Showing papers in "Methods in Enzymology in 2008"

Use of dynasore, the small molecule inhibitor of dynamin, in the regulation of endocytosis.

Abstract: The large GTPase dynamin is essential for clathrin-dependent coated-vesicle formation. Dynasore is a cell-permeable small molecule that inhibits the GTPase activity of dynamin1, dynamin2 and Drp1, the mitochondrial dynamin. Dynasore was discovered in a screen of approximately 16,000 compounds for inhibitors of the dynamin2 GTPase. Dynasore is a noncompetitive inhibitor of dynamin GTPase activity and blocks dynamin-dependent endocytosis in cells, including neurons. It is fast acting (seconds) and its inhibitory effect in cells can be reversed by washout. Here we present a detailed synthesis protocol for dynasore, and describe a series of experiments used to analyze the inhibitory effects of dynasore on dynamin in vitro and to study the effects of dynasore on endocytosis in cells.

Nitrite and Nitrate Measurement by Griess Reagent in Human Plasma: Evaluation of Interferences and Standardization

Abstract: Nitrite and nitrate represent the final products of nitric oxide (NO) oxidation pathways, and their hematic concentrations are frequently assessed as an index of systemic NO production However, their intake with food can influence their levels Nitrite and nitrate could have a role by producing NO, because nitrite can release NO after reaction with deoxyhemoglobin and dietary nitrate can be reduced substantially to nitrite by commensal bacteria in the oral cavity Different methods have been applied for nitrite/nitrate detection, with the most commonly used being the spectrophotometric assay based on the Griess reagent However, a reference methodology for these determinations is still missing and many possible interferences have been reported This chapter assesses how different experimental conditions can influence the results when detecting nitrite and nitrate in human plasma by the Griess assay and provides a simple method characterized by high reproducibility and minimized interferences by plasma constituents

An optimized three-dimensional in vitro model for the analysis of angiogenesis.

Abstract: Angiogenesis is the formation of new blood vessels from the existing vasculature. It is a multistage process in which activated endothelial cells (EC) degrade basement membrane, sprout from the parent vessel, migrate, proliferate, align, undergo tube formation, and eventually branch and anastomose with adjacent vessels. Here we describe a three-dimensional in vitro assay that reproduces each of these steps. Human umbilical vein endothelial cells (HUVEC) are cultured on microcarrier beads, which are then embedded in a fibrin gel. Fibroblasts cultured on top of the gel provide factors that synergize with bFGF and VEGF to promote optimal sprouting and tube formation. Sprouts appear around day 2, lumen formation begins at day 4, and at day 10 an extensive anastomosing network of capillary-like tubes is established. The EC express a similar complement of genes as angiogenic EC in vivo and undergo identical morphologic changes during tube formation. This model, therefore, recapitulates in vivo angiogenesis in several critical aspects and provides a system that is easy to manipulate genetically, can be visualized in real time, and allows for easy purification of angiogenic EC for downstream analysis.

Messenger RNA half-life measurements in mammalian cells.

Abstract: The recognition of the importance of mRNA turnover in regulating eukaryotic gene expression has mandated the development of reliable, rigorous, and "user-friendly" methods to accurately measure changes in mRNA stability in mammalian cells. Frequently, mRNA stability is studied indirectly by analyzing the steady-state level of mRNA in the cytoplasm; in this case, changes in mRNA abundance are assumed to reflect only mRNA degradation, an assumption that is not always correct. Although direct measurements of mRNA decay rate can be performed with kinetic labeling techniques and transcriptional inhibitors, these techniques often introduce significant changes in cell physiology. Furthermore, many critical mechanistic issues as to deadenylation kinetics, decay intermediates, and precursor-product relationships cannot be readily addressed by these methods. In light of these concerns, we have previously reported transcriptional pulsing methods based on the c-fos serum-inducible promoter and the tetracycline-regulated (Tet-off) promoter systems to better explain mechanisms of mRNA turnover in mammalian cells. In this chapter, we describe and discuss in detail different protocols that use these two transcriptional pulsing methods. The information described here also provides guidelines to help develop optimal protocols for studying mammalian mRNA turnover in different cell types under a wide range of physiologic conditions.

In vitro three dimensional collagen matrix models of endothelial lumen formation during vasculogenesis and angiogenesis.

Abstract: Discovery and comprehension of detailed molecular signaling pathways underlying endothelial vascular morphogenic events including endothelial lumen formation are key steps in understanding their roles during embryonic development, as well as during various disease states. Studies that used in vitro three-dimensional (3D) matrix endothelial cell morphogenic assay models, in conjunction with in vivo studies, have been essential to identifying molecules and explaining their related signaling pathways that regulate endothelial cell morphogenesis. We present methods to study molecular mechanisms controlling EC lumen formation in 3D collagen matrices. In vitro models representing vasculogenesis and angiogenesis, whereby EC lumen formation and tube morphogenesis readily occur, are described. We also detail different methods of gene manipulation in ECs and their application to analyze critical signaling events regulating EC lumen formation.

Biochemical methods to monitor autophagy-related processes in yeast.

Abstract: An increasing number of reports have elucidated the importance of macroautophagy in cell physiology and pathology. Macroautophagy occurs at a basal level and participates in the turnover of cytoplasmic constituents including long-lived proteins to maintain cellular homeostasis, but it also serves as an adaptive response to protect cells from various intra- or extracellular stresses. In addition, macroautophagy plays a role in development and aging and acts to protect against cancer, microbial invasion, and neurodegeneration. The machinery involved in carrying out this process, the autophagy-related (Atg) proteins were identified and characterized in various fungal systems, in particular because of the powerful tools available for genetic manipulation and the relative abundance of good biochemical assays in these model organisms. The analysis of these Atg proteins has allowed us to begin to understand the molecular mechanism of this process. Furthermore, many of the autophagy genes are functionally conserved in higher eukaryotes, including mammals, allowing the findings in fungi to be applied to other systems. Here, we discuss three biochemical methods to measure autophagy-related activities and to examine individual steps of the corresponding process. These methods rely on the detection of different modification states of certain marker proteins. Processing of the precursor form of the resident vacuolar hydrolase aminopeptidase I (Ape1) is applicable to fungi, whereas cleavage of the GFP-Atg8 and Pex14-GFP chimeras can be used in a wide array of systems.

Chemistry of Nitric Oxide and Related Species

Abstract: Nitric oxide (NO) has essential roles in a remarkable number of diverse biological processes. The reactivity of NO depends upon its physical properties, such as its small size, high diffusion rate, and lipophilicity (resulting in its accumulation in hydrophobic regions), and also on its facile but selective chemical reactivity toward a variety of cellular targets. NO also undergoes reactions with oxygen, superoxide ions, and reducing agents to give products that themselves show distinctive reactivity toward particular targets, sometimes with the manifestation of toxic effects, such as nitrosative stress. These include nitroxyl (HNO), the oxides NO2/N2O4, and N2O3, peroxynitrite, and S-nitrosothiols (RSNO). HNO is attracting considerable attention due to its pharmacological properties, which appear to be distinct from those of NO, and that may be significant in the treatment of heart failure.

The quantitative Pho8Delta60 assay of nonspecific autophagy.

Abstract: The measurement of autophagic flux is critical in understanding the regulation of autophagy. The Pho8Delta60 assay employs a very sensitive enzymatic assay that provides a high signal-to-noise ratio and allows for precise quantification of autophagic flow in yeast. Pho8, alkaline phosphatase, is a resident vacuolar enzyme that is delivered to the vacuole membrane through a portion of the secretory pathway. The assay utilizes a genetically engineered version of Pho8 that lacks the N-terminal transmembrane domain that allows for translocation into the endoplasmic reticulum. Accordingly, Pho8Delta60 remains in the cytosol and is delivered to the vacuole only through autophagy. Once in the vacuole lumen, the C-terminal propeptide is proteolytically removed, which results in activation. Thus, the alkaline phosphatase activity reflects the amount of the cytosol delivered to the vacuole through nonspecific autophagy.

Chapter 2. Chick embryo chorioallantoic membrane models to quantify angiogenesis induced by inflammatory and tumor cells or purified effector molecules.

Abstract: Angiogenesis plays a critical role in many normal physiological processes as well as in tumor neovascularization associated with cancer progression. Among various animal model systems designed to study the mechanisms underlying angiogenesis, chick embryo models have been useful tools in analyzing the angiogenic potential of purified factors and intact cells. The chorioallantoic membrane (CAM), a specialized, highly vascularized tissue of the avian embryo, serves as an ideal indicator of the anti- or pro-angiogenic properties of test compounds. In this chapter, we describe a number basic chick embryo CAM models of angiogenesis. A special emphasis is on the model system employing three-dimensional (3D) collagen grafts planted on the CAM, referred herein as onplants. This collagen onplant model allows for unambiguous quantification of angiogenesis and also for in-depth analysis of the cellular and biochemical mechanisms by which specific cells of different origin or purified effector molecules induce or inhibit the angiogenic process.

Methods for distinguishing apoptotic from necrotic cells and measuring their clearance.

Abstract: Three major morphological types of cell death can be distinguished: type I (apoptotic cell death), type II (autophagic cell death), and type III (necrotic cell death). Details of the pathways of apoptotic and autophagic cell death have been described, and distinct biochemical markers have been identified. However, no distinct surface or biochemical markers of necrotic cell death have been identified yet, and only negative markers are available. These include absence of apoptotic parameters (caspase activation, cytochrome c release, and oligonucleosomal DNA fragmentation) and differential kinetics of cell death markers (phosphatidylserine exposure and cell membrane permeabilization). Moreover, a confounding factor is that apoptotic cells in the absence of phagocytosis proceed to secondary necrosis, which has many morphological features of primary necrotic cells. Secondary necrotic cells have already gone through an apoptotic stage, and so it is generally advisable in cell death research to perform time kinetics of cell death parameters. This chapter concentrates on methods that can distinguish apoptosis from necrosis on three different levels (morphological, biochemical, and analysis of cell-cell interactions) and emphasizes that only a combination of several techniques can correctly characterize cell death type. First, we describe analysis of apoptotic versus necrotic morphology by time-lapse microscopy, flow fluorocytometry, and transmission electron microscopy. We also discuss various biochemical techniques for analysis of cell surface markers (phosphatidylserine exposure versus cell permeability by flow fluorocytometry), cellular markers such as DNA fragmentation (flow fluorocytometry), caspase activation, Bid cleavage, and cytochrome c release (Western blotting). Next, we describe how primary and secondary necrotic cells can be distinguished by analysis of supernatant for caspases, HMGB1, and release of cytokeratin 18. Finally, we discuss cell-cell interactions during cell death and describe a quantitative method for examining dead cell clearance by flow fluorocytometry. A selection of techniques that can be used to study internalization mechanisms used by phagocytes to engulf dying cells is also presented, such as scanning and transmission electron microscopy and fluorescence microscopy.

The chemistry of peroxynitrite: implications for biological activity.

Abstract: In biological systems, nitric oxide (NO) combines rapidly with superoxide (O2-) to form peroxynitrite ion (ONOO-), a substance that has been implicated as a culprit in many diseases. Peroxynitrite ion is essentially stable, but its protonated form (ONOOH, pKa = 6.5 to 6.8) decomposes rapidly via homolysis of the O-O bond to form about 28% free NO2 and OH radicals. At physiological pH and in the presence of large amounts of bicarbonate, ONOO- reacts with CO2 to produce about 33% NO2 and carbonate ion radicals (CO3-) in the bulk of the solution. The quantitative role of OH/CO3(-) and NO2 radicals during the decomposition of peroxynitrite (ONOOH/ONOO-) under physiological conditions is described in detail. Specifically, the effect of the peroxynitrite dosage rate on the yield and distribution of the final products is demonstrated. By way of an example, the detailed mechanism of nitration of tyrosine, a vital aromatic amino acid, is delineated, showing the difference in the nitration yield between the addition of authentic peroxynitrite and its continuous generation by NO and O2- radicals.

DNA damage response and apoptosis.

Abstract: A number of methods have been developed to examine the morphologic, biochemical, and molecular changes that happen during the DNA damage response that may ultimately lead to death of cells through various mechanisms that include apoptosis. When cells are exposed to ionizing radiation or chemical DNA-damaging agents, double-stranded DNA breaks (DSB) are generated that rapidly result in the phosphorylation of histone variant H2AX. Because phosphorylation of H2AX at Ser 139 correlates well with each DSB, phospho-H2AX is a sensitive marker to used to examine the DNA damage and its repair. Apoptotic cells are characterized on the basis of their reduced DNA content and morphologic changes, including nuclear condensation, which can be detected by flow cytometry (sub-G1 DNA content), trypan blue, or Hoechst staining. The appearance of phosphatidylserine on the plasma membrane with annexin V-fluorochrome conjugates indicates the changes in plasma membrane composition and function. By combining it with propidium iodide staining, this method can also be used to distinguish early versus late apoptotic or necrotic events. The activation of caspases is another well-known biochemical marker of apoptosis. Finally, the Bcl-2 family of proteins and the mitochondria that play a critical role in DNA damage-induced apoptosis can be examined by translocation of Bax and cytochrome c in and out of mitochondria. In this chapter, we discuss the most commonly used techniques used in our laboratory for determining the DNA damage response leading to apoptosis.

The Aortic Ring Model of Angiogenesis

Abstract: Angiogenesis is regulated by a complex cascade of cellular and molecular events. The entire process can be reproduced in vitro by culturing rat or mouse aortic explants in three-dimensional biomatrices under chemically defined conditions. Angiogenesis in this system is driven by endogenous growth factors released by the aorta and its outgrowth. Sprouting endothelial cells closely interact with pericytes, macrophages, and fibroblasts in an orderly sequence of morphogenetic events that recapitulates all stages of angiogenesis. This model can be used to study the basic mechanisms of the angiogenic process and to test the efficacy of proangiogenic or antiangiogenic compounds. Aortic cultures can be evaluated with a range of morphologic and molecular techniques for the study of gene expression. In this chapter we describe basic protocols currently used in our laboratory to prepare, quantify, and analyze this assay.

Real-time and quantitative imaging of mammalian stress granules and processing bodies.

Abstract: Nuclear mRNA domains such as nucleoli, speckles, Cajal bodies, and gems demonstrate that RNA function and morphology are inextricably linked; granular mRNA structures are self-generated in tandem with metabolic activity. Similarly, cytoplasmic compartmentalization of mRNA into mRNP structures such as stress granules (SGs) and processing bodies (PBs) reiterate the link between function and structure; the assembly of SGs and PBs requires mRNA released from disassembling polysomes on translational arrest. SGs contain mRNA still associated with some of the translational machinery, specifically 40S subunits and a subset of translation initiation factors including eIF3, eIF4F, eIF4B, and PABP. PBs also contain mRNA and eIF4E but lack other preinitiation factors and contain instead a number of proteins associated with mRNA decay such as DCP1a, DCP2, hedls/GE-1, p54/RCK. Many other proteins (e.g., argonaute, FAST, RAP-55, TTP) and microRNAs are present in both SGs and PBs, sometimes shepherding specific mRNA transcripts between the translation and decay machineries. Recently, we described markers and methods to visualize SGs and PBs in fixed cells (Kedersha and Anderson, 2007), but understanding the dynamic nature of SGs and PBs requires live cell imaging. This presents unique challenges, because it requires the overexpression of fluorescently tagged SG/PB marker proteins, which can shift the mRNA equilibrium toward SGs or PBs, thus obscuring the result. We describe stably expressed, fluorescently tagged SG and PB markers that exhibit similar behavior to their endogenous counterparts, thus allowing real-time imaging of SGs and PBs.

Analysis of Citrulline, Arginine, and Methylarginines Using High‐Performance Liquid Chromatography

Abstract: Citrulline is a product of arginine degradation by nitric oxide synthase and is a precursor for arginine synthesis in animal cells. After arginine is incorporated into proteins, it may undergo methylation to form N(G)-monomethylarginine, which may be converted to asymmetric dimethylarginine and symmetric dimethylarginine. The degradation of these methylated proteins produces free methylarginines. This chapter focuses on the analysis of these amino acids in biological samples (including plasma/serum, urine, cell culture medium, and tissues) using high-performance liquid chromatography that involves precolumn derivatization with o-phthaldialdehyde. Fluorescence is monitored at excitation and emission wavelengths of 340 and 455 nm, respectively. Detection limits are 5 nM for amino acids. The assays are linear between 1 and 100 microM for citrulline and arginine and between 0.1 and 10 microM for methylarginines. These chromatographic methods are highly sensitive, specific, accurate, and easily automated and provide a useful tool to study the regulation of the arginine-nitric oxide pathway.

Characterization of EHT 1864, a novel small molecule inhibitor of Rac family small GTPases.

Abstract: There is now considerable experimental evidence that aberrant activation of Rho family small GTPases promotes uncontrolled proliferation, invasion, and metastatic properties of human cancer cells Therefore, there is considerable interest in the development of small molecule inhibitors of Rho GTPase function However, to date, most efforts have focused on inhibitors that block Rho GTPase function indirectly, either by targeting enzymes involved in post-translational processing or downstream protein kinase effectors We have reported the identification and characterization of the EHT 1864 small molecule as an inhibitor of Rac family small GTPases, placing Rac1 in an inert and inactive state and then impairing Rac1-mediated functions in vivo Our work suggests that EHT 1864 selectively inhibits Rac1 downstream signaling and cellular transformation by a novel mechanism involving guanine nucleotide displacement This chapter provides the details for some of the biochemical and biological methods used to characterize the mode of action of EHT 1864 on Rac1 and its impact on Rac1-dependent cellular functions

Imaging of reactive oxygen species and nitric oxide in vivo in plant tissues.

Abstract: During the last decades there has been a growing interest in the study of reactive oxygen species (ROS) and nitric oxide (NO) production in plant tissues and their role in signaling and cellular response to biotic and abiotic stress conditions. Despite growing molecular data on this subject, less attention has been paid to the topological distribution of ROS and NO production in plant tissues. Knowledge of the contribution of different cells to the accumulation of ROS and NO is important to get deeper insights into the cellular response of plants to adverse conditions. This chapter focuses on the imaging of ROS and NO accumulation in vivo in plant tissues by confocal laser microscopy using specific fluorescent probes.

The Ras inhibitor farnesylthiosalicylic acid (Salirasib) disrupts the spatiotemporal localization of active Ras: a potential treatment for cancer.

Abstract: Chronic activation of Ras proteins by mutational activation or by growth factor stimulation is a common occurrence in many human cancers and was shown to induce and be required for tumor growth. Even if additional genetic defects are present, “correction” of the Ras defect has been shown to reverse Ras‐dependent tumorigenesis. One way to block Ras protein activity is by interfering with their spatiotemporal localization in cellular membranes or in membrane microdomains, a prerequisite for Ras signaling and biological activity. Detailed reports describe the use of this method in studies employing farnesylthiosalicylic acid (FTS, Salirasib), a Ras farnesylcysteine mimetic, which selectively disrupts the association of chronically active Ras proteins with the plasma membrane. FTS competes with Ras for binding to Ras‐escort proteins, which possess putative farnesyl‐binding domains and interact only with the activated form of Ras proteins, thereby promoting Ras nanoclusterization in the plasma membrane and robust signals. This chapter presents three‐dimensional time‐lapse images that track the FTS‐induced inhibition of membrane‐activated Ras in live cells on a real‐time scale. It also describes a mechanistic model that explains FTS selectivity toward activated Ras. Selective blocking of activated Ras proteins results in the inhibition of Ras transformation in vitro and in animal models, with no accompanying toxicity. Phase I clinical trials have demonstrated a safe profile for oral FTS, with minimal side effects and promising activity in hematological malignancies. Salirasib is currently undergoing trials in patients with pancreatic cancer and with nonsmall cell lung cancer, with or without identified K‐Ras mutations. The findings might indicate whether with the disruption of the spatiotemporal localization of oncogenic Ras proteins and the targeting of prenyl‐binding domains by anticancer drugs is worth developing as a means of cancer treatment.

Synthesis and biophysical characterization of stabilized alpha-helices of BCL-2 domains.

Abstract: Rational design of compounds to mimic the functional domains of BCL-2 family proteins requires chemical reproduction of the biologic complexity afforded by the relatively large and folded surfaces of BCL-2 homology (BH) domain peptide alpha-helices. Because the intermolecular handshakes of BCL-2 proteins are so critical to controlling cellular fate, we undertook the development of a toolbox of peptidic ligands that harness the natural potency and specificity of BH alpha-helices to interrogate and potentially medicate the deregulated apoptotic pathways of human disease. To overcome the classic deficiencies of peptide reagents, including loss of bioactive structure in solution, rapid proteolytic degradation in vivo, and cellular impermeability, we developed a new class of compounds based on hydrocarbon stapling of BH3 death domain peptides. Here we describe the chemical synthesis of Stabilized Alpha-Helices of BCL-2 domains or SAHBs, and the analytical methods used to characterize their secondary structure, proteolytic stability, and cellular penetrance.

Assessment of superoxide production and NADPH oxidase activity by HPLC analysis of dihydroethidium oxidation products.

Abstract: Assessment of low-level superoxide in nonphagocytic cells is crucial for assessing redox-dependent signaling pathways and the role of enzymes such as the NADPH oxidase complex. However, most superoxide probes present inherent limitations. Particularly, assessment of dihydroethidium (DHE) fluorescence is limited regarding a lack of possible quantification and simultaneous detection of its two main products: 2-hydroxyethidium, more specific for superoxide, and ethidium, which reflects H2O2-dependent pathways involving metal proteins. HPLC separation and analysis of those two main products have been described. This chapter reports procedures used for the validation of superoxide measurements in vascular system. Superoxide assessment was performed for cultured cells and tissue fragments incubated with DHE, followed by acetonitrile extraction and HPLC run, with simultaneous fluorescence detection of 2-hydroxyethidium and ethidium and ultraviolet detection of remaining DHE. It also describes procedures for DHE-based NADPH oxidase activity assays using HPLC or fluorometry. Such methods can enhance accuracy and allow better quantitation of vascular superoxide measurements.

Fluorescence approaches to quantifying biomolecular interactions.

Abstract: This review is conceived as an introductory text to aid in the understanding and conception of fluorescence-based measurements of biomolecular interactions. The major fluorescence observables are introduced briefly. Next, the criteria that are involved in the choice of the fluorescent probe are discussed in terms of their advantages and disadvantages for different types of experiments. The last sections deal with the experimental design for fluorescence-based assays aimed at detecting different types of biomolecular interactions. Included in our examples are protein-ligand interactions, protein-nucleic acid interactions, aqueous phase protein-protein interactions and protein interactions in or at the cell membrane. We hope that this introduction will be of use to students and researchers considering the use of fluorescence in their work.

Identification and characterization of endoplasmic reticulum stress-induced apoptosis in vivo.

Abstract: The endoplasmic reticulum (ER) is recognized primarily as the site of synthesis and folding of secreted and membrane-bound proteins. The ER provides stringent quality control systems to ensure that only correctly folded, functional proteins are released from the ER and that misfolded proteins are degraded. The efficient functioning of the ER is essential for most cellular activities and for survival. Stimuli that interfere with ER function can disrupt ER homeostasis, impose stress to the ER, and subsequently cause accumulation of unfolded or misfolded proteins in the ER lumen. ER transmembrane proteins detect the onset of ER stress and initiate highly specific signaling pathways collectively called the "unfolded protein response" (UPR) to restore normal ER functions. However, if ER homeostasis cannot be reestablished in response to intense or prolonged ER stress, the UPR induces ER stress-associated apoptosis to protect the organism by removing the stressed cells that produce misfolded or malfunctioning proteins. This chapter summarizes current understanding of ER stress-induced apoptosis and reliable methods to examine ER stress and apoptosis in mammalian cells. Since the liver is the major organ dealing with metabolic or pathological stress and is responsible for the detoxification of chemical compounds, the experimental protocols described here focus on identification and characterization of ER stress-induced apoptosis in mouse liver.

Using fluorophore-labeled oligonucleotides to measure affinities of protein-DNA interactions

Abstract: Changes in fluorescence emission intensity and anisotropy can reflect changes in the environment and molecular motion of a fluorophore. Researchers can capitalize on these characteristics to assess the affinity and specificity of DNA-binding proteins using fluorophore-labeled oligonucleotides. While there are many advantages to measuring binding using fluorescent oligonucleotides, there are also some distinct disadvantages. Here we describe some of the relevant issues for the novice, illustrating key points using data collected with a variety of labeled oligonucleotides and the relaxase domain of F plasmid TraI. Topics include selection of a fluorophore, experimental design using a fluorometer equipped with an automatic titrating unit, and analysis of direct binding and competition assays.

Lysosomes in apoptosis.

Abstract: Lysosomes are specialized organelles for protein recycling and as such are involved in the terminal steps of autophagy. However, it has become evident that lysosomes also play an important role in the progression of apoptosis. This latter function seems to be dependent on lysosomal proteases, which need to be released into the cytosol for apoptosis to be efficient. Among the lysosomal proteases, the most abundant are the cysteine cathepsins and the aspartic protease cathepsin D, which seem to be the major apoptosis mediators. This chapter reviews the methods used to study lysosomes and lysosomal proteases.

Site-selective Red-Edge effects.

Abstract: Observation of Red Edge effects is the basis of unique methodology that allows combination of site-photoselection with dynamics of molecular relaxations. The important dynamic information on molecular level can be obtained even by simple recording of steady-state fluorescence using the lifetime as the time marker. The extension to time domain allows distinguishing these relaxations from other dynamic processes that influence the excited-state energies. In this Chapter I briefly discuss the background of this technique and concentrate on quantitative measure of these effects and on importance of their distinction from ground-state heterogeneity. The peculiarity of Trp emission in proteins and the optimal selection of fluorescence probes are discussed. The Red Edge excitations influence dramatically the excited-state reactions that are coupled with dielectric relaxations and this opens a new fascinating prospect for protein and biomembrane studies.

Endothelial cell adhesion and migration.

Abstract: Endothelial cell adhesion and migration is fundamental to a number of physiologic processes, including vascular development and angiogenesis. It has been investigated in a variety of contexts, including tumorigenesis, wound healing, tissue engineering, and biomaterial design. The chemical and mechanical extracellular environments are critical regulators of these processes, affecting integrin-matrix binding, cell adhesion strength, and cell migration. Understanding the synergy between matrix chemistry and mechanics will ultimately lead to precise control over adhesion and migration. Moreover, a better understanding of endothelial cell adhesion is critical for development of therapeutics and biomaterials for the treatment of endothelial cell dysfunction and the progression of vascular disease. This chapter will focus on the specific interactions between endothelial cells and the extracellular matrix that mediate adhesion and migration. Several engineering methods used to probe and quantify endothelial cell adhesion and migration will be discussed.

Assembly, purification, and assay of the activity of the ASC pyroptosome.

Abstract: Pyroptosis is an inflammatory form of cell death mediated by caspase-1. Until recently, little was known about the mechanism by which caspase-1 is specifically activated to induce pyroptosis. Using biochemical and time-lapse confocal bioimaging approaches, it has been shown that caspase-1 is activated during pyroptosis by a large supramolecular assembly termed the pyroptosome. Biochemical and mass spectroscopic analyses revealed that the pyroptosome assembly is an oligomer of dimers of the adaptor protein ASC. Only one distinct pyroptosome is formed in each cell when macrophages or monocytes are stimulated with proinflammatory stimuli, which rapidly recruits and activates caspase-1, resulting in pyroptosis. This chapter describes methods for real-time observation and recording of the pyroptosome assembly process in live THP-1 monocytes. It also describes biochemical methods for the assembly, purification, and assay of the ASC pyroptosome from the THP-1 cell line, which could be adapted for use with other cell lines containing ASC, such as primary mouse macrophages. Finally, it describes methods for the in vitro reconstitution of a functional ASC pyroptosome from the recombinant ASC protein produced in Escherichia coli.

Interactions of NO with Hemoglobin: From Microbes to Man

Abstract: Hemoglobins are found in organisms from every major phylum and subserve life‐sustaining respiratory functions across a broad continuum. Sustainable aerobic respiration in mammals and birds relies on the regulated delivery of oxygen (O 2 ) and nitric oxide (NO) bioactivity by hemoglobin, through reversible binding of NO and O 2 to hemes as well as S ‐nitrosylation of cysteine thiols (SNO synthase activity). In contrast, bacterial and yeast flavohemoglobins function in vivo as denitrosylases (O 2 nitroxylases), and some multimeric, invertebrate hemoglobins function as deoxygenases (Cys‐dependent NO dioxygenases), which efficiently consume rather than deliver NO and O 2 , respectively. Analogous mechanisms may operate in plants. Bacteria and fungi deficient in flavohemoglobin show compromised virulence in animals that results from impaired resistance to NO, whereas animals and humans deficient in S ‐nitrosylated Hb exhibit altered vasoactivity. NO‐related functions of hemoglobins center on reactions with ferric (FeIII) heme iron, which is exploited in enzymatic reactions that address organismal requirements for delivery or detoxification of NO and O 2 . Delivery versus detoxification of NO/O 2 is largely achieved through structural changes and amino acid rearrangements within the heme pockets, thereby influencing the propensity for heme/cysteine thiol redox coupling. Additionally, the behavior exhibited by hemoglobin in vivo may be profoundly dependent both on the abundance of NO and O 2 and on the allosteric effects of heterotropic ligands. Here we review well‐documented examples of redox interactions between NO and hemoglobin, with an emphasis on biochemical mechanisms and physiological significance.

Interactive relations between nitric oxide (NO) and carbon monoxide (CO): heme oxygenase-1/CO pathway is a key modulator in NO-mediated antiapoptosis and anti-inflammation.

Abstract: Nitric oxide (NO) and carbon monoxide (CO) are synthesized from l-arginine and heme by the catalytic reaction of NO synthase (NOS) and heme oxygenase (HO). NO, a highly reactive free radical, plays an important role in the regulation of vascular and immune function, antiapoptosis, and neurotransmission by producing cGMP, nitrosyl iron complexes, and S-nitrosothiols. CO, a more stable molecule, exerts similar biological activities to those of NO by cGMP production, p38 mitogen-activated protein kinase activation, and nuclear factor-kappaB activation. NO induces the suppression of apoptosis and inflammation in hepatocytes and macrophages by an elevation in HO-1 and CO production, and these effects were not observed in mice lacking HO-1 as well as in cells treated with a HO-1 inhibitor. These evidences indicate that the HO-1/CO pathway is a key player in NO-mediated cytoprotection and anti-inflammation. This chapter reviews new advances in the interactive relations between iNOS/NO and HO-1/CO pathways in the regulation of apoptosis and inflammation.

In vivo real-time measurement of nitric oxide in anesthetized rat brain.

Abstract: During the last two decades nitric oxide (.NO) gas has emerged as a novel and ubiquitous intercellular modulator of cell functions. In the brain, .NO is implicated in mechanisms of synaptic plasticity but it is also involved in cell death pathways underlying several neurological diseases. Because of its hydrophobicity, small size, and rapid diffusion properties, the rate and pattern of .NO concentration changes are critical determinants for the understanding of its diverse actions in the brain. .NO measurement in vivo has been a challenging task due to its low concentration, short half-life, and high reactivity with other biological molecules, such as superoxide radical, thiols, and heme proteins. Electrochemical methods are versatile approaches for detecting and monitoring various neurotransmitters. When associated with microelectrodes inserted into the brain they provide high temporal and spatial resolution, allowing measurements of neurochemicals in physiological environments in a real-time fashion. To date, electrochemical detection of .NO is the only available technique that provides a high sensitivity, low detection limit, selectivity, and fast response to measure the concentration dynamics of .NO in vivo. We have used carbon fiber microelectrodes coated with two layers of Nafion and o-phenylenediamine to monitor the rate and pattern of .NO change in the rat brain in vivo. The analytical performance of microelectrodes was assessed in terms of sensitivity, detection limit, and selectivity ratios against major interferents: ascorbate, dopamine, noradrenaline, serotonin, and nitrite. For the in vivo recording experiments, we used a microelectrode/micropipette array inserted into the brain using a stereotaxic frame. The characterization of in vivo signals was assessed by electrochemical and pharmacological verification. Results support our experimental conditions that the measured oxidation current reflects variations in the .NO concentration in brain extracellular space. We report results from recordings in hippocampus and striatum upon stimulation of N-methyl-d-aspartate-subtype glutamate receptors. Moreover, the kinetics of .NO disappearance in vivo following pressure ejection of a .NO solution is also addressed.