Interaction of the integrin receptors with ligands determines the molecular basis of integrin –dependent cell adhesion. Integrin ligands are typically large proteins with relatively low binding affinities. This makes direct ligand-binding kinetic measurements somewhat difficult. Here we examine several real-time methods, aimed to overcome these experimental limitations and to distinguish the regulation of integrin conformation and affinity. This chapter includes: the use of a small ligand-mimetic probe for studies of inside-out regulation of integrin affinity and unbending, real-time cell aggregation and disaggregation kinetics to probe integrin conformational states and the number of integrin-ligand bonds, as well as the real-time monitoring of ligand -induced epitopes under signaling through G-protein-coupled receptors, and others. Experimental data obtained using these novel methods are summarized in terms of the current model of integrin activation.
Ligand-receptor interaction; Ligand mimetic; Real-time kinetics; Cells adhesion; Inside-out signal; Monoclonal antibodies; Quantitative approaches
Proteomic analyses of protein-electrophile adducts generally employ affinity capture of the adduct moiety, which enables global analyses, but is poorly suited to targeted studies of specific proteins. We describe a targeted molecular-probe approach to study modifications of the molecular chaperone heat-shock protein 90 (Hsp90), which regulates diverse client proteins. Noncovalent affinity capture with a biotinyl analog of the HSP90 inhibitor geldanamycin enables detection of the native protein isoforms Hsp90α and Hsp90β and their phosphorylated forms. We applied this probe to map and quantify adducts formed on Hsp90 by 4-hydroxynonenal (HNE) in RKO cells. This approach was also applied to measure the kinetics of site-specific adduction of selected Hsp90 residues. A protein-selective affinity capture approach is broadly applicable for targeted analysis of electrophile adducts and their biological effects.
electrophile; Hsp90; affinity probe; geldanamycin; kinetic analysis
Genomic structural variation (SV) can be thought of on a continuum from a single base pair insertion/deletion (INDEL) to large megabase-scale rearrangements involving insertions, deletions, duplications, inversions or translocations of whole chromosomes or chromosome arms. These variants can occur in coding or non-coding DNA, they can be inherited or arise sporadically in the germline or somatic cells. Many of these events are segregating in the population and can be considered common alleles while others are new alleles and thus rare events. All species studied to date harbor structural variants and these may be benign, contributing to phenotypes such as sensory perception and immunity, or pathogenic resulting in genomic disorders including DiGeorge/velocardiofacial, Smith-Margenis, Williams-Beuren and Prader-Willi syndromes. As structural variants are identified, validated and their significance, origin and prevalence elucidated it is of critical importance that this data be collected and collated in a way that can be easily accessed and analyzed.
This chapter will describe current structural variation online resources (see Figure 1 and Table 1), highlight the challenges in capturing, storing and displaying SV data, and discuss how dbVar and DGVa, the genomic structural variation databases developed at NCBI and EBI respectively, were designed to address these issues.
CNV; INDEL; SV; dbVar; DGVa; DGV
Cellular proteins are critically involved in all steps of the human immunodeficiency virus type 1 (HIV-1) life cycle. Disruption of host functions essential for virus replication or discovery of new proteins that block viral replication may provide novel antiviral approaches. In recent years, genetic selections for and against genes carried by retroviral vectors have become increasingly powerful, allowing for the isolation of cells with altered susceptibility to virus infection. Screens of complementary DNA (cDNA) libraries, for clones able to induce resistance to infection by recombinant human immunodeficiency virus 1 (HIV-1) genomes, has proved to be an excellent tool to identify new interfering factors. The restriction factors TRIM5α (1), the Zinc Finger Antiviral Protein (ZAP) (2) as well as the dominant negative factor N-86-HnRNPU (3) have all been discovered by means of such genetic screens. Here we report the strategy and techniques to prepare a library and isolate HIV antiviral genes, using the identification of N-86-HnRNPU as an example.
HIV-1; cDNA libraries; Genetic Screen; restriction factors
Our laboratory specializes in directed protein evolution, i.e. evolution of proteins under defined selective pressures in the laboratory. Our target genes are encoded in ColE1 plasmids to facilitate the generation of libraries in vivo. We have observed that when random mutations are not restricted to the coding sequence of the target genes, directed evolution results in a strong positive selection of plasmid origin of replication (ori) mutations. Surprisingly, this is true even during evolution of new biochemical activities, when the activity that is being selected was not originally present. The selected plasmid ori mutations are diverse and produce a range of plasmid copy numbers, suggesting a complex interplay between ori and coding mutations rather than a simple enhancement of level of expression of the target gene. Thus, plasmid dosage may contribute significantly to evolution by fine-tuning levels of activity. Here we present examples illustrating these observations as well as our methods for efficient quantification of plasmid copy number.
recombinant gene expression; ColE1 plasmid; plasmid copy number; GFP; ALKBH2; transformation; mutagenesis; R-loop; RNA I; RNA II; directed evolution; MMS; MNNG
The mitochondrial regulation of cell death involves conditions that result in the release of proapoptotic factors, such as cytochrome c, Smac-DIABLO, AIF, OMI/HtrA2, and others, by disruption of the outer mitochondrial membrane (OMM) permeability barrier that is controlled by pro- and antiapoptotic proteins of the Bcl-2 family. One of the mechanisms contributing to the OMM permeabilization is dependent on the interaction of proapoptotic Bcl-2 family proteins and other factors straight with the OMM. Another mechanism is initiated by the permeability transition of the inner mitochondrial membrane (IMM), leading to an increase in the matrix volume and reorganization of the IMM structure, which in turn, influence the OMM permeability barrier. The OMM also provides surface for the assembly of the apoptosome, where the mitochondria-derived proapoptotic factors induce caspase activation. Fluorescence measurements have been devised for evaluation of the barrier function of both OMM and IMM and of the downstream effectors of the factors released from the mitochondria to the cytosol. Many of these measurements are real-time, quantitative, and can be conveniently performed in a fluorometer cuvette containing suspensions of permeabilized cells or isolated mitochondria. This chapter provides a step-by-step manual for the measurements of the mitochondrial membrane potential, retention of Ca2+ and cytochrome c, matrix volume, and caspase activation and discusses protocols for discrimination between different mechanisms of the OMM permeabilization.
Apoptosis; Mitochondrial membrane permeability; Fluorometer; Mitochondrial membrane potential; Western blot
The use of behavioral testing has become an invaluable tool for assessing the efficacy of therapeutics for a variety of disorders of the central nervous system. This chapter will describe in detail several behavioral paradigms to evaluate the efficacy of PPAR agonists to modulate cognitive impairments in rodent models. When used together as a battery these procedures allow for a global assessment of cognition. These tests are explained in detail below, and include: (1) Novel Object Recognition (NOR), (2) Morris Water Maze (MWM), (3) Delay Match to Place (DMP), and (4) Cue Strategy.
PPAR agonists; Cognition; Novel object recognition; Morris water maze; Delay match to place; Cue strategy
Human genomics research has produced vast amounts of data that can be applied to or used to inform pharmacogenomic studies. The Internet is an extremely useful resource for pharmacogenomics as many Web sites provide access to data from genomic and clinical studies or host tools which can be used to interpret findings or generate hypotheses. Human genetic variation can now easily be explored or visualized through genome browsers and Web-based repositories which store the details of millions of human germ-line and somatic genetic variants. Gene expression data from many different tissue and cell types are available through Web-based repositories, and human genetic variants that associate with mRNA expression can be identified using Web data portals. Pharmacogenetic associations can be explored through publically available data repositories and the functionality of genetic variants predicted through Web-based bioinformatic tools. Furthermore, resources relating to currently used genetic tests are available online. Large clinical and population studies, many linked to medical records, can be queried for the availability of biospecimens or data. In the future, as the amount of genomic and associated clinical data increases, there is little doubt that Web-based resources will continue to evolve and overcome barriers hindering their efficient use, leading to systems-based approaches to pharmacogenomics.
Genome browser; Genetic variation; Genotypes; Gene expression; eQTLs; Genetic association studies; GWAS; Biorepositories; Bioinformatic tools
Determining the preferred substrate cleavage sequence of proteases is an important step towards understanding their roles in cancer development and progression. Knowledge of this sequence can aid in the design of new experimental tools for study as well as aid in the identification of endogenous protease substrates and signaling pathways. Various investigators have demonstrated a number of techniques in order to uncover these sequences, but most can be very time consuming. We have designed and successfully implemented a complete diverse ACC tetrapeptide positional scanning synthetic combinatorial library that allows for the rapid screening of proteases to determine their preferred residues at positions P1-P4. These sequences can be readily verified through kinetic measurements on single peptide substrates and utilized to further knowledge of the role of proteases in cancer.
Peptide Library; Substrate Profiling; Protease Specificity; PS-SCL; ACC
Clinical proteomics encompasses a multitude of experimental approaches, tools and techniques based on proteomics technology that are directly aimed to accelerate and improve diagnosis and treatment of human diseases. Surface-enhanced laser desorption ionization time-of-flight (SELDI-TOF) mass spectrometry is a variant of matrix-enhanced laser desorption ionization (MALDI) that makes use of chemically-modified surfaces to reduce the complexity of biological samples prior to separation in the mass analyzer. Compared to other proteomic techniques, SELDI has several important advantages such as ability to analyze complex biological samples with minimal pre-processing, ease of handling and high throughput. Importantly, once the biomarker or combination of biomarkers with potential clinical value has been established, validation analyses can be conducted in close proximity to clinical settings which is important for establishing the utility of new diagnostics in clinical decision making and perhaps future theranostic interventions. This chapter provides protocols for experimental design and methodology aimed at 1) UUdiscovering biologically relevant biomarkers in amniotic fluid using SELDI-TOF; 2) validating the clinical utility of the biomarkers as new diagnostics; 3) translating the biomarker findings into pathophysiological phenomena to provide further insight and extend the current understanding of the disease process. Many of the principles described herein for amniotic fluid could be generalized to studies involving other types of biological samples and other clinical questions.
Biomarkers; proteomics; SELDI; mass spectrometry; proteomics; profile; diagnostics
The ability to identify the donor stem cells following transplantation into injured hearts is critical. This is particularly important in evaluating stem cell survival and lineage differentiation into mature cardiovascular cells. Several approaches have been employed for tracking the donor stem cells, including fluorescent dyes and fluorescent protein gene transfer. Here, we will induce a protocol using lentivirus-mediated green fluorescent protein (GFP) to monitor the fate of donor stem cells following transplantation.
Green fluorescent protein (GFP); Lentivirus; Stem cells; Myocardial infarction (MI)
Cardiac progenitor cells (CPC) are a unique pool of progenitor cells residing in the heart that play an important role in cardiac homeostasis and physiological cardiovascular cell turnover during acute myocardial infarction (MI). Transplanting CPC into the heart has shown promise in two recent clinical trials of cardiac repair (SCIPIO & CADUCEUS). CSCs were originally isolated directly from enzymatically digested hearts followed by cell sorting using stem cell markers. However, long exposure to enzymatic digestion can affect the integrity of stem cell markers on the cell surface and also compromise stem cell function. Here, we describe a two-step procedure in which a large number of intact cardiac progenitor cells can be purified from small amount of heart tissue.
Cardiac progenitor cells; Cardiosphere; Magnetic-activated cell sorting (MACS)
Detection of transcription factors expressed in immune cell populations, particularly in subpopulations that are represented at low frequencies in lymphoid and nonlymphoid organs, presents a particular challenge when using traditional methods such as western blot analysis. Therefore, development of flow cytometry-based methods which allow identification of transcription factors in specific immune cell populations is of main interest. Here we developed and optimized a protocol for rapid and convenient detection of the transcription factor BCL11B (CTIP2) in T lymphocyte subpopulations using flow cytometry. The method employs saponin and Tween 20 both during the fixation and permeabilization steps, and we demonstrate that it is efficient for three independent antibody clones against distinctive BCL11B epitopes. In addition, we prove that the method preserves the staining of surface markers.
transcription factors; BCL11B/CTIP2; T lymphocytes; CD4+ T lymphocytes; flow cytometry
Breakdown of the blood-brain barrier (BBB) is present in several neurological disorders such as stroke, brain tumors, and multiple sclerosis. Non-invasive evaluation of BBB breakdown is important for monitoring disease progression and evaluating therapeutic efficacy in such disorders. One of the few techniques available for non-invasively and repeatedly localizing and quantifying BBB damage is magnetic resonance imaging (MRI). This usually involves the intravenous administration of a gadolinium-containing MR contrast agent such as Gd-DTPA, followed by dynamic contrast-enhanced MR imaging (DCE-MRI) of brain and blood, and analysis of the resultant data to derive indices of blood-to-brain transfer. There are two advantages to this approach. First, measurements can be made repeatedly in the same animal; for instance, they can be made before drug treatment and then again after treatment to assess efficacy. Secondly, MRI studies can be multiparametric. That is, MRI can be used to assess not only a blood-to-brain transfer or influx rate constant (Ki or K1) by DCE-MRI but also complementary parameters such as: 1) cerebral blood flow (CBF), done in our hands by arterial spin-tagging (AST) methods; 2) magnetization transfer (MT) parameters, most notably T1sat, which appear to reflect brain water-protein interactions plus BBB and tissue dysfunction; 3) the apparent diffusion coefficient of water (ADCw) and/or diffusion tensor, which is a function of the size and tortuosity of the extracellular space; and 4) the transverse relaxation time by T2-weighted imaging, which demarcates areas of tissue abnormality in many cases. The accuracy and reliability of two of these multiparametric MRI measures, CBF by AST and DCE-MRI determined influx of Gd-DTPA, have been established by nearly congruent quantitative autoradiographic (QAR) studies with appropriate radiotracers. In addition, some of their linkages to local pathology have been shown via corresponding light microscopy and fluorescence imaging. This chapter describes: 1) multiparametric MRI techniques with emphasis on DCE-MRI and AST-MRI; 2) the measurement of the blood-to-brain influx constant and CBF; and 3) the role of each in determining BBB permeability.
Apparent diffusion coefficient; Arterial spin tagging; Blood-brain barrier; Cerebral blood flow; Cerebral ischemia; Gd-DTPA; Hemorrhagic transformation; Influx constant; Look-Locker; Magnetic resonance contrast agents; Magnetization transfer; Patlak plot; Quantitative autoradiography; Rat; T1; T1sat; T1WI; T2; TOMROP
In mucins, glycosylation is complex and the most predominant posttranslational modification. Since mucins exhibit differential glycosylation pattern under physiological and pathological conditions, analysis of mucin glycans is important for understanding their specific functions during pathological conditions like cancer. Given the complexity of mucin glycans, several sophisticated analytical tools such as HPLC, mass spectrometry, and lectin sandwich assays are employed for glyco-analysis of mucins. However the specialized expertise and instrumentation required for such analysis are beyond the reach of an average cancer biology laboratory. We described in this chapter the utility of the simple electrophoresis/immunoblotting method to examine the mucin glycan epitopes, using specific antibodies and lectins.
Mucin; Mucin glycans; Mucin-associated carbohydrate glycans; Tumor-associated carbohydrate antigen; Mucin immunoprecipitation; Western blot/Western transfer; Lectins
DNA unwinding and polymerization are complex processes involving many intermediate species in the reactions. Our understanding of these processes is limited because the rates of the reactions or the existence of intermediate species is not apparent without specially designed experimental techniques and data analysis procedures. In this chapter we describe how pre-steady state single-turnover measurements analyzed by model-based methods can be used for estimating the elementary rate constants. Using the hexameric helicase and the DNA polymerase from bacteriophage T7 as model systems, we provide stepwise procedures for measuring the kinetics of the reactions they catalyze based on radioactivity and fluorescence. We also describe analysis of the experimental measurements using publicly available models and software gfit (http://gfit.sf.net).
Hexameric helicase; Replication; DNA unwinding; T7 bacteriophage; DNA polymerase; DNA synthesis; Strand displacement; Primer extension; gfit; global regression analysis
Measurement of fluorescence and phosphorescence in vivo is readily used to quantify the concentration of specific species that are relevant to photodynamic therapy. However, the tools to make the data quantitatively accurate vary considerably between different applications. Sampling of the signal can be done with point samples, such as specialized fiber probes or from bulk regions with either imaging or sampling, and then in broad region image-guided manner. Each of these methods is described below, the application to imaging photosensitizer uptake is discussed, and developing methods to image molecular responses to therapy are outlined.
Fluorescence; measurement; quantification; molecular; fluorescent; photosensitizer; imaging; instrumentation; system; fiber; spectroscopy
Atomic force microscopy (AFM) has become a conventional tool for elucidation of the molecular mechanisms of protein aggregation and, specifically, for analysis of assembly pathways, architecture, aggregation state, and heterogencity of oligomeric intermediates or mature fibrils. AFM imaging provides useful information about particle dimensions, shape, and substructure with nanometer resolution. Conventional AFM methods have been very helpful in the analysis of polymorphic assemblies formed in vitro from homogeneous proteins or peptides. However, AFM imaging on its own provides limited insight into conformation or composition of assemblies produced in the complex environment of a cell, or prepared from a mixture of proteins as a result of cross-seeding. In these cases, its combination with fluorescence microscopy (AFFM) increases its resolution.
Amyloids; Assembly; Atomic force microscopy; Atomic force fluorescence microscopy; Immunofluorescence; Oligomers
p53, a guardian of the genome, exerts its tumor suppression activity by regulating a large number of downstream targets involved in cell cycle arrest, DNA repair, apoptosis, and cellular senescence. Although p53-mediated apoptosis is able to kill cancer cells, a role for cellular senescence in p53-dependent tumor suppression is becoming clear. Mouse studies showed that activation of p53-induced premature senescence promotes tumor regression in vivo. However, p53-mediated cellular senescence also leads to aging-related phenotypes, such as tissue atrophy, stem cell depletion, and impaired wound healing. In addition, several p53 isoforms and two p53 homologs, p63 and p73, have been shown to play a role in cellular senescence and/or aging. Importantly, p53, p63, and p73 are necessary for the maintenance of adult stem cells. Therefore, understanding the dual role the p53 protein family in cancer and aging is critical to solve cancer and longevity in the future. In this chapter, we provide an overview on how p53, p63, p73, and their isoforms regulate cellular senescence and aging.
Aging; p53; p63; p73; Senescence
Tumor cell motility and invasion rely on actin cytoskeleton rearrangements mediated by the activation of RhoGTPase signaling pathways. Invadopodia are membrane-degrading protrusions that mediate extracellular matrix degradation. Here, we provide procedures for imaging RhoGTPase biosensors in tumor cells during the formation of invadopodia and matrix degradation.
RhoGTPase; FRET; Biosensors; Invadopodia
The liver is the largest internal organ in mammals, serving a wide spectrum of vital functions. Loss of liver function due to drug toxicity or viral infection is a major cause of death in the United States. The development of Bioartificial Liver (BAL) devices and the demand for pharmaceutical and cosmetic toxicity screening require the development of long-term hepatocyte culture techniques. However, primary hepatocytes rapidly lose their cuboidal morphology and liver-specific functions over a few days in culture. Accumulation of stress fibers, loss of metabolic function, and cell death are known phenomena. In recent years, several techniques were developed that can support high levels of liver-specific gene expression, metabolic and synthetic function for several weeks in culture. These include the collagen double-gel configuration, hepatocyte spheroids, coculture with endothelial cells, and micropatterned cocultures with 3T3-J2 fibroblasts. This chapter covers the current status of hepatocyte culture techniques, including: hepatocyte isolation, media formulation, oxygen supply, heterotypic cell–cell interactions, and basic functional assays.
Liver; Hepatocytes; Metabolism; Oxygen; Coculture; Culture medium; Non-parenchymal cells
G protein-coupled receptors (GPCR) are a superfamily of receptors that are vital in a wide array of physiological processes. Modulation of GPCR signaling has been an intensive area of therapeutic study, mainly due to the diverse pathophysiological significance of GPCRs. Pepducins are cell-penetrating lipidated peptides designed to target the intracellular loops of the GPCR of interest. Pepducins can function as agonists or antagonists of their cognate receptor, making them highly useful compounds for the study of GPCR signaling. Pepducins have been used to control platelet-dependent hemostasis and thrombosis, tumor growth, invasion, and angiogenesis, as well as to improve sepsis outcomes in mice. Pepducins have been successfully designed against a wide variety of GPCRs including the protease-activated receptors (PAR1, 2, 4), the chemokine receptors (CXCR1, 2, 4), the sphingosine-1-phosphate receptor (S1P3), the adrenergic receptor (ADRA1B), and have the potential to help reveal the functions of intractable GPCRs. Pharmacokinetic, pharmacodynamic, and biodistribution studies have showed that pepducins are widely distributed throughout the body except the brain and possess appropriate drug-like properties for use in vivo. Here, we discuss the delivery, pharmacology, and biodistribution of pepducins, as well as the effects of pepducins in models of inflammation, cardiovascular disease, cancer, and angiogenesis.
Pepducin; GPCR; Inflammation; Sepsis; Thrombosis; Cancer; Angiogenesis; PAR1; PAR4; CXCR1; CXCR2; CXCR4
Glial cells, traditionally viewed as passive elements in the CNS, are now known to have many essential functions. Many of these functions have been revealed by work on retinal glial cells. This work has been conducted almost exclusively on ex vivo preparations and it is essential that retinal glial cell functions be characterized in vivo as well. To this end, we describe an in vivo rat preparation to assess the functions of retinal glial cells. The retina of anesthetized, paralyzed rats is viewed with confocal microscopy and laser speckle flowmetry to monitor glial cell responses and retinal blood flow. Retinal glial cells are labeled with the Ca2+ indicator dye Oregon Green 488 BAPTA-1 and the caged Ca2+ compound NP-EGTA by injection of the compounds into the vitreous humor. Glial cells are stimulated by photolysis of caged Ca2+ and the activation state of the cells assessed by monitoring Ca2+ indicator dye fluorescence. We find that, as in the ex vivo retina, retinal glial cells in vivo generate both spontaneous and evoked intercellular Ca2+ waves. We also find that stimulation of glial cells leads to the dilation of neighboring retinal arterioles, supporting the hypothesis that glial cells regulate blood flow in the retina. This in vivo preparation holds great promise for assessing glial cell function in the healthy and pathological retina.
Glial cell; Müller cell; Astrocyte; Retina; In vivo preparation; Intracellular calcium; Calcium wave; Blood flow; Confocal microscopy; Laser speckle flowmetry
High-performance liquid chromatography (HPLC) is extremely useful for the study of proteins and the characterization of their posttranslational modifications. Here we describe a method that utilizes cation-exchange HPLC to separate multiply acetylated histone H3 species on the basis of their charge and hydrophilicity. This high-resolution method allows for the separation of histone H3 species that differ by as few as one acetyl group, and is compatible with subsequent analysis by a variety of techniques, including mass spectrometry and western blotting.
Cation-exchange chromatography; PolyCAT A; Acetylation; HPLC; Histone H3; Acetic anhydride
Islet transplantation is the most exciting treatment option for individuals afflicted with Type 1 diabetes. However, the severe shortage of human pancreas and the need to use risky immunosuppressive drugs to prevent transplant rejection remain two major obstacles for the routine use of islet transplantation in diabetic patients. Successful development of a bioartificial pancreas using the approach of microencapsulation with perm-selective coating of islets with biopolymers for graft immunoisolation holds tremendous promise for diabetic patients because it has great potential to overcome these two barriers. In this chapter, we provide a detailed description of the microencapsulation process.
Islets; Alginate; Microencapsulation; Immunoisolation; Diabetes; Transplantation