with taut+/- and wild-type mice. Furthermore, taut-/- mice show a variety of pathological features, for example, subtle derangement of renal osmoregulation, changes in neuroreceptor expression, and loss of long-term potentiation in the striatum, and they develop clinically relevant age-dependent disorders, for example, visual, auditory, and olfactory dysfunctions, unspecific hepatitis, and liver fibrosis. Taurine-deficient animal models such as acutely dietary-manipulated foxes and cats, pharmacologically induced taurine-deficient rats, and taurine transporter knockout mouse are powerful tools allowing identification of the mechanisms and complexities of diseases mediated by impaired taurine transport and taurine depletion (Chapman et al., 1993; Heller-Stilb et al., 2002; Huxtable, 1992; Lake, 1993; Moise et al., 1991; Novotny et al., 1991; Pion et al., 1987; Timbrell et al., 1995; Warskulat et al., 2004, 2006b). Taurine, which is the most abundant amino acid in many tissues, is normally found in intracellular concentrations of 10 to 70 mmol/kg in mammalian heart, brain, skeletal muscle, liver, and retina (Chapman et al., 1993; Green et al., 1991; Huxable, 1992; Timbrell et al., 1995). These high taurine levels are maintained by an ubiquitous expression of Na(+)-dependent taurine transporter (TAUT) in the plasma membrane (Burg, 1995; Kwon and Handler, 1995; Lang et al., 1998; Liu et al., 1992; Ramamoorthy et al., 1994; Schloss et al., 1994; Smith et al., 1992; Uchida et al., 1992; Vinnakota et al., 1997; Yancey et al., 1975). Taurine is not incorporated into proteins. It is involved in cell volume regulation, neuromodulation, antioxidant defense, protein stabilization, stress responses, and via formation of taurine-chloramine in immunomodulation (Chapman et al., 1993; Green et al., 1991; Huxtable, 1992; Timbrell et al., 1995). On the basis of its functions, taurine may protect cells against various types of injury (Chapman et al., 1993; Green et al., 1991; Huxtable, 1992; Kurz et al., 1998; Park et al., 1995; Stapleton et al., 1998; Timbrell et al., 1995; Welch and Brown, 1996; Wettstein and Haussinger, 1997). In order to examine the multiple taurine functions, murine models have several intrinsic advantages for in vivo research compared to other animal models, including lower cost, maintenance, and rapid reproduction rate. Further, experimental reagents for cellular and molecular studies are widely available for the mouse. In particular, mice can be easily genetically manipulated by making transgene and knockout mice. This chapter focuses on the phenotype of the TAUT-deficient murine model (taut-/-; Heller-Stilb et al., 2002), which may help researchers elucidate the diverse roles of taurine in development and maintenance of normal organ functions and morphology.

Regulation of Hypoxia‐Inducible Factors During Inflammation

Abstract: The microenvironment of inflamed and injured tissue is characterized by low levels of oxygen and glucose and high levels of inflammatory cytokines, reactive oxygen, and nitrogen species and metabolites. The transcription factor complex hypoxia-inducible factor (HIF)-1 is regulated by hypoxia as well as by a broad variety of inflammatory mediators. In cells of the innate and adaptive immune system, HIF-1 is upregulated by bacterial and viral compounds, even under normoxic conditions. This upregulation prepares these cells to migrate to and to function in hypoxic and inflamed tissues. Once extravasated from the vasculature, the activity of cells is further enhanced by stimulation of HIF-1 by proinflammatory cytokines like interleukin (IL)-1beta (beta) and tumor necrosis factor (TNF) alpha (alpha), and locally expressed tissue factors. Crosstalk between hypoxic induction of HIF-1 and other signaling pathways activated by inflammation ensures a cell type-specific and stimulus-adequate cellular response. Prolonged activation of HIF-1 under conditions of inflammation, however, may contribute to the survival of damaged tissue and cells, thus promoting the development of tumors.

Plant gene networks in osmotic stress response: from genes to regulatory networks.

Abstract: Because of their sessile nature, plants grown in a dynamic climate have evolved a range of adaptations that enable them to survive in various environmental stress conditions during growth and development. Plants respond to environmental stresses at both cellular and molecular levels by altering the expression of many genes via a complexity of signaling pathways. These pathways begin with signal perception and end with the expression of stress-responsive target genes. Ultimately, the selective upregulation of target genes leads to the alteration of physiological response so as to confer tolerance of the stress. In the signal transduction network, various regulatory and functional proteins function collectively to ensure survival of the plants. This chapter summarizes the methodology used to dissect gene regulatory networks involved in the response to osmotic stresses, such as drought and high salinity.

Extraction and analysis of sterols in biological matrices by high performance liquid chromatography electrospray ionization mass spectrometry

Abstract: We describe the development of a high performance liquid chromatography mass spectrometry (HPLC-MS) method that allows the identification and quantitation of sterols in mammalian cells and tissues. Bulk lipids are extracted from biological samples by a modified Bligh/Dyer procedure in the presence of eight deuterated sterol standards to allow subsequent quantitation and determination of extraction efficiency. Sterols and other lipids are resolved by HPLC on a reverse-phase C18 column using a binary gradient of methanol and water, both containing 5mM ammonium acetate. Sterol identification is performed using an Applied Biosystems (Foster City, CA) 4000 QTRAP mass spectrometer equipped with a TurboV electrospray ionization source and operated in the positive (+) selected reaction monitoring (SRM) mode. The total run time of the analysis is 30 min. Sterols are quantitated by comparison of the areas under the elution curves derived from the detection of endogenous compounds and isotopically labeled standards. The sensitivity of the method for sterol detection ranges between 10 and 2000 fmol on-column. Cultured RAW 264.7 mouse macrophages contain many different sterols, including the liver X receptor (LXR) ligand 24,25-epoxycholesterol. Tissues such as mouse brain also contain large numbers of sterols, including 24(s)-hydroxycholesterol, which is involved in cholesterol turnover in the brain. The extraction procedure described is flexible and can be tailored to sample type or information sought. The instrumental analysis method is similarly adaptable and offers high selectivity and sensitivity.

Comparative genomic and protein sequence analyses of a complex system controlling bacterial chemotaxis.

Abstract: Molecular machinery governing bacterial chemotaxis consists of the CheA–CheY two‐component system, an array of specialized chemoreceptors, and several auxiliary proteins. It has been studied extensively in Escherichia coli and, to a significantly lesser extent, in several other microbial species. Emerging evidence suggests that homologous signal transduction pathways regulate not only chemotaxis, but several other cellular functions in various bacterial species. The availability of genome sequence data for hundreds of organisms enables productive study of this system using comparative genomics and protein sequence analysis. This chapter describes advances in genomics of the chemotaxis signal transduction system, provides information on relevant bioinformatics tools and resources, and outlines approaches toward developing a computational framework for predicting important biological functions from raw genomic data based on available experimental evidence.

Structure-specific, quantitative methods for analysis of sphingolipids by liquid chromatography-tandem mass spectrometry: "inside-out" sphingolipidomics.

Abstract: Due to the large number of highly bioactive subspecies, elucidation of the roles of sphingolipids in cell structure, signaling, and function is beginning to require that one perform structure‐specific and quantitative (i.e., “sphingolipidomic”) analysis of all individual subspecies, or at least of those are relevant to the biologic system of interest. As part of the LIPID MAPS Consortium, methods have been developed and validated for the extraction, liquid chromatographic (LC) separation, and identification and quantitation by electrospray ionization (ESI), tandem mass spectrometry (MS/MS) using an internal standard cocktail that encompasses the signaling metabolites (e.g., ceramides, ceramide 1‐phosphates, sphingoid bases, and sphingoid base 1‐phosphates) as well as more complex species (sphingomyelins, mono‐ and di‐hexosylceramides). The number of species that can be analyzed is growing rapidly with the addition of sulfatides and other complex sphingolipids as more internal standards become available. This review describes these methods as well as summarizes others from the published literature. Sphingolipids are an amazingly complex family of compounds that are found in all eukaryotes as well as some prokaryotes and viruses. The size of the sphingolipidome (i.e., all of the individual molecular species of sphingolipids) is not known, but must be immense considering mammals have over 400 headgroup variants (for a listing, see http://www.sphingomap.org ), each of which is comprised of at least a few—and, in some cases, dozens—of lipid backbones. No methods have yet been developed that can encompass so many different compounds in a structurally specific and quantitative manner. Nonetheless, it is possible to analyze useful subsets of the sphingolipidome, such as the backbone sphingolipids involved in signaling (sphingoid bases, sphingoid base 1‐phosphates, ceramides, and ceramide 1‐phosphates) and metabolites at important branchpoints, such as the partitioning of ceramide into sphingomyelins, glucosylceramides, galactosylceramides, and ceramide 1‐phosphate versus turnover to the backbone sphingoid base. This review describes methodology that has been developed as part of the LIPID MAPS Consortium ( www.lipidmaps.org ) as well as other methods that can be used for sphingolipidomic analysis to the extent that such is currently feasible. The focus of this review is primarily mammalian sphingolipids; hence, if readers are interested in methods to study other organisms, they should consult the excellent review by Stephen Levery in another volume of Methods in Enzymology (Levery, 2005), which covers additional species found in plants, fungi, and other organisms. It should be noted from the start that although many analytical challenges remain in the development of methods to analyze the full “sphingolipidome,” the major impediment to progress is the limited availability of reliable internal standards for most of the compounds of interest. Because it is an intrinsic feature of mass spectrometry that ion yields tend to vary considerably among different compounds, sources, methods, and instruments, an analysis that purports to be quantitative will not be conclusive unless enough internal standards have been added to correct for these variables. Ideally, there should be some way of standardizing every compound in the unknown mixture; however, that is difficult, if not impossible, to do because the compounds are not available, and the inclusion of so many internal standards generates a spectrum that may be too complex to interpret. Therefore, a few representative internal standards are usually added, and any known differences in the ion yields of the analytes of interest versus the spiked standard are factored into the calculations. Identification of appropriate internal standards has been a major focus of the LIPID MAPS Consortium, and the methods described in this review are based on the development of a certified (i.e., compositionally and quantitatively defined by the supplier) internal standard cocktail that is now commercially available (Avanti Polar Lipids, Alabaster, AL). For practical and philosophical reasons, an internal standard cocktail was chosen over the process of an investigator adding individual standards for only the analytes of interest. On the practical level, addition of a single cocktail minimizes pipetting errors as well as keeping track of whether each internal standard is still usable (e.g., has it degraded while in solution?). Philosophically, the internal standard cocktail was chosen because an underlying premise of systems analysis asserts that, due to the high relevancy of unexpected interrelationships involving more distant components, one can only understand a biological system when factors outside the primary focus of the experiment have also been examined. Indeed, the first payoffs of “omics” and systems approaches involve the discoveries of interesting compounds in unexpected places when a “sphingolipidomic” analytical method was being used as routine practice instead of a simpler method that would have only measured the compound initially thought to be important (Zheng et al., 2006). Thus, routine addition of a broad internal standard cocktail at the outset of any analysis maximizes the opportunity for such discoveries, both at the time the original measurements are made and when one decides to return to the samples later, which can fortunately be done for many sphingolipids because they remain relatively stable in storage.

Transient kinetics, fluorescence, and FRET in studies of initiation of translation in bacteria.

Abstract: Initiation of mRNA translation in prokaryotes requires the small ribosomal subunit (30S), initiator fMet-tRNAfMet, three initiation factors, IF1, IF2, and IF3, and the large ribosomal subunit (50S) During initiation, the 30S subunit, in a complex with IF3, binds mRNA, IF1, IF2·GTP, and fMet-tRNAfMet to form a 30S initiation complex which then recruits the 50S subunit to yield a 70S initiation complex, while the initiation factors are released Here we describe a transient kinetic approach to study the timing of elemental steps of 30S initiation complex formation, 50S subunit joining, and the dissociation of the initiation factors from the 70S initiation complex Labeling of ribosomal subunits, fMet-tRNAfMet, mRNA, and initiation factors with fluorescent reporter groups allows for the direct observation of the formation or dissociation of complexes by monitoring changes in the fluorescence of single dyes or fluorescence resonance energy transfer (FRET) between two fluorophores Subunit joining was monitored by light scattering or by FRET between dyes attached to the ribosomal subunits The kinetics of chemical steps, that is, GTP hydrolysis by IF2 and peptide bond formation following the binding of aminoacyl-tRNA to the 70S initiation complex, were measured by the quench-flow technique The methods described here are based on results obtained with initiation components from Escherichia coli but can be adopted for mechanistic studies of initiation in other prokaryotic or eukaryotic systems

In vivo measurement by FRET of pathway activity in bacterial chemotaxis.

Abstract: The two‐component pathway in Escherichia coli chemotaxis has become a paradigm for bacterial signal processing. Genetics and biochemistry of the pathway as well as physiological responses have been studied in detail. Despite its relative simplicity, the chemotaxis pathway is renowned for its ability to amplify and integrate weak signals and for its robustness against various kinds of perturbations. All this information inspired multiple attempts at mathematical analysis and computer modeling, but a quantitative understanding of the pathway was hampered by our inability to follow the signal processing in vivo . To address this problem, we developed assays based on fluorescence resonance energy transfer (FRET) and bioluminescence resonance energy transfer (BRET) that enabled us to monitor activity‐dependent protein interactions in real time directly in living cells. Here, we describe quantitative applications of these assays in cell populations and on a single‐cell level to study the interaction of the phosphorylated response regulator CheY with its phosphatase CheZ. Since this interaction defines the rate of CheY dephosphorylation, which at steady state equals the rate of CheY phosphorylation, it can be used to characterize intracellular kinase activity and thus to analyze properties of the chemotaxis signaling network.

Mass spectrometric identification and characterization of RNA-modifying enzymes.

Abstract: Posttranscriptional modifications are characteristic structural features of RNA molecules. To study the functional roles played by RNA modifications, it is necessary to identify the genes and enzymes that are responsible for their biosynthesis. Many uncharacterized genes for RNA modifications still remain buried in the genomes of model organisms. We describe here a systematic genomewide screening method that uses a reverse genetic approach combined with mass spectrometry, which we have named "ribonucleome analysis," to identify uncharacterized genes that are involved in generating RNA modifications.

Shotgun Lipidomics by Tandem Mass Spectrometry under Data‐Dependent Acquisition Control

Abstract: Data‐dependent acquisition of full MS/MS spectra from all detectable (or, alternatively, preselected) lipid precursors produces a rich data set, whose subsequent interpretation by the dedicated software LipidInspector emulates the simultaneous acquisition of an unlimited number of precursor and neutral loss scans in a single analysis. Using logical operations, emulated scans can be combined into highly specific data interpretation routines (termed Boolean scans) enabling in‐depth structural characterization of fragmented precursors. Alternatively, a small number of preselected precursors can be fragmented regardless of their relative intensities in survey spectra, hence emulating selected reaction monitoring (SRM) analysis that attains both high detection specificity and sensitivity. Although the data‐dependent acquisition approach is, in principle, cross‐platform, it benefits from the high mass resolution capacity of hybrid tandem mass spectrometers with time‐of‐flight and, especially, Fourier transform or Orbitrap analyzers.

Assembly and analysis of eukaryotic translation initiation complexes.

Abstract: The canonical initiation process is the most complex aspect of translation in eukaryotes. It involves the coordinated interactions of at least 11 eukaryotic initiation factors, 40S and 60S ribosomal subunits, mRNA, and aminoacylated initiator tRNA (Met-tRNA(i)(Met)), as well as binding and hydrolysis of GTP and ATP. The factor requirements for many individual steps in this process, including scanning, initiation codon recognition, and ribosomal subunit joining, have until recently been obscure. We established the factor requirements for these steps by reconstituting the initiation process in vitro from individual purified components of the translation apparatus and developed approaches to explain the mechanism of individual steps and the roles of individual factors and to characterize the structure of initiation complexes. Here we describe protocols for the purification of native initiation factors and for expression and purification of active recombinant forms of all single subunit initiation factors, for the reconstitution of the initiation process, and for determination of the position of ribosomal complexes on mRNA by primer extension inhibition ("toe printing"). We also describe protocols for site-directed ultraviolet (UV) cross-linking to determine the interactions of individual nucleotides in mRNA with components of the initiation complex and for directed hydroxyl radical probing to determine the position of initiation factors on the ribosome.

lambda-Red genetic engineering in Salmonella enterica serovar Typhimurium

Abstract: The use of the recombination system from bacteriophage lambda, lambda-Red, allows for PCR-generated fragments to be targeted to specific chromosomal locations in sequenced genomes. A minimal region of homology of 30 to 50 bases flanking the fragment to be inserted is all that is required for targeted mutagenesis. Procedures for creating specific insertions, deletions, and site-directed changes are described.

Detection of enzymatic activity of transfer RNA modification enzymes using radiolabeled tRNA substrates.

Abstract: The presence of modified ribonucleotides derived from adenosine, guanosine, cytidine, and uridine is a hallmark of almost all cellular RNA, and especially tRNA The objective of this chapter is to describe a few simple methods that can be used to identify the presence or absence of a modified nucleotide in tRNA and to reveal the enzymatic activity of particular tRNA-modifying enzymes in vitro and in vivo The procedures are based on analysis of prelabeled or postlabeled nucleotides (mainly with [(32)P] but also with [(35)S], [(14)C] or [(3)H]) generated after complete digestion with selected nucleases of modified tRNA isolated from cells or incubated in vitro with modifying enzyme(s) Nucleotides of the tRNA digests are separated by two-dimensional (2D) thin-layer chromatography on cellulose plates (TLC), which allows establishment of base composition and identification of the nearest neighbor nucleotide of a given modified nucleotide in the tRNA sequence This chapter provides useful maps for identification of migration of approximately 70 modified nucleotides on TLC plates by use of two different chromatographic systems The methods require only a few micrograms of purified tRNA and can be run at low cost in any laboratory

Liquid chromatography mass spectrometry for quantifying plasma lysophospholipids: potential biomarkers for cancer diagnosis.

Abstract: Cancer is a complex disease with many genetic and epigenetic aberrations that result in development of tumorigenic phenotypes. While many factors contribute to the etiology of cancer, emerging data implicate lysophospholipids acting through specific cell-surface, and potentially intracellular, receptors in acquiring the transformed phenotype propagated during disease. Lysophospholipids bind to and activate specific cell-surface G protein-coupled receptors (GPCRs) that initiate cell growth, proliferation, and survival pathways, and show altered expression in cancer cells. In addition, a number of enzymes that increase lysophospholipid production are elevated in particular cell lineages and cancer patients' cells, whereas in a subset of patients, the enzymes degrading lysophospholipids are decreased. Thus, ideal conditions are established to increase lysophospholipids in the tumor microenvironment. Indeed, ascites from ovarian cancer patients, which reflects both the tumor environment and a tumor-conditioned media, exhibits markedly elevated levels of specific lysophospholipids as well as one of the enzymes involved in production of lysophospholipids: autotaxin (ATX). The potential sources of lysophospholipids in the tumor microenvironment include tumor cells and stroma, such as mesothelial cells, as well as inflammatory cells and platelets activated by the proinflammatory tumor environment. If lysophospholipids diffuse from the tumor microenvironment into the bloodstream and persist, they have the potential to serve as early diagnostic markers as well as potential monitors of tumor response to therapy. Many scientific and technical challenges need to be resolved to determine whether lysophospholipids or the enzymes producing lysophospholipids alone or in combination with other markers have the potential to contribute to early diagnosis. Breast cancer is the most frequently diagnosed cancer among women. Mammography is associated with morbidity and has a high false positive and false negative rate. Thus, there is a critical need for biomarkers that can contribute to reduced false positive and false negative diagnoses, and to identify, stage, and/or predict prognosis of this disease to improve patient management. Here we describe a technical approach that can be applied to human blood plasma to measure the concentration of growth factor-like lysophospholipids contained in circulation. Using liquid chromatography mass spectrometry (LC/MS/MS), we quantified the amount of lysophosphatidic acid (16:0, 18:0, 18:1, 18:2, and 20:4), lysophosphatidylinositol (18:0), lysophosphatidylserine (18:1), lysophosphatidylcholine (16:0, 18:0, 18:1, 18:2, and 20:4), sphingosine-1-phosphate, and sphingosylphosphorylcholine species from human female plasma samples with malignant, benign, or no breast tumor present. Other methods described here include handling patient blood samples, lipid extraction, and factors that affect lysophospholipid production and loss during sample handling.

Application of the transfer model to understand how naturally occurring osmolytes affect protein stability.

Abstract: A primary thermodynamic goal in protein biochemistry is to attain a predictive understanding of the energetic changes responsible for solvent‐induced folding and unfolding. This chapter demonstrates the use of Tanford's transfer model to predict solvent‐dependent cooperative protein folding/unfolding free energy changes ( m values). This approach provides a thermodynamic description of these free energy changes in terms of individual contributions from the peptide backbone and residue side chains. The quantitative success of the transfer model has been hindered for many years because of unresolved issues involving proper measurement of the group transfer‐free energies of amino acid side chains and the peptide backbone unit. This chapter demonstrates what is necessary to design experiments properly so that reliable values of group transfer‐free energies are obtainable. It then demonstrates how to derive a prediction of the m value for the description of protein folding/unfolding cooperativity and that the calculated values using the transfer model agree quite well with experimentally measured values.