MedWorm.com provides a medical RSS filtering service. Over 6000 RSS medical sources are combined and output via different filters. This feed contains the latest items from the 'Springer protocols feed by Biochemistry' source. Fri, 19 Mar 2010 17:18:04 +0100 http://www.medworm.com/index.php?rid=3353697&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-670-2_9 Prion diseases, also called transmissible spongiform encephalopathies (TSEs), are a group of neurodegenerative disorders affecting animals and humans. No effective treatments are currently available for the diseases, vCJD in particular. It is believed that the formation of protease-resistant insoluble prion protein (PrPSc), which is the main component of amyloidal deposits, from the cellular prion protein (PrPC), is essential for the progression of the disease. Therefore, both PrPSc and PrPC are currently being used as potential drug targets. (Source: Springer protocols feed by Biochemistry) Springer protocols feed by Biochemistry info http://www.medworm.com/rss/comments.php?id=3353697 Thu, 11 Mar 2010 17:39:16 +0100 3353697 http://www.medworm.com/index.php?rid=3353696&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-670-2_8 We have designed a novel high-throughput (HTP) kinase assay using an array-based surface plasmon resonance (SPR) apparatus. For high flexibility and performance, the kinase assay procedure is divided into an in vitro phosphorylation part and a phospho-detection part on a sensor chip. Not only biotinylated peptides but also recombinant proteins fused with FLAG-GST tandem tag can be used as native substrates. The substrate is selectively captured by a capture antibody immobilized on a sensor chip, and phospho-tyrosine (pTyr) residues are detected by an anti-pTyr antibody. The level of tyrosine phosphorylation is calculated from the capture level of the substrates and the binding level of the anti-pTyr antibody monitored by SPR. A wide dynamic range and real-time monitoring of SPR contribute ... Springer protocols feed by Biochemistry info http://www.medworm.com/rss/comments.php?id=3353696 Thu, 11 Mar 2010 17:39:16 +0100 3353696 http://www.medworm.com/index.php?rid=3353695&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-670-2_7 Sensitive detection of small molecules using surface plasmon resonance (SPR) presents significant challenges as the antigen cannot serve as a signal generator because of its low mass; efficient binding of the target requires the binding event to be spaced from the sensor surface through a specialist linker conjugation. Competitive immunoassay of steroid hormones can be performed by conjugation through a rationally designed linker system at positions distant from existing antigenic functional groups. The binding signal from the primary antibody can then be further enhanced by sequential addition of secondary antibody or conjugated gold nanoparticles which can produce 13-fold signal enhancements through both their mass and co-operative plasmon coupling. (Source: Springer protocols feed by Bi... Springer protocols feed by Biochemistry info http://www.medworm.com/rss/comments.php?id=3353695 Thu, 11 Mar 2010 17:39:16 +0100 3353695 http://www.medworm.com/index.php?rid=3353694&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-670-2_6 Direct assay of small molecules by SPR in general is troublesome and at least tedious procedures have to be applied. Competition experiments offer an attractive alternative. A small ligand known to bind to the analyte is immobilized on an SPR sensor surface, and the binding of the larger analyte in the presence of compounds under investigation in a concentration range is assayed. The resulting inhibition curves of the equilibrium SPR signal as function of the compound concentration can be analyzed to yield thermodynamic binding constants for the interaction in solution between analyte and the compounds under investigation. An additional advantage of this method is that series of compounds can be analyzed using the same sensor surface, so there is no immobilization needed for each compound.... Springer protocols feed by Biochemistry info http://www.medworm.com/rss/comments.php?id=3353694 Thu, 11 Mar 2010 17:39:16 +0100 3353694 http://www.medworm.com/index.php?rid=3353693&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-670-2_5 Surface plasmon resonance (SPR) is a robust method to detect and quantify macromolecular interactions; however, to measure binding interactions, one component must be immobilized on a sensor surface. This is typically achieved using covalent immobilization via free amines or thiols, or noncovalent immobilization using high-affinity interactions such as biotin/streptavidin or antibody/antigen. In this chapter we describe a robust method to covalently immobilize His6 fusion proteins on the sensor surface for SPR analysis. (Source: Springer protocols feed by Biochemistry) Springer protocols feed by Biochemistry info http://www.medworm.com/rss/comments.php?id=3353693 Thu, 11 Mar 2010 17:39:16 +0100 3353693 http://www.medworm.com/index.php?rid=3353692&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-670-2_4 Surface plasmon resonance (SPR) is a highly sensitive method for the detection of molecular interactions. One interacting partner is immobilized on the sensor chip surface while the other is injected across the sensor surface. This chapter focuses on high-affinity immobilization of protein substrates for affinity and kinetic analyses using biotin/streptavidin interaction and GST/anti-GST-antibody interaction. (Source: Springer protocols feed by Biochemistry) Springer protocols feed by Biochemistry info http://www.medworm.com/rss/comments.php?id=3353692 Thu, 11 Mar 2010 17:39:16 +0100 3353692 http://www.medworm.com/index.php?rid=3353691&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-670-2_3 Surface plasmon resonance (SPR) is one of the leading tools in biomedical research. The challenge in its use is the controlled positioning of one of the components of an interaction on a carefully designed surface. Many attempts in interaction analysis fail due to the non-functional or unsuccessful immobilization of a reactant onto the complex matrix of that surface. The most common technique for linking ligands covalently to a hydrophilic solid surface is amine coupling via reactive esters. In this chapter detailed methods and problem discussions will be given to assist in fast decision analysis to optimize immobilization and regeneration. Topics in focus are different coupling techniques for small and large molecules, streptavidin–biotin sandwich immobilization, and optimizing rege... Springer protocols feed by Biochemistry info http://www.medworm.com/rss/comments.php?id=3353691 Thu, 11 Mar 2010 17:39:16 +0100 3353691 http://www.medworm.com/index.php?rid=3353690&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-670-2_2 This chapter presents an introduction to the kinetic analysis of SPR biosensor data for the determination of affinity and kinetic rate constants of biomolecular interactions between an immobilized and a soluble binding partner. The need to be aware of and critically test the assumptions underlying the analysis models is emphasized and the consequences for the experimental design are discussed. The two most common sources of deviation in SPR surface binding kinetics from the ideal pseudo-first-order binding kinetics of bimolecular reactions are mass transport limitations and the heterogeneity of the surface sites. These problems are intrinsic to the use of a biosensor surface for characterizing interactions. The effect of these factors on the observed binding kinetics, and strategies to acc... Springer protocols feed by Biochemistry info http://www.medworm.com/rss/comments.php?id=3353690 Thu, 11 Mar 2010 17:39:16 +0100 3353690 http://www.medworm.com/index.php?rid=3353689&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-670-2_1 Surface plasmon resonance (SPR) analysis is rather unique in that it allows assay of binding constants (affinity) and kinetic analysis of binding phenomena. This introductory chapter deals with some specific features that are relevant to many diverse applications. The role and impact of kinetics in biomolecular interactions is highlighted. A concise description of the physical principles of the SPR phenomenon is given from a practical point of view, such that some possibilities and limitations of the method can be rationalized, e.g., depth of the evanescent field. A specific condition that may come forward in kinetic analysis is mass transport limitation (MTL). A practical model is presented, which allows estimation of the extent of MTL. Based on this model it can be rationalized whether M... Springer protocols feed by Biochemistry info http://www.medworm.com/rss/comments.php?id=3353689 Thu, 11 Mar 2010 17:39:15 +0100 3353689 http://www.medworm.com/index.php?rid=3353688&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-670-2_19 Surface plasmon resonance is widely used to study binding interactions with proteins, potentially yielding information on kinetics, thermodynamics and active concentrations. However, the technology cannot identify the involved interaction partners. Mass spectrometry, on the other hand, can be used for specific identification of proteins in amounts comparable to the levels that can be captured on a Biacore SPR sensorchip. Here we present protocols for capturing, washing and eluting proteins from Biacore instruments as well as for robust sample preparation for sensitive mass spectrometric identification. (Source: Springer protocols feed by Biochemistry) Springer protocols feed by Biochemistry info http://www.medworm.com/rss/comments.php?id=3353688 Thu, 11 Mar 2010 17:39:15 +0100 3353688 http://www.medworm.com/index.php?rid=3353687&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-670-2_18 The combination of surface plasmon resonance (SPR) and mass spectrometry (MS) creates a comprehensive protein investigation approach wherein SPR is employed for protein quantification and MS is utilized to structurally characterize the proteins. In such, MS utterly complements the SPR detection and reveals intrinsic protein structural modifications that go unregistered via the SPR detection. Protein complexes and non-specific binding can also be delineated via the SPR-MS approach. Described here are the protocols and know-how for successful and reproducible integration of SPR and MS. The individual steps of the entire SPR-MS process are illustrated via an example showing analysis of myoglobin from human plasma. (Source: Springer protocols feed by Biochemistry) Springer protocols feed by Biochemistry info http://www.medworm.com/rss/comments.php?id=3353687 Thu, 11 Mar 2010 17:39:15 +0100 3353687 http://www.medworm.com/index.php?rid=3353686&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-670-2_17 The G-protein coupled receptor rhodopsin is a classical example of a seven transmembrane helix receptor; it is photoexcited and transmits this light signal to a G-protein mediated cascade. Many components of this receptor-triggered cascade can be purified in their native forms from natural sources making this system most suitable for biophysical studies. A central aspect of cellular signal transduction routes is to understand protein–protein interactions in a quantitative way. Surface plasmon resonance (SPR) spectroscopy is a biosensor-based technique that allows investigating molecular interactions by determining kinetic parameters. We here show how dark-adapted rhodopsin can be immobilized on the sensor chip surface. A laser device implemented in the SPR system allowed us to trigge... Springer protocols feed by Biochemistry info http://www.medworm.com/rss/comments.php?id=3353686 Thu, 11 Mar 2010 17:39:15 +0100 3353686 http://www.medworm.com/index.php?rid=3353685&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-670-2_16 Surface analytical tools as surface plasmon resonance (SPR) have become increasingly important in biomedical research since they offer high detection sensitivity compared to traditional biomedical methods. For the use of SPR as a biomedical research tool there is a need to immobilize the reactants to a solid sensor surface. It is nowadays fairly straightforward to immobilize various reactants and hydrophilic proteins to a solid sensor surface and SPR has successfully been used in several applications using such proteins when studying various protein interactions. When using SPR for the analysis of transmembrane proteins the immobilization onto the solid surface becomes more difficult. Transmembrane proteins are more sensitive to the surroundings and need to be incorporated into a structure... Springer protocols feed by Biochemistry info http://www.medworm.com/rss/comments.php?id=3353685 Thu, 11 Mar 2010 17:39:15 +0100 3353685 http://www.medworm.com/index.php?rid=3353684&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-670-2_15 Surface plasmon resonance (SPR) spectroscopy is emerging as a useful tool for determination of molecular interactions in real time. Studies on the molecular pathogenesis of amyloidoses have shown that the plasma membrane plays an important role in amyloidogenesis and cytotoxicity induced by amyloidogenic proteins. By immobilizing lipid bilayers on a sensor chip surface, SPR spectroscopy has been employed to examine the binding of amyloidogenic proteins, such as amyloid β protein (Aβ), to a variety of lipid membranes, and it provided new insights into the molecular interactions between these amyloidogenic proteins and membranes. In this chapter, we describe the application of SPR spectroscopy to the determination of the binding of Aβ to lipid membranes. (Source: Springer prot... Springer protocols feed by Biochemistry info http://www.medworm.com/rss/comments.php?id=3353684 Thu, 11 Mar 2010 17:39:15 +0100 3353684 http://www.medworm.com/index.php?rid=3353683&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-670-2_14 Surface plasmon resonance (SPR) employs the optical principle of SPR to measure changes in mass on a sensor chip surface in real time. Surface chemistry has been developed which enables the immobilization of lipid bilayers and determination of protein–membrane interactions in real time. Antimicrobial peptides are being increasingly recognized as potential candidate antibacterial drugs in the face of the rapidly emerging bacterial resistance to conventional antibiotics in recent years. However, a precise understanding of the relationship between antimicrobial peptide structure and their cytolytic function in a range of organisms is still lacking. This is a result of the complex nature of the interactions of antimicrobial peptides with the cell membrane, the mechanism of which can vary... Springer protocols feed by Biochemistry info http://www.medworm.com/rss/comments.php?id=3353683 Thu, 11 Mar 2010 17:39:15 +0100 3353683 http://www.medworm.com/index.php?rid=3353682&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-670-2_13 Qualitative and quantitative aspects of protein interactions with membranes may be studied by optical sensors. Biacore offers two dedicated chips for working with lipids and membranes: the L1 and HPA sensor chips. The L1 chip is the most frequently used in protein–membrane interaction studies and it allows the capture of intact liposomes. This chapter describes the protocol for immobilization of liposomes on L1 sensor chips and discusses some of the experimental considerations. An alternative approach that utilizes a streptavidin-coated sensor chip (SA sensor chip) is described for cases when it is not possible to use an L1 chip. (Source: Springer protocols feed by Biochemistry) Springer protocols feed by Biochemistry info http://www.medworm.com/rss/comments.php?id=3353682 Thu, 11 Mar 2010 17:39:15 +0100 3353682 http://www.medworm.com/index.php?rid=3353681&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-670-2_12 Surface plasmon resonance has become one of the most important techniques for studying bimolecular interactions. Most of the researchers are using it to study protein–protein interactions, but in recent years membrane model systems have also become available and this makes it possible to study protein–membrane interactions as well. In this review chapter we describe possible ways to prepare lipid membrane surfaces on various sensor chips and some of the experimental considerations one has to take into account when performing such experiments. (Source: Springer protocols feed by Biochemistry) Springer protocols feed by Biochemistry info http://www.medworm.com/rss/comments.php?id=3353681 Thu, 11 Mar 2010 17:39:15 +0100 3353681 http://www.medworm.com/index.php?rid=3353680&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-670-2_11 When designing DNA biosensors, the immobilization of specific DNA probes is one of the most essential parts. Unfortunately, many of the existing strategies (e.g., adsorption, complexation, and entrapment) can only be used on standard microscope slides, while for more tailored surfaces alternative strategies are still required. In the case of gold surfaces, the self-assembly of mixed DNA/alkanethiol films is a very common approach to directly couple single-stranded DNA probes. The quality of these mixed films greatly depends on different parameters including the sensitivity and the detection limit. We have shown a positive relation between the length of the used carbon spacer and the general performance of the DNA biosensor. In this chapter an extended protocol for the controlled immobiliza... Springer protocols feed by Biochemistry info http://www.medworm.com/rss/comments.php?id=3353680 Thu, 11 Mar 2010 17:39:15 +0100 3353680 http://www.medworm.com/index.php?rid=3353679&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-670-2_10 Surface plasmon resonance is a valuable tool to determine the affinity between glycoconjugates and sugar-binding proteins such as plant and animal lectins. The main interest of using such an approach is that neither the lectins – which are proteins – nor their ligands – natural compounds such as glycoproteins, oligosaccharides, polysaccharides, or synthetic glycoconjugates such as glycoclusters or neoglycoproteins – require any tag. Because lectins bear several binding sites, they behave like immunoglobulin eliciting avidity phenomena. This peculiarity may lead to erroneous results if special conditions are not applied. We obtained best and reproducible results when the lectin was immobilized and its ligands were used as soluble analytes. With heterogeneous glycocon... Springer protocols feed by Biochemistry info http://www.medworm.com/rss/comments.php?id=3353679 Thu, 11 Mar 2010 17:39:15 +0100 3353679 http://www.medworm.com/index.php?rid=2962538&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-454-8_15 Chondroitin/dermatan sulfate (CS/DS) glycosaminoglycans (GAGs) are present in high levels in connective tissue where they play roles as structural molecules and in protein-binding interactions. Recent developments in the techniques for analysis of CS/DS using capillary electrophoresis (CE) have enabled progress in the understanding of changes in CS/DS structure that accompany connective tissue diseases including osteoarthritis. Key to these developments is the ability to extract CS/DS GAGs from small quantities of connective tissue. This chapter describes a method for connective tissue GAG extraction, derivatization, and workup for subsequent capillary electrophoretic and/or mass spectrometric analysis. (Source: Springer protocols feed by Biochemistry) Springer protocols feed by Biochemistry info http://www.medworm.com/rss/comments.php?id=2962538 Fri, 30 Oct 2009 00:00:00 +0100 2962538 http://www.medworm.com/index.php?rid=2962537&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-454-8_14 Glycosylation is highly sensitive to the biochemical environment and plays a key role in development and disease manifestation. Moreover, glycan biosynthesis depends on several highly competitive processes; thus, variations in the concentration of specific glycosyltransferases produce different products. For this reason, monitoring changes in glycosylation may be a more specific and sensitive approach to biomarker discovery and possibly disease diagnosis. Glycans in serum are of particular interest as approximately half of all proteins are glycosylated. We have developed the methods for profiling the glycans in human serum to identify glycan biomarker. Global release methods were used including chemical and enzymatic to access O-linked and N-linked glycans, respectively. Glycans were relea... Springer protocols feed by Biochemistry info http://www.medworm.com/rss/comments.php?id=2962537 Fri, 30 Oct 2009 00:00:00 +0100 2962537 http://www.medworm.com/index.php?rid=2962536&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-454-8_13 N- Glycosylation of proteins is recognized as one of the most common post-translational modifications. Until recently it was believed that N-glycosylation occurred exclusively in eukaryotes until the discovery of the general protein glycosylation pathway (Pgl) in Campylobacter jejuni. We have developed a new glycomics strategy based on lectin-affinity capture of lipid-linked oligosaccharides (LLOs) coupled to capillary electrophoresis mass spectrometry. The LLO intermediates of the C. jejuni Pgl pathway were used to validate the methodology and to better characterize the bacterial model system for protein N-glycosylation. This method provides a rapid, non-radioactive approach for the characterization of intermediates in polysaccharide biosynthesis and is a useful tool for glycoengineering ... Springer protocols feed by Biochemistry info http://www.medworm.com/rss/comments.php?id=2962536 Fri, 30 Oct 2009 00:00:00 +0100 2962536 http://www.medworm.com/index.php?rid=2962535&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-454-8_12 Metabolomics is essentially the study of all low molecular weight molecules in a biological system under defined conditions. In glycomics, there is much potential to gain insight into the biosynthesis of novel glycoconjugate structures by probing the metabolome for substrates that are suspected, or known, to be involved in the biosynthetic processes. Recently, we employed the use of hydrophilic interaction liquid chromatography–mass spectrometry (HILIC–MS) in a focused metabolomic study of sugar-nucleotides relevant to the biosynthesis of highly novel carbohydrate modifications on the flagellin of Campylobacter sp. We exploited the unique selectivity of the HILIC–MS method for discriminating between closely related sugar-nucleotide intermediates and allowed their subseque... Springer protocols feed by Biochemistry info http://www.medworm.com/rss/comments.php?id=2962535 Fri, 30 Oct 2009 00:00:00 +0100 2962535 http://www.medworm.com/index.php?rid=2962534&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-454-8_11 Glycomics which is the study of saccharides and genes responsible for their formation requires the continuous development of rapid and sensitive methods for the identification of glycan structures. It involves glycoanalysis which relies upon the development of methods for determining the structure and interactions of carbohydrates. For the application of functional glycomics to microbial virulence, carbohydrates and their associated metabolic and carbohydrate processing enzymes and respective genes can be identified and exploited as targets for drug discovery, glyco-engineering, vaccine design, and detection and diagnosis of diseases. Glycomics also encompasses the detailed understanding of carbohydrate–protein interactions and this knowledge can be applied to research efforts focuse... Springer protocols feed by Biochemistry info http://www.medworm.com/rss/comments.php?id=2962534 Fri, 30 Oct 2009 00:00:00 +0100 2962534 http://www.medworm.com/index.php?rid=2962533&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-454-8_10 To develop a novel high-throughput tool for monitoring carbohydrate–protein interactions, carbohydrate or glycoprotein microarrays have been prepared for binding with lectins. The interaction events are marked by attachment of fluorescent dyes and gold nanoparticles. The attachment of the fluorescent dyes and gold nanoparticles is achieved by standard avidin–biotin chemistry. The detection principle is fluorescence or resonance light scattering (RLS). The electroless deposition of silver onto the gold particles has been employed for RLS signal enhancement. Well-defined recognition systems, three monosaccharides (Man-α, Glc-β, and Gal-β) or three glycoproteins (Asf, RNase A, and RNase B) with two lectins (ConA and RCA120), are chosen here to establish the microar... Springer protocols feed by Biochemistry info http://www.medworm.com/rss/comments.php?id=2962533 Fri, 30 Oct 2009 00:00:00 +0100 2962533 http://www.medworm.com/index.php?rid=2715424&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-977-2_19 Microscopy has been one of the most direct and powerful tools since the beginning of biological research. Continued advances such as confocal and two-photon fluorescence microscopy and fluorescent proteins now make imaging useful at a variety of spatial scales (molecules, circuits, cells, tissues, and even whole embryos) and temporal scales (<seconds to days). Zebrafish is uniquely poised to benefit from these continued technological improvements because of its inherent suitability for both imaging and genetics. This chapter presents an approach called “in toto imaging”. The goal of in toto imaging is to image and track every single cell movement and division that forms a tissue or organ. This approach is powerful for understanding how cell lineage, shape changes, and moveme... Springer protocols feed by Biochemistry info http://www.medworm.com/rss/comments.php?id=2715424 Thu, 20 Aug 2009 11:31:54 +0100 2715424 http://www.medworm.com/index.php?rid=2715423&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-977-2_18 The zebrafish provides an ideal model for the study of vertebrate organogenesis, including the formation of the digestive tract and its associated organs. Despite optical transparency of embryos, the internal position of the developing digestive system and its close juxtaposition with the yolk initially made morphological analysis relatively challenging, particularly during the first 3 d of development. However, methodologies have been successfully developed to address these problems and comprehensive morphologic analysis of the developing digestive system has now been achieved using a combination of light and fluorescence microscope approaches—including confocal analysis—to visualize wholemount and histological preparations of zebrafish embryos. Furthermore, the expanding numb... Springer protocols feed by Biochemistry info http://www.medworm.com/rss/comments.php?id=2715423 Thu, 20 Aug 2009 11:31:54 +0100 2715423 http://www.medworm.com/index.php?rid=2715422&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-977-2_17 The zebrafish is a favorite model organism to study tissue morphogenesis during development at a subcellular level. This largely results from the fact that zebrafish embryos are transparent and thus accessible to various imaging techniques, such as confocal and two-photon excitation (2PE) microscopy. In particular, 2PE microscopy has been shown to be useful for imaging deep cell layers within the embryo and following tissue morphogenesis over long periods. This chapter describes how to use 2PE microscopy to study morphogenetic movements during early zebrafish embryonic development, providing a general blueprint for its use in zebrafish. (Source: Springer protocols feed by Biochemistry) Springer protocols feed by Biochemistry info http://www.medworm.com/rss/comments.php?id=2715422 Thu, 20 Aug 2009 11:31:54 +0100 2715422 http://www.medworm.com/index.php?rid=2715421&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-977-2_16 Zebrafish are ideally suited for the live imaging of early immune cell compartments. Macrophages that initially appear on the yolk surface prior to the onset of circulation are the first functional immune cells within the embryo, predating the emergence of the first granulocytic cells—the heterophilic neutrophils. Both cell types have been shown in zebrafish to contribute to a robust early innate immune system, capable of clearing systemic infections and participating in wound healing. Early imaging of these cells within zebrafish relied on differential interference contrast (DIC) optics because of their superficial locations in the embryo and the optical transparency of embryonic tissues. Recently, the creation of a number of transgenic reporter lines possessing fluorescently marked... Springer protocols feed by Biochemistry info http://www.medworm.com/rss/comments.php?id=2715421 Thu, 20 Aug 2009 11:31:54 +0100 2715421 http://www.medworm.com/index.php?rid=2715420&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-977-2_15 Understanding the dynamics of developmental and cellular processes requires documentation of their changes with appropriate temporal and spatial resolution. Furthermore, simultaneous recording from a population of embryos under identical conditions allows statistical estimates of precision and variability to be made. This chapter describes a protocol for time-lapse microscopy of multiple embryos in parallel developing under tightly controlled conditions. This method is currently best suited to follow tissue-scale morphogenetic movements with temporal resolution in the minute range, for hours or even days. Applications of the method include the comparison of the dynamics of a process of interest between groups of wild-type embryos and their mutant siblings or between embryos treated with di... Springer protocols feed by Biochemistry info http://www.medworm.com/rss/comments.php?id=2715420 Thu, 20 Aug 2009 11:31:54 +0100 2715420 http://www.medworm.com/index.php?rid=2715419&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-977-2_14 We describe a method for a reporter assay based on a fluorescence intensity readout that uses transient techniques in zebrafish to easily deliver the reporter assay components. In addition, we describe a rigorously controlled strategy for determining the bona fide miRNA binding sites in the 3′UTR of mRNAs. (Source: Springer protocols feed by Biochemistry) Springer protocols feed by Biochemistry info http://www.medworm.com/rss/comments.php?id=2715419 Thu, 20 Aug 2009 11:31:54 +0100 2715419 http://www.medworm.com/index.php?rid=2715418&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-977-2_13 In expression microarray experiments, post-hybridization procedures involving data acquisition and analysis are mainly computational and hence are described as “dry-lab procedures.” The aim of these procedures is to acquire the fluorescent signals representing the relative abundance of tens of thousands of RNA species in samples with biological interest, and transform them into meaningful data with biological significance. These procedures begin with image and data acquisition followed by data normalization and processing, and thereafter primary and secondary data analyses. Although the actual data analysis is carried out after the wet-lab procedures, the strategy for data analysis should be planned prior to wet lab procedures when designing the experiment, to ensure effective ... Springer protocols feed by Biochemistry info http://www.medworm.com/rss/comments.php?id=2715418 Thu, 20 Aug 2009 11:31:54 +0100 2715418 http://www.medworm.com/index.php?rid=2715417&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-977-2_12 The availability of microarray technology for zebrafish research has enabled the expression of tens of thousands of genes to be studied simultaneously in one experiment. The experiment usually involves measuring and comparing the relative abundance of tens of thousands of mRNA species in experimental samples obtained from mutant versus wild-type embryos, disease versus normal tissues, embryos/fish of different developmental stages, physiologic states, or from multiple treatments and/or time-points. A microarray experiment comprised of several stages can be divided into two distinct parts (i.e., the “wet-lab” and the “dry-lab”). The success of a microarray experiment hinges on the “wet-lab” procedures, which include technology that allows for generation o... Springer protocols feed by Biochemistry info http://www.medworm.com/rss/comments.php?id=2715417 Thu, 20 Aug 2009 11:31:54 +0100 2715417 http://www.medworm.com/index.php?rid=2715416&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-977-2_9 We describe transgenic methods to express the Escherichia coli nfsB in a tissue restricted manner in the zebrafish. This bacterial gene encodes a nitroreductase (NTR) enzyme that can render prodrugs such as metronidazole (Met) cytotoxic. Using the expression of NTR fused to a fluorescent protein, one can simultaneously make cells susceptible to prodrug treatment and visualize cell ablation as it occurs. (Source: Springer protocols feed by Biochemistry) Springer protocols feed by Biochemistry info http://www.medworm.com/rss/comments.php?id=2715416 Thu, 20 Aug 2009 11:31:54 +0100 2715416 http://www.medworm.com/index.php?rid=2715415&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-977-2_11 Although a common approach in large vertebrate embryos such as chick or frog, manipulation at the tissue level is only rarely applied to zebrafish embryos. Despite its relatively small size, the zebrafish embryo can be readily used for micromanipulations such as tissue and organ primordium transplantation, explantation, and microbead implantation, to study inductive tissue interactions and tissue autonomy of pleiotropic, mutant phenotypes or to isolate tissue for organotypic and primary cell culture or RNA isolation. Since this requires special handling techniques, tools, and tricks, which are rarely published and thus difficult to apply without hands-on demonstration, this article provides detailed instructions and protocols on tissue micromanipulation. The goal is to introduce a broader ... Springer protocols feed by Biochemistry info http://www.medworm.com/rss/comments.php?id=2715415 Thu, 20 Aug 2009 11:31:54 +0100 2715415 http://www.medworm.com/index.php?rid=2715414&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-977-2_10 A method is described that allows the introduction by electroporation of either small dyes or larger RNA, DNA, or morpholino constructs into single cells or small groups of cells in zebrafish embryos or larvae. The dye or construct is delivered to cells via a patch-like microelectrode that also delivers the electroporation stimulus train. This technique allows the experimenter to target cells of their choice at a particular time of development and at a particular location in the embryo, and is useful for fate mapping, analysing neuronal organisation, ectopic expression and gene knockdown experiments. (Source: Springer protocols feed by Biochemistry) Springer protocols feed by Biochemistry info http://www.medworm.com/rss/comments.php?id=2715414 Thu, 20 Aug 2009 11:31:54 +0100 2715414 http://www.medworm.com/index.php?rid=2715413&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-977-2_8 In the past, microinjection of plasmid DNA into early embryos represented the state of the art to generate transgenic zebrafish. However, this approach suffers significant drawbacks (mosaic distribution of the injected transgene, late transgene integration at high copy numbers, low transgenesis frequency), making the generation of transgenic lines a laborious task. Coinjection of I-SceI meganuclease with a reporter construct flanked by I-SceI sites overcomes these problems by earlier transgene integration into the host genome. Here, we provide an optimized protocol for I-SceI meganuclease-mediated transgenesis in zebrafish. This simple protocol provides a reliable method to transiently test tissue-specific reporter expression of meganuclease constructs in injected embryos (F0). Furthermore... Springer protocols feed by Biochemistry info http://www.medworm.com/rss/comments.php?id=2715413 Thu, 20 Aug 2009 11:31:54 +0100 2715413 http://www.medworm.com/index.php?rid=2715412&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-977-2_7 Transgenesis using bacterial artificial chromosomes (BAC) allows greater fidelity in directing desirable expression of transgenes. Application of this technology in the optically transparent zebrafish with fluorescent protein reporters enables unparalleled visual analysis of gene expression in a living organism. We have developed a streamlined procedure of directly selecting multiple BAC clones based on public sequence databases followed by rapid modification with green fluorescent protein or red fluorescent protein for transgenic analysis in zebrafish. In this chapter, experimental procedures for BAC DNA preparation and generation of transgenic zebrafish lines by microinjection are described. (Source: Springer protocols feed by Biochemistry) Springer protocols feed by Biochemistry info http://www.medworm.com/rss/comments.php?id=2715412 Thu, 20 Aug 2009 11:31:54 +0100 2715412 http://www.medworm.com/index.php?rid=2715411&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-977-2_6 The Tol2 transposon system can create insertions in the zebrafish genome efficiently. By using this system, the gene trap and enhancer trap methods have been developed. The gene trap and enhancer trap constructs contain the green fluorescent protein (GFP) reporter gene or the yeast Gal4 transcription activator gene. By creating random integrations of these constructs in the genome, transgenic fish expressing the GFP gene or the Gal4 gene in specific cells, tissues or organs are generated. These fish are valuable resources for developmental biology. Especially, the Gal4-expressing transgenic fish can be used to ectopically express any gene of interest placed downstream of the Gal4 recognition sequence, UAS, and thereby allow visualization, modification or ablation of the Gal4-expressing cel... Springer protocols feed by Biochemistry info http://www.medworm.com/rss/comments.php?id=2715411 Thu, 20 Aug 2009 11:31:54 +0100 2715411 http://www.medworm.com/index.php?rid=2715410&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-977-2_5 Transgenesis is an important methodology for studying the function of genes and genomes in model plants and animals. For the model vertebrate zebrafish, methods using the Tol2 transposable element have been developed for this purpose. With these methods, the function of the transgene can be analyzed in both transient and stable transgenic fish. Recently, cis-sequences necessary for transposition of the Tol2 element were revealed. This enabled development of transposon vectors containing minimal DNA sequences that are easily manipulated. More recently, several transposon vectors containing the Gateway sequence were created and reported. These are useful because any foreign sequences can be cloned into a transposon vector fairly easily and rapidly. This chapter describes the features of thes... Springer protocols feed by Biochemistry info http://www.medworm.com/rss/comments.php?id=2715410 Thu, 20 Aug 2009 11:31:54 +0100 2715410 http://www.medworm.com/index.php?rid=2715409&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-977-2_4 In recent decades, laboratories throughout the world generated several thousand mutant, transgenic, and wild-type zebrafish lines and more lines continue to be produced. At the same time, relatively little effort has been expended to develop reliable, high-throughput, standardized, long-term cryopreservation storage methods, even though laboratories and the research community as a whole struggle to maintain the large number of lines alive. Safe and reliable methods for maintaining these valuable genetic resources are vital for future biomedical research. (Source: Springer protocols feed by Biochemistry) Springer protocols feed by Biochemistry info http://www.medworm.com/rss/comments.php?id=2715409 Thu, 20 Aug 2009 11:31:54 +0100 2715409 http://www.medworm.com/index.php?rid=2715408&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-977-2_3 Chemical mutagenesis using N-ethyl-N-nitrosourea is the current method of choice for dense mutagenesis in zebrafish. Methods are available for both pre-meiotic and post-meiotic sperm mutagenesis; in this chapter, pre-meiotic mutagenesis is described. Mutated males are crossed with untreated females to create an F1 generation that is heterozygous for the mutations. The F1 females can be screened directly by making haploid embryos using in vitro fertilization (IVF) with ultraviolet (UV)-irradiated sperm. This approach requires substantially fewer fish and less aquarium space than the classical F2 generation screen and is feasible for a small research group. Production of haploid embryos is described in detail. (Source: Springer protocols feed by Biochemistry) Springer protocols feed by Biochemistry info http://www.medworm.com/rss/comments.php?id=2715408 Thu, 20 Aug 2009 11:31:54 +0100 2715408 http://www.medworm.com/index.php?rid=2715407&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-977-2_2 We describe a quantitative PCR (qPCR)-based assay as a quick way to assess the infectivity after each round of viral production and injection. Most of the required equipment is commercially available and commonly present in most research laboratories. (Source: Springer protocols feed by Biochemistry) Springer protocols feed by Biochemistry info http://www.medworm.com/rss/comments.php?id=2715407 Thu, 20 Aug 2009 11:31:54 +0100 2715407 http://www.medworm.com/index.php?rid=2715406&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-977-2_1 ENU (N-ethyl-N-nitrosourea) mutagenesis is a widely accepted and proven method to introduce random point mutations in the genome. Because there are no targeted knockout strategies available for zebrafish so far, random mutagenesis is currently the preferred method in both forward and reverse genetic approaches. To obtain high-density mutagenized zebrafish, six consecutive ENU treatments are applied at weekly intervals to adult male zebrafish by bathing them in ENU solution. With this procedure an average germ line mutation load of one mutation every 1.0 × 105–1.5 × 105 basepairs is reached routinely in our lab. (Source: Springer protocols feed by Biochemistry) Springer protocols feed by Biochemistry info http://www.medworm.com/rss/comments.php?id=2715406 Thu, 20 Aug 2009 11:31:53 +0100 2715406 http://www.medworm.com/index.php?rid=2875136&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-483-4_10 We present the details of the measurement of the duplex survival probability under force, and show that dissociation time scales for duplexes that are perfectly complimentary differ by greater than approximately two orders of magnitude from those containing a single sequence mismatch, offering opportunities for sequence detection. (Source: Springer protocols feed by Biochemistry) Springer protocols feed by Biochemistry info http://www.medworm.com/rss/comments.php?id=2875136 Sun, 31 May 2009 23:00:00 +0100 2875136 http://www.medworm.com/index.php?rid=2777776&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-483-4_4 Plastic microfluidic devices are fabricated with an array of pseudo-valves for two-dimensional (2D) protein separation. The devices are made by compression molding; the mold is created by electroplating on a glass master fabricated by photolithography. Each device consists of one channel for isoelectric focusing (IEF) and multiple parallel channels for polyacrylamide gel electrophoresis (PAGE). The IEF channel (first dimension) is orthogonal to the PAGE channels (second dimension). Microfluidic pseudo-valves are created at the intersections of orthogonal channels by photodefinable, in situ gel polymerization. These valves enable the introduction of two types of separation media into orthogonal channels for performing 2D protein separation in the device. The presence of the pseudo-valves pr... Springer protocols feed by Biochemistry info http://www.medworm.com/rss/comments.php?id=2777776 Sun, 31 May 2009 23:00:00 +0100 2777776 http://www.medworm.com/index.php?rid=2777775&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-483-4_37 Targeted cancer therapy allows the delivery of therapeutic agents to cancer cells without incurring undesirable side effects on the neighboring healthy tissues. Over the past decade, there has been an increasing interest in the development of advanced cancer therapeutics using targeted nanoparticles. Here we describe the preparation of drug-encapsulated nanoparticles formulated with biocompatible and biodegradable poly(d,l-lactic-co-glycolic acid)-block-poly(ethylene glycol) (PLGA-b-PEG) copolymer and surface functionalized with the A10 2-fluoropyrimidine ribonucleic acid aptamers that recognize the extracellular domain of prostate-specific membrane antigen (PSMA), a well-characterized antigen expressed on the surface of prostate cancer cells. We show that the self-assembled nanoparticles ... Springer protocols feed by Biochemistry info http://www.medworm.com/rss/comments.php?id=2777775 Sun, 31 May 2009 23:00:00 +0100 2777775 http://www.medworm.com/index.php?rid=2777774&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-483-4_38 Examining messenger RNA (mRNA) expression is useful for the determination of cell and tissue conditions. Many methods of determining mRNA expression require total RNA extraction or cell fixation. These processes cause difficulties in examining mRNA expression in single living cells without causing cell death. We think that analysis of specific mRNA expression in single living cells will become important in cell biology. In this chapter, we present a method to examine mRNA expression of single living cells without killing the cells. The single-cell nanoprobe (SCN) method uses atomic force microscopy (AFM) to extract mRNA. We also present examples of β-actin mRNA detection and multiple mRNA detection from single living cells. (Source: Springer protocols feed by Biochemistry) Springer protocols feed by Biochemistry info http://www.medworm.com/rss/comments.php?id=2777774 Sun, 31 May 2009 23:00:00 +0100 2777774 http://www.medworm.com/index.php?rid=2777773&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-483-4_40 Zinc finger nucleases (ZFNs) are custom-designed molecular scissors, engineered to cut at specific DNA sequences. ZFNs combine the zinc finger proteins (ZFPs) with the nonspecific cleavage domain of the FokI restriction enzyme. The DNA-binding specificity of ZFNs can be easily altered experimentally. This easy manipulation of the ZFN recognition specificity enables one to deliver a targeted double-strand break (DSB) to a genome. The targeted DSB stimulates local gene targeting by several orders of magnitude at that specific cut site via homologous recombination (HR). Thus, ZFNs have become an important experimental tool to make site-specific and permanent alterations to genomes of not only plants and mammals but also of many other organisms. Engineering of custom ZFNs involves many steps. ... Springer protocols feed by Biochemistry info http://www.medworm.com/rss/comments.php?id=2777773 Sun, 31 May 2009 23:00:00 +0100 2777773 http://www.medworm.com/index.php?rid=2777772&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-483-4_39 We present a method for reverse transfection using a gold colloid (GC) as a nanoscaffold by generating nanoclusters of the DNA/reagentcomplex on a glass surface, which could then be used for the regulation of the particle size of the complex and delivery of DNA into nuclei. With this method, we have found that the conjugation of gold nanoparticles (20 nm in particle size) to the pEGFP-N1/Jet–PEI complex resulted in an increase in the intensity of fluorescence of enhanced green fluorescent protein (EGFP) (based on the efficiency of transfection) from human mesenchymal stem cells (hMSCs), as compared with the control without GC. In this manner, we constructed a method for reverse transfection using GC to deliver genes into the cells effectively. (Source: Springer protocols feed by Bioc... Springer protocols feed by Biochemistry info http://www.medworm.com/rss/comments.php?id=2777772 Sun, 31 May 2009 23:00:00 +0100 2777772 http://www.medworm.com/index.php?rid=2777771&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-483-4_41 Artificial transcription factors (ATFs) are designed to mimic natural transcription factors in the control of gene expression and are comprised of domains for DNA binding and gene regulation. ATF domains are modular, interchangeable, and can be composed of protein-based or nonpeptidic moieties, yielding DNA-interacting regulatory molecules that can either activate or inhibit transcription. Sequence-specific targeting is a key determinant in ATF activity, and DNA-binding domains such as natural zinc fingers and synthetic polyamides have emerged as useful DNA targeting molecules. Defining the comprehensive DNA binding specificity of these targeting molecules for accurate manipulations of the genome can be achieved using cognate site identifier DNA microarrays to explore the entire sequence s... Springer protocols feed by Biochemistry info http://www.medworm.com/rss/comments.php?id=2777771 Sun, 31 May 2009 23:00:00 +0100 2777771 http://www.medworm.com/index.php?rid=2396690&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-261-8_12 Visualization of protein–protein interactions in vivo offers a powerful tool to resolve spatial and temporal aspects of cellular functions. Bimolecular fluorescence complementation (BiFC) makes use of nonfluorescent fragments of green fluorescent protein or its variants that are added as “tags” to target proteins under study. Only upon target protein interaction is a fluorescent protein complex assembled and the site of interaction can be monitored by microscopy. In this chapter, we describe the method and tools for use of BiFC in the yeast Saccharomyces cerevisiae. (Source: Springer protocols feed by Biochemistry) Springer protocols feed by Biochemistry info http://www.medworm.com/rss/comments.php?id=2396690 Fri, 06 Jun 2008 04:00:00 +0100 2396690 http://www.medworm.com/index.php?rid=2396689&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-261-8_3 The transport of membrane-bound proteins through post-Golgi compartments depends on the coordinated function of multiple genes that direct the recognition and routing of protein cargoes to their final cellular destination. As many of these sorting components are nonessential for viability, genome-wide screening of the yeast gene-deletion mutant collection provides a useful strategy for their identification. The potential of this approach is limited only by the availability of transport assays suitable for the high-throughput screening of yeast colony arrays. Two large-scale phenotypic screens to identify novel transport genes are described here. The fluorescence-based Calcofluor white assay identifies mutants with altered plasma membrane localization of the chitin synthase Chs3, which recy... Springer protocols feed by Biochemistry info http://www.medworm.com/rss/comments.php?id=2396689 Fri, 06 Jun 2008 04:00:00 +0100 2396689 http://www.medworm.com/index.php?rid=2396688&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-261-8_2 A variety of Saccharomyces cerevisiae strain libraries allow for systematic analysis of strains bearing gene deletions, repressible genes, overexpressed genes, or modified genes on a genome-wide scale. Here we introduce a method for culturing yeast strains in 96-well format to achieve log-phase growth and a high-throughput technique for generating whole-cell protein extracts from these cultures using sodium dodecyl sulfate and heat lysis. We subsequently describe a procedure to analyze these whole-cell extracts by immunoblotting for alkaline phosphatase and carboxypeptidase yscS to identify strains with defects in protein transport pathways or protein glycosylation. These methods should be readily adaptable to many different areas of interest. (Source: Springer protocols feed by Biochemist... Springer protocols feed by Biochemistry info http://www.medworm.com/rss/comments.php?id=2396688 Fri, 06 Jun 2008 04:00:00 +0100 2396688 http://www.medworm.com/index.php?rid=2396687&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-261-8_4 Genetic approaches have revealed more than 50 genes involved in the delivery of soluble zymogens like carboxypeptidase Y (CPY) to the lysosome-like vacuole in Saccharomyces cerevisiae. At least 20 of these genes function in transport between the prevacuolar endosome-like compartment (PVC) and the vacuole. To gain biochemical access to these functions, the authors developed a cell-free assay that measures transport-coupled proteolytic maturation of soluble zymogens in vitro. A polycarbonate filter with a defined pore size is used to lyse yeast spheroplasts after pulse-chase radiolabeling. Differential centrifugation enriches for PVCs containing proCPY (p2CPY) in a 125,000g membrane pellet and is used as donor membranes. Nonradiolabeled spheroplasts are also lysed with a polycarbonate filter... Springer protocols feed by Biochemistry info http://www.medworm.com/rss/comments.php?id=2396687 Fri, 06 Jun 2008 04:00:00 +0100 2396687 http://www.medworm.com/index.php?rid=2396686&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-261-8_5 The import of precursor proteins into mitochondria represents a cell biological process that is absolutely required for the survival of an eukaryotic cell. A complex chain of reactions needs to be followed to achieve a successful transport of mitochondrial proteins from the cytosol through the double membrane system to their final destination. In order to elucidate the details of the translocation process, in vitro import assays have been developed that are based on the incubation of isolated active mitochondria with natural or artificial precursor proteins containing the appropriate targeting information. Although most of the protein components of the import machinery have been identified and functionally characterized using this basic system, the definition of the molecular mechanisms re... Springer protocols feed by Biochemistry info http://www.medworm.com/rss/comments.php?id=2396686 Fri, 06 Jun 2008 04:00:00 +0100 2396686 http://www.medworm.com/index.php?rid=2396685&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-261-8_6 Import of proteins is of vital importance for the biogenesis of mitochondria. The vast majority of mitochondrial proteins is encoded within the nuclear genome and translocated into various mitochondrial compartments after translation in the cytosol as preproteins. Even in rather primitive eukaryotes like yeasts, these are 700 to 1,000 different proteins, whereas only a handful of proteins is encoded in the mitochondrial DNA. In vitro import studies are important tools to understand import mechanisms and pathways. Using isolated mitochondria and radioactively labeled precursor proteins, it was possible to identify several import machineries and pathways consisting of a large number of components during the last decades. (Source: Springer protocols feed by Biochemistry) Springer protocols feed by Biochemistry info http://www.medworm.com/rss/comments.php?id=2396685 Fri, 06 Jun 2008 04:00:00 +0100 2396685 http://www.medworm.com/index.php?rid=2396684&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-261-8_8 Mitochondrial biogenesis requires the contribution of two genomes and of two compartmentalized protein synthesis systems (nuclear and mitochondrial). Mitochondrial protein synthesis is unique on many respects, including the use of a genetic code with deviations from the universal code, the use of a restricted number of transfer RNAs, and because of the large number of nuclear encoded factors involved in assembly of the mitochondrial biosynthetic apparatus. The mitochondrial biosynthetic apparatus is involved in the actual synthesis of a handful of proteins encoded in the mitochondrial DNA. The budding yeast Saccharomyces cerevisiae and the fission yeast Schizosaccharomyces pombe are excellent models to identify and study factors required for mitochondrial translation. For that purpose, in ... Springer protocols feed by Biochemistry info http://www.medworm.com/rss/comments.php?id=2396684 Fri, 06 Jun 2008 04:00:00 +0100 2396684 http://www.medworm.com/index.php?rid=2396683&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-261-8_7 Mitochondria are essential organelles of eukaryotic cells. The biogenesis of mitochondria depends on the coordinated function of two separate genetic systems: one in the nucleus and one in the organelle. The study of mitochondria requires the analysis of both genetic systems and their protein products. In this chapter, we focus on the translation and sorting of mitochondrially encoded proteins into the mitochondrial inner membrane in the baker’s yeast Saccharomyces cerevisiae. The starting point is the labeling of these proteins, followed by some of the methods developed to investigate their topology and membrane incorporation. The methods described here can be applied also to the study of other aspects of organelle biogenesis such as folding, assembly, and degradation of proteins. (... Springer protocols feed by Biochemistry info http://www.medworm.com/rss/comments.php?id=2396683 Fri, 06 Jun 2008 04:00:00 +0100 2396683 http://www.medworm.com/index.php?rid=2396682&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-261-8_9 Biogenesis of the mitochondrial respiratory chain enzymes involves the coordinated action of the mitochondrial and nuclear genomes. As a matter of fact, the structural subunits forming these multimeric enzymes are encoded in both genomes. In addition, the assistance of nuclear encoded factors, termed assembly factors, is necessary to allow for the expression of the mitochondrial DNA-encoded subunits and to facilitate their maturation, membrane insertion, and further assembly into the corresponding enzymatic complex. These processes involve transient interactions among the newly synthesized mitochondrial products and specific assembly factors. The identification and characterization of these interactions can be achieved by the method described here, consisting of pulling down tagged version... Springer protocols feed by Biochemistry info http://www.medworm.com/rss/comments.php?id=2396682 Fri, 06 Jun 2008 04:00:00 +0100 2396682 http://www.medworm.com/index.php?rid=2396681&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-261-8_13 We present here an overview of the principles involved in doing quantitative fluorescence microscopy. We illustrate these with examples drawn from our work with the Golgi apparatus and endosomes in cultured mammalian cells. The principles themselves can be applied to any system. (Source: Springer protocols feed by Biochemistry) Springer protocols feed by Biochemistry info http://www.medworm.com/rss/comments.php?id=2396681 Fri, 06 Jun 2008 04:00:00 +0100 2396681 http://www.medworm.com/index.php?rid=2396680&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-261-8_14 In this chapter the authors describe automated imaging methods to quantify the transport rates of transmembrane as well as soluble cargo, and to evaluate the integrity of the Golgi complex. The quantification of cargo transport rates serves as an example of fluorescence intensity-based assays, the quantification of the Golgi complex integrity—as an example of morphology-based assays. These quantitative assays could be applied for single experiments as well as for middle- and high-throughput screening approaches. Each of these assays can be used to appreciate effects caused by gene silencing by RNAi, cDNA overexpression or application of chemical compounds. For each assay the authors discuss protocols for sample preparation, parameters for automated image acquisition, strategies of im... Springer protocols feed by Biochemistry info http://www.medworm.com/rss/comments.php?id=2396680 Fri, 06 Jun 2008 04:00:00 +0100 2396680 http://www.medworm.com/index.php?rid=2396679&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-261-8_15 Correlative microscopy is a hybrid method that allows the localization of events observed under visible, ultraviolet, or infrared light, at molecular and submolecular levels, combining two microscopy techniques. However, the main limitation of correlative microscopy is to develop a labeling technique that can be easily used first in light and then in electron microscopy. Laser etching is a well-established method to create precisely designed shapes or volumes in various materials including glass. We have applied this technique to develop a new correlative light and electron microscopy method and to apply it in our study of the Golgi apparatus. The location of the cell of interest is laser-inscribed into the glass allowing a simple follow-up in light and fluorescence microscopy. Furthermore... Springer protocols feed by Biochemistry info http://www.medworm.com/rss/comments.php?id=2396679 Fri, 06 Jun 2008 04:00:00 +0100 2396679 http://www.medworm.com/index.php?rid=2396678&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-261-8_17 Intracellular transport between compartments within the cell is generally mediated by membrane vesicles. Their formation is initiated by activation of small GTPases that then recruit cytosolic proteins to the membrane surface to form a coat, interact with cargo proteins, and deform the lipid bilayer. Liposomes proved to be a useful tool to study the molecular mechanisms of these processes in vitro. To analyze the involvement of membrane proteins, the cytosolically exposed sequences may be coupled chemically to reactive lipids in the membrane. Here we describe the use of such peptidoliposomes presenting lipid-coupled cytosolic tails of cargo proteins for the in vitro analysis of the membrane recruitment of AP-1 adaptors in the process of forming AP-1/clathrin coats. AP-1 recruitment is medi... Springer protocols feed by Biochemistry info http://www.medworm.com/rss/comments.php?id=2396678 Fri, 06 Jun 2008 04:00:00 +0100 2396678 http://www.medworm.com/index.php?rid=2396677&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-261-8_16 There have been many indications that kinases play a role in signaling the transport of proteins through the secretory pathway. Specifically, the serine/threonine kinase Akt affects the transport of cholesterol regulatory components from the endoplasmic reticulum (ER) to the Golgi. However, elucidating the target of Akt in this process has proven to be challenging. Here we describe an approach devised to investigate the Akt target(s) based on examining a potential candidate. (Source: Springer protocols feed by Biochemistry) Springer protocols feed by Biochemistry info http://www.medworm.com/rss/comments.php?id=2396677 Fri, 06 Jun 2008 04:00:00 +0100 2396677 http://www.medworm.com/index.php?rid=2396676&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-261-8_18 Lipid-mixing assay is now commonly used to study protein, temperature and ion-dependent membrane fusion events. This assay has been crucial to demonstrate the ability of neuronal and non-neuronal soluble NSF attachment receptor (SNARE) to promote spontaneous fusion of liposomes. This lipid-mixing assay is based on the fluorescence resonance energy transfer (FRET) capability between a donor fluorescent lipid and a quenching lipid. When fusion between donor fluorescent liposomes and nonfluorescent acceptor liposome occurred, FRET decreases. This assay allows a real-time reading of SNARE-mediated liposome fusion. (Source: Springer protocols feed by Biochemistry) Springer protocols feed by Biochemistry info http://www.medworm.com/rss/comments.php?id=2396676 Fri, 06 Jun 2008 04:00:00 +0100 2396676 http://www.medworm.com/index.php?rid=2396675&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-261-8_19 A prerequisite for understanding the cellular functions of an unknown protein is the establishment of its subcellular localization. As increasing numbers of novel proteins of the biosynthetic pathway are currently being identified, accessible new methods are required to facilitate their localization. Differentiating rat pheochromocytoma (PC12) cells reorganize their biosynthetic membrane compartments as they develop neurite-like processes. The authors recently showed that polarization of these cells involves the expansion of the intermediate compartment (IC) between the rough endoplasmic reticulum (RER) and the Golgi apparatus. Tubules emerging from the vacuolar parts of the IC move to the developing neurites accumulating in their growth cones, whereas the vacuoles, like RER and Golgi, rem... Springer protocols feed by Biochemistry info http://www.medworm.com/rss/comments.php?id=2396675 Fri, 06 Jun 2008 04:00:00 +0100 2396675 http://www.medworm.com/index.php?rid=2396674&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-261-8_20 The epithelial to mesenchymal transition (EMT) is the breakdown of epithelial cell morphology that gives way to a more mobile, mesenchymal phenotype. Although this process is fundamental to the development of multicellular organisms, it is also a key occurrence in many diseases, including cancers of epithelial origin E-cadherin is a central component of adherens junctions (AJs), which act as structural and signaling hubs in epithelial cells that oppose EMT. The loss of E-cadherin from the plasma membrane is an early indication of EMT and a marker of poor prognosis in many cancers making the trafficking of E-cadherin an area of great interest. Recent work from the authors’ laboratory has established the role of type Iγ phosphatidylinositol 4-phosphate 5-kinase (PIPKIγ) in ... Springer protocols feed by Biochemistry info http://www.medworm.com/rss/comments.php?id=2396674 Fri, 06 Jun 2008 04:00:00 +0100 2396674 http://www.medworm.com/index.php?rid=2396673&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-261-8_21 Controlled nucleocytoplasmic localization regulates activity of NFκB as well as other transcription factors. Analysis of the nucleocytoplasmic protein shuttling has been greatly facilitated by the use of leptomycin B (LMB), an inhibitor of CRM1-dependent nuclear export. The authors have previously shown that LMB inhibits NFκB activity in human neutrophils by increasing the nuclear accumulation of NFκB inhibitor, IκBα. In this chapter, the authors describe a protocol that uses LMB to study the nucleocytoplasmic shuttling of IκBα in human macrophage-like U937 cells, thus inhibiting NFκB activity. This protocol should be readily adaptable to analyze the nucleocytoplasmic shuttling of other proteins in human leukocytes. (Source: Springer protocol... Springer protocols feed by Biochemistry info http://www.medworm.com/rss/comments.php?id=2396673 Fri, 06 Jun 2008 04:00:00 +0100 2396673 http://www.medworm.com/index.php?rid=2396672&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-261-8_22 Accurate measurement of synaptic vesicle exocytosis and endocytosis is crucial to understanding the molecular basis of synaptic transmission. The fusion of a pH-sensitive green fluorescent protein (pHluorin) to various synaptic vesicle proteins has allowed the study of synaptic vesicle recycling in real time. Two such probes, synaptopHluorin and sypHy, have been imaged at synapses of hippocampal neurons in culture. The combination of these reporters with techniques for molecular interference, such as RNAi allows for the study of molecules involved in synaptic vesicle recycling. Here the authors describe methods for the culture and transfection of hippocampal neurons, imaging of pHluorin-based probes at synapses and analysis of pHluorin signals down to the resolution of individual synaptic ... Springer protocols feed by Biochemistry info http://www.medworm.com/rss/comments.php?id=2396672 Fri, 06 Jun 2008 04:00:00 +0100 2396672 http://www.medworm.com/index.php?rid=2396671&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-261-8_23 The internalization of activated receptor tyrosine kinases (RTKs) by endocytosis and their subsequent down regulation in lysosomes plays a critical role in regulating the duration and intensity of downstream signaling events. Uncoupling of the RTK cMet from ligand-induced degradation was recently shown to correlate with sustained receptor signaling and increased cell tumorigenicity, suggesting that the corruption of these endocytic mechanisms could contribute to increased cMet signaling in metastatic cancers. To understand how cMet signaling for normal cell growth is controlled by endocytosis and how these mechanisms are dysregulated in metastatic cancers, we developed flow cytometry-based assays to examine cMet internalization. (Source: Springer protocols feed by Biochemistry) Springer protocols feed by Biochemistry info http://www.medworm.com/rss/comments.php?id=2396671 Fri, 06 Jun 2008 04:00:00 +0100 2396671 http://www.medworm.com/index.php?rid=2396670&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-261-8_24 Receptor trafficking is essential to the delivery of nutrients and to the proper regulation of signaling pathways in mammalian cells. Numerous transmembrane receptors undergo clathrin-mediated endocytosis, followed by sorting in the early endosome. The low-density lipoprotein (LDL) receptor-related protein (LRP) is a multiligand endocytic receptor and a member of the LDL receptor family. At the cell surface, it binds to and continuously internalizes numerous ligands including lipoproteins, proteases, protease inhibitors, growth factors, and β-amyloid precursor protein via clathrin-mediated endocytosis. Its rapid endocytosis rate allows efficient clearance of extracellular and transmembrane ligands. Once internalized into the early or sorting endosome, LRP ligands are delivered to lyso... Springer protocols feed by Biochemistry info http://www.medworm.com/rss/comments.php?id=2396670 Fri, 06 Jun 2008 04:00:00 +0100 2396670 http://www.medworm.com/index.php?rid=2396669&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-261-_26 Insulin regulates the glucose uptake in muscle and adipose cells by acutely modulating the amount of the GLUT4 glucose transporter in the plasma membrane. The steady-state cell surface distribution of a membrane protein is an equilibrium between exocytosis and endocytosis. The authors study the effect of insulin on GLUT4 using quantitative immunofluorescence microscopy adapted to single-cell analysis. They use an HA-GLUT4-GFP reporter molecule as a surrogate of GLUT4 trafficking. Insulin induces an increase of GLUT4 in the plasma membrane by both increasing GLUT4 exocytosis and decreasing its endocytosis. Quantitative immunofluorescence techniques such as those described in this review can be used to study the trafficking of virtually any membrane protein. (Source: Springer protocols feed ... Springer protocols feed by Biochemistry info http://www.medworm.com/rss/comments.php?id=2396669 Fri, 06 Jun 2008 04:00:00 +0100 2396669 http://www.medworm.com/index.php?rid=2396668&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-261-8_25 Neurons transmit information by exocytosis of synaptic vesicles (SV), which contain neurotransmitter. Exocytosis is followed by efficient retrieval of the plasma membrane by endocytosis to generate a new SV. SV retrieval supports multiple cycles of synaptic transmission. Over the years, styryl dyes have been widely used to probe the mechanism of SV recycling in the processes of cultured neurons. The styryl dye method is complementary to electrophysiological measurements or genetic reporter approaches. Owing to their ease to culture, cerebellar granule neurons provide a robust neuronal model system for the assay. These cells are readily transfected with various DNA constructs to study the function of exocytic or endocytic proteins in SV recycling. (Source: Springer protocols feed by Biochem... Springer protocols feed by Biochemistry info http://www.medworm.com/rss/comments.php?id=2396668 Fri, 06 Jun 2008 04:00:00 +0100 2396668 http://www.medworm.com/index.php?rid=2396667&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-261-8_27 Postprandial blood glucose homeostasis is regulated by an insulin-stimulated increase in glucose transport into muscle and fat tissues via glucose transporter isoform 4 (GLUT4). In the basal state, this constitutively recycling membrane protein predominantly resides intracellularly. In order to achieve the insulin-stimulated increase in glucose flux, GLUT4 increases its cell surface abundance at the expense of preformed intracellular depots. By confocal microscopy of cultured L6 muscle cells stably expressing myc-tagged GLUT4 (L6-GLUT4myc), we can visualize the two arms of GLUT4 traffic: exocytosis (movement to the cell surface) and endocytosis (internalization from the cell surface). (Source: Springer protocols feed by Biochemistry) Springer protocols feed by Biochemistry info http://www.medworm.com/rss/comments.php?id=2396667 Fri, 06 Jun 2008 04:00:00 +0100 2396667 http://www.medworm.com/index.php?rid=2396666&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-261-8_28 Animal mitochondria are refractory to transformation. This fact has hampered the study of the oxidative phosphorylation system biogenesis by genetic manipulation of the mitochondrial DNA (mtDNA). In humans, a larger variety of mutants have been obtained from patients with mitochondrial diseases, but still we lack a great portion of the range of potential mutants and there is a major obstacle: Animal models cannot be derived from human mtDNA mutants. Until now the only source of mtDNA mutants in mouse was restricted to some drug-resistant-specific cell lines in which a given mtDNA mutation provided growth advantage in the presence of the inhibitor for a specific complex. To overcome these limitations, the authors have developed a protocol that allows the systematic generation of cells harbo... Springer protocols feed by Biochemistry info http://www.medworm.com/rss/comments.php?id=2396666 Fri, 06 Jun 2008 04:00:00 +0100 2396666 http://www.medworm.com/index.php?rid=1928124&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-398-1_31 Computational methods for predicting protein interaction partners are becoming increasingly popular. Many of them are mature enough to be widely used by molecular biologists who can look for proteins related to the protein of interest in order to infer information about its context in the cell. In this chapter we describe the use of the mirrortree set of programs and related software for predicting protein interactions. They are all based on the idea that interacting or functionally related proteins tend to show similar phylogenetic trees due to coevolution. The basic mirrortree program can be used to calculate the similarity between the phylogenetic trees implicit in the multiple sequence alignments of two protein families. The ECID database contains protein interactions and relationships... Springer protocols feed by Biochemistry info http://www.medworm.com/rss/comments.php?id=1928124 Sun, 01 Jun 2008 04:00:00 +0100 1928124 http://www.medworm.com/index.php?rid=1928123&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-398-1_30 Prism (protein interactions by structural matching) is a system that employs a novel prediction algorithm for protein–protein interactions. It adopts a bottom-up approach that combines structure and sequence conservation in protein interfaces. The algorithm seeks possible binary interactions between proteins through structure similarity and evolutionary conservation of known interfaces. It is composed of a database containing protein interface structures derived from the Protein Data Bank (PDB) and predicted protein–protein interactions. It also provides related information about the proteins and an interactive protein interface viewer. In the current version, 3799 structurally nonredundant interfaces are used to predict the interactions among 6170 proteins. A substantial numbe... Springer protocols feed by Biochemistry info http://www.medworm.com/rss/comments.php?id=1928123 Sun, 01 Jun 2008 04:00:00 +0100 1928123 http://www.medworm.com/index.php?rid=1928122&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-398-1_3 An understanding of gene function requires a complementation of gene and gene expression analysis by the systematic analysis of proteins. Progress in plant proteomics has been lagging behind animal and microbial proteomics due to the lack of plant genome data and the problems involved in successful protein extraction from plant material. With the sequencing of more and more plant genomes, this slow progress will soon be overcome. The moss Physcomitrella patens is a model organism in the field of plant functional genomics. P. patens is the first seedless plant for which the complete genome was sequenced. Genome annotation is currently in progress. While identification of proteins requires knowledge of all coding genes of the organism under study, gene annotation and functional characterizat... Springer protocols feed by Biochemistry info http://www.medworm.com/rss/comments.php?id=1928122 Sun, 01 Jun 2008 04:00:00 +0100 1928122 http://www.medworm.com/index.php?rid=1928121&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-398-1_29 Structural designability is the number of ways it is possible to encode for structure. A protein’s designability has been equated with the size of sequence space encoding for the protein’s structure, a measure that reflects the structure’s robustness to mutation. Current evidence suggests that designability is fundamental to our understanding of the evolvability and distribution of structures in nature and is a significant factor associated with human disease. Here, we describe definitions and principles underlying the concept of designability and discuss its relation to disease. (Source: Springer protocols feed by Biochemistry) Springer protocols feed by Biochemistry info http://www.medworm.com/rss/comments.php?id=1928121 Sun, 01 Jun 2008 04:00:00 +0100 1928121 http://www.medworm.com/index.php?rid=1928120&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-398-1_28 Genome sequencing projects have resulted in a rapid accumulation of predicted protein sequences. With experimentally verified information on protein function lagging far behind, computational methods are used for functional annotation of proteins. Here we describe a number of protocols for protein sequence and structure analysis that can be used to infer function of uncharacterized proteins. These protocols rely on publicly available computational resources and tools and can be utilized by anyone with an Internet access. (Source: Springer protocols feed by Biochemistry) Springer protocols feed by Biochemistry info http://www.medworm.com/rss/comments.php?id=1928120 Sun, 01 Jun 2008 04:00:00 +0100 1928120 http://www.medworm.com/index.php?rid=1928119&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-398-1_27 Proteins perform many important tasks in living organisms, such as catalysis of biochemical reactions, transport of nutrients, and recognition and transmission of signals. The plethora of aspects of the role of any particular protein is referred to as its “function.” One aspect of protein function that has been the target of intensive research by computational biologists is its subcellular localization. Proteins must be localized in the same subcellular compartment to cooperate toward a common physiological function. Aberrant subcellular localization of proteins can result in several diseases, including kidney stones, cancer, and Alzheimer’s disease. To date, sequence homology remains the most widely used method for inferring the function of a protein. However, the applic... Springer protocols feed by Biochemistry info http://www.medworm.com/rss/comments.php?id=1928119 Sun, 01 Jun 2008 04:00:00 +0100 1928119 http://www.medworm.com/index.php?rid=1928118&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-398-1_26 Biomedical knowledge is to a very large extent represented only in textual form. To make this knowledge accessible to humans and/or further automatic processing, text mining applications have been developed. At the end of this chapter we present an overview of the most important open access applications and their functionality. The main part of the paper is devoted to the major problems with which all such applications have to deal. The first problem is terminology processing, i.e., recognizing biomedical terms and identifying their meanings, at least to a certain degree. The second problem is to bring together information units that are distributed over more than one sentence. The task of coreference resolution consists of identifying the entities to which the text refers in different sen... Springer protocols feed by Biochemistry info http://www.medworm.com/rss/comments.php?id=1928118 Sun, 01 Jun 2008 04:00:00 +0100 1928118 http://www.medworm.com/index.php?rid=1928117&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-398-1_25 Protein sequence alignment is the task of identifying evolutionarily or structurally related positions in a collection of amino acid sequences. Although the protein alignment problem has been studied for several decades, many recent studies have demonstrated considerable progress in improving the accuracy or scalability of multiple and pairwise alignment tools, or in expanding the scope of tasks handled by an alignment program. In this chapter, we review state-of-the-art protein sequence alignment and provide practical advice for users of alignment tools. (Source: Springer protocols feed by Biochemistry) Springer protocols feed by Biochemistry info http://www.medworm.com/rss/comments.php?id=1928117 Sun, 01 Jun 2008 04:00:00 +0100 1928117 http://www.medworm.com/index.php?rid=1928116&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-398-1_24 With genome sequencing projects producing huge amounts of sequence data, database sequence similarity search has become a central tool in bioinformatics to identify potentially homologous sequences. It is thus widely used as an initial step for sequence characterization and annotation, phylogeny, genomics, transcriptomics, and proteomics studies. Database similarity search is based upon sequence alignment methods also used in pairwise sequence comparison. Sequence alignment can be global (whole sequence alignment) or local (partial sequence alignment) and there are algorithms to find the optimal alignment given particular comparison criteria. However, as database searches require the comparison of the query sequence with every single sequence in the database, heuristic algorithms have been... Springer protocols feed by Biochemistry info http://www.medworm.com/rss/comments.php?id=1928116 Sun, 01 Jun 2008 04:00:00 +0100 1928116 http://www.medworm.com/index.php?rid=1928115&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-398-1_23 MassSorter is a software tool that sorts, systemizes, and analyzes data from peptide mass fingerprinting (PMF) experiments on proteins with known amino acid sequences. Several experiments can be simultaneously analyzed for sequence coverage and posttranslational modifications occurring during sample handling, induced chemical modifications, and unexpected cleavages. Experimental m/z values are compared with m/z values from an in silico digestion, taking modifications into account. Filters can be defined by users for marking autolytic protease peaks and other contaminating peaks. MassSorter functions as a database of all the detected peptides. It includes tools for visualization of the results, such as sequence coverage, accuracy plots, statistics, and 3D models. (Source: Springer protocols... Springer protocols feed by Biochemistry info http://www.medworm.com/rss/comments.php?id=1928115 Sun, 01 Jun 2008 04:00:00 +0100 1928115 http://www.medworm.com/index.php?rid=1928114&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-398-1_22 One fundamental problem in proteomics study is to identify proteins and determine their expression levels in cells. Coupled with advanced liquid chromatography, tandem mass spectrometry has become the standard tool for peptide sequencing. In the past decade, many different algorithms and software packages have been developed to support high-throughput proteomics studies. This chapter reviews and compares the computational methods and software for the interpretation of tandem mass spectra. We also present techniques to assess the reliability of peptide identification. Finally, future directions and new research paradigms in tandem mass spectrometry are discussed. (Source: Springer protocols feed by Biochemistry) Springer protocols feed by Biochemistry info http://www.medworm.com/rss/comments.php?id=1928114 Sun, 01 Jun 2008 04:00:00 +0100 1928114 http://www.medworm.com/index.php?rid=1928113&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-398-1_21 Driven by advances in mass spectrometry and analytical chemistry, coupled with the expanding number of completely sequenced genomes, proteomics is becoming a widely exploited technology for characterizing the proteins found in living systems. As proteomics becomes increasingly more high-throughput there is a parallel need for storage of the large quantities of data generated, to support data exchange and allow further analyses. The capture and storage of such data, along with subsequent release and dissemination, not only aid in sharing of the data throughout the proteomics community but also provide scientific insights into the observations between different laboratories, instruments, and software. Growing numbers of resources offer a range of approaches for the capture, storage, and diss... Springer protocols feed by Biochemistry info http://www.medworm.com/rss/comments.php?id=1928113 Sun, 01 Jun 2008 04:00:00 +0100 1928113 http://www.medworm.com/index.php?rid=1928112&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-398-1_20 Many fundamental processes involve protein–protein interactions. Recent advances in technology make it possible to perform large–scale, genome–wide interaction mapping experiments that result in an always increasing amount of data. Protein–protein interaction databases are thus becoming a major resource for investigating biological networks and pathways. In this chapter we describe the Molecular INTeraction database (MINT). The MINT database aims at storing, in a structured format, information about protein–protein interactions (PPIs) by extracting experimental details from work published in peer–reviewed journals. (Source: Springer protocols feed by Biochemistry) Springer protocols feed by Biochemistry info http://www.medworm.com/rss/comments.php?id=1928112 Sun, 01 Jun 2008 04:00:00 +0100 1928112 http://www.medworm.com/index.php?rid=1928111&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-398-1_2 Within ecosystems microorganisms coexist and interact. Knowledge of these interactions is of great importance in the fields of ecology, food production, and medicine. Such interactions often involve the synthesis of antibiotic secondary metabolites. Different kinds of s molecules or direct contacts are other forms of microbial interactions. Recently, modern molecular methods such as microarrays and proteomics have been employed to investigate such interactions. In this chapter, the use of proteomics for studies of microbial interactions is discussed. The choice of experimental setup is dependent on the aims of the specific study. One aspect of competition between microbes can be simulated by treatment of one microbe with antibiotics produced by a competing microbe. A more complicated appro... Springer protocols feed by Biochemistry info http://www.medworm.com/rss/comments.php?id=1928111 Sun, 01 Jun 2008 04:00:00 +0100 1928111 http://www.medworm.com/index.php?rid=1928110&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-398-1_19 The PRIDE database has been developed to allow the proteomics community to share publicly, or within private collaborations, the vast volume of data generated by proteomics laboratories across the globe. These data are being generated at an expanding rate as increasingly sophisticated technologies become available. Compounding this problem, the infrastructure and techniques used to generate these data vary in terms of the instrumentation used, the protein sequence databases searched, the search engines employed, and the automatic or manual filtering of identifications following the initial automated search. The PRIDE project provides an infrastructure to solve these problems, including a generic, standards-based format that can be annotated to capture data generated using any proteomics pi... Springer protocols feed by Biochemistry info http://www.medworm.com/rss/comments.php?id=1928110 Sun, 01 Jun 2008 04:00:00 +0100 1928110 http://www.medworm.com/index.php?rid=1928109&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-398-1_18 We describe the origins and overall concepts behind these standards, as well as the individual efforts that are ongoing in the field of mass spectrometry proteomics and protein interactions. (Source: Springer protocols feed by Biochemistry) Springer protocols feed by Biochemistry info http://www.medworm.com/rss/comments.php?id=1928109 Sun, 01 Jun 2008 04:00:00 +0100 1928109 http://www.medworm.com/index.php?rid=1928108&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-398-1_17 Glycoproteins constitute a large fraction of the proteome. The fundamental role of protein glycosylation in cellular development, growth, and differentiation, tissue development, and in host–pathogen interactions is by now widely accepted. Proteome–wide characterization of glycoproteins is a complex task and is currently achieved by mass spectrometry–based methods that enable identification of glycoproteins and localization, classification, and analysis of individual glycan structures on proteins. In this chapter we briefly introduce a range of analytical technologies for recovery and analysis of glycoproteins and glycopeptides. Combinations of affinity–enrichment techniques, chemical and biochemical protocols, and advanced mass spectrometry facilitate detailed glyc... Springer protocols feed by Biochemistry info http://www.medworm.com/rss/comments.php?id=1928108 Sun, 01 Jun 2008 04:00:00 +0100 1928108 http://www.medworm.com/index.php?rid=1928107&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-398-1_16 We describe a method for the specific isolation of representative N-terminal peptides of proteins and their proteolytic fragments. Their isolation is based on a gel-free, peptidecentric proteomics approach using the principle of diagonal chromatography. We will indicate that the introduction of an altered chemical property to internal peptides holding a free α-N-terminus results in altered column retention of these peptides, thereby enabling the isolation and further characterization by mass spectrometry of N-terminal peptides. Besides pointing to changes in protein expression levels when performing such proteome surveys in a differential modus, protease specificity and substrate repertoires can be allocated since both are specified by neo-N-termini generated after a protease cleavag... Springer protocols feed by Biochemistry info http://www.medworm.com/rss/comments.php?id=1928107 Sun, 01 Jun 2008 04:00:00 +0100 1928107 http://www.medworm.com/index.php?rid=1928106&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-398-1_15 In the present chapter we detail how mass spectrometry (MS) can be used to characterize noncovalent complexes, especially multimeric proteins and protein/ligand complexes. This original application of MS, also called “supramolecular MS” or “nondenaturing MS,” appeared in the early 1990s and has continuously evolved since then. Nondenaturing MS is now fully integrated in structural biology programs and in drug discovery platforms. Indeed, appropriate sample preparation and fine tuning of the instrument make it possible to transfer weak assemblieswithout disruption fromsolution into the gas phase of themass spectrometer. In this chapterwe detail experimental conditions (sample preparation, optimization of instrumental parameters, etc.) required for the detection of no... Springer protocols feed by Biochemistry info http://www.medworm.com/rss/comments.php?id=1928106 Sun, 01 Jun 2008 04:00:00 +0100 1928106 http://www.medworm.com/index.php?rid=1928105&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-398-1_14 Protein arrays make possible the functional screening of large numbers of immobilized proteins in parallel. To facilitate the supply of proteins and to avoid their deterioration on storage, we describe our protein in situ array (PISA) method for production of protein arrays in a single step directly from PCR DNA, using cell-free transcription and translation. In PISA, the in vitro-generated proteins are immobilized, as they are formed, on the surface of wells, beads, or slides coated with a protein-capturing reagent. In our preferred method, proteins are tagged with a double-hexahistidine sequence that binds strongly to Ni-NTA-coated surfaces. Advantages of PISA include avoiding bacterial expression and protein purification and making functional protein arrays available as required from ge... Springer protocols feed by Biochemistry info http://www.medworm.com/rss/comments.php?id=1928105 Sun, 01 Jun 2008 04:00:00 +0100 1928105 http://www.medworm.com/index.php?rid=1928104&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-398-1_13 We describe our eukaryotic system, in which rabbit reticulocyte extracts are used for cell free transcription/translation and cDNA is recovered by in situ RT-PCR performed on the selected complexes. (Source: Springer protocols feed by Biochemistry) Springer protocols feed by Biochemistry info http://www.medworm.com/rss/comments.php?id=1928104 Sun, 01 Jun 2008 04:00:00 +0100 1928104 http://www.medworm.com/index.php?rid=1928103&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-398-1_12 Signaling pathways transduce extracellular stimuli from the membrane to the nucleus. Constitutive and thus inappropriate stimulation of these kinase cascades is associated with and observed in a majority of tumors. The transduction of signals in these pathways is achieved through protein–protein interactions regulated by changes in the phosphorylation status of key members. Therefore, the analysis of the interactions formed or broken in response to mitogenic stimulation is an important step toward understanding the molecular mechanisms of carcinogenesis. Today, mass spectrometry-based proteomics is one of the most widely used methods to unravel the molecular protein interaction networks that underlie these signaling cascades. This approach is powerful, but usually results in long lis... Springer protocols feed by Biochemistry info http://www.medworm.com/rss/comments.php?id=1928103 Sun, 01 Jun 2008 04:00:00 +0100 1928103 http://www.medworm.com/index.php?rid=1928102&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-398-1_11 Networks of interacting protein control physiological processes in all living cells. Considerable effort has recently been invested in understanding protein interactions under normal and diseased conditions. One approach to elucidate the composition of protein complexes is native fractionation followed by immunological or MS-based identification of individual compounds. Native fractionation, in contrast to widespread affinity-based purification methods, allows analysis of protein interactions at the endogenous expression level and within a physiological context. In this chapter we describe a protocol for native fractionation of membrane-bound protein complexes from isolated porcine rod outer segments (ROSs). Protein complexes from isolated ROS membranes were solubilized using the nonionic ... Springer protocols feed by Biochemistry info http://www.medworm.com/rss/comments.php?id=1928102 Sun, 01 Jun 2008 04:00:00 +0100 1928102 http://www.medworm.com/index.php?rid=1928101&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-398-1_10 We describe how to generate high content pretransformed cDNA libraries, and perform an efficient yeast mating screen for protein-protein interactions with any bait protein of interest. (Source: Springer protocols feed by Biochemistry) Springer protocols feed by Biochemistry info http://www.medworm.com/rss/comments.php?id=1928101 Sun, 01 Jun 2008 04:00:00 +0100 1928101 http://www.medworm.com/index.php?rid=1928100&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-398-1_1 Recent technical innovations in mass spectrometry-based techniques have resulted in a range of highly sensitive and versatile instruments for high-throughput, high-sensitive, proteome-scale profiling. This wide diversity of instrumentation commercially available for mass spectrometry-based proteomics makes the choice of instrumentation sometimes difficult. The choice of instruments depends on the biological problem and the proteomic strategy chosen for protein identification. This chapter will give a short overview of the instruments routinely used in proteomic laboratories and the technical criteria that should be considered before instrument selection. (Source: Springer protocols feed by Biochemistry) Springer protocols feed by Biochemistry info http://www.medworm.com/rss/comments.php?id=1928100 Sun, 01 Jun 2008 04:00:00 +0100 1928100 http://www.medworm.com/index.php?rid=1554953&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-242-7_26 DNA microarray studies offer a robust method for nonbiased analysis of whole genome messenger ribonucleic acid expression patterns. A growing number of studies have applied this experimental approach to studies on ethanol either in cell culture of animal models of ethanol exposure or self-administration. Expression profiling has identified novel gene networks responding to ethanol or differing across animal strains with differing responses to ethanol. Recent studies have shown benefit for meta-analysis of microarray data across different laboratories. Gene network analysis offers unique opportunities for understanding the molecular mechanisms of ethanol responses, toxicity and addiction. Eventually, such work may generate novel targets for future pharmacotherapy. To fully capitalize on the... Springer protocols feed by Biochemistry info http://www.medworm.com/rss/comments.php?id=1554953 Fri, 02 May 2008 04:00:00 +0100 1554953 http://www.medworm.com/index.php?rid=1539288&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-242-7_6 The avian embryo has proven utility for studying ethanol's damaging effects upon the embryo. Chicken and quail are long-established models for developmental biology research; much of what we know regarding limb, craniofacial, neural crest, hindbrain, and cardiac morphogenesis was first established with avian models. These models also are for popular mechanistic studies of teratogens, including ethanol. Avian models have been used to explore ethanol's effects on neurogenesis, cardiogenesis, intracellular signaling, neurobehavior, and apoptosis. Presented here are several of these methodologies for adaptation by interested researchers. (Source: Springer protocols feed by Biochemistry) Springer protocols feed by Biochemistry info http://www.medworm.com/rss/comments.php?id=1539288 Fri, 02 May 2008 04:00:00 +0100 1539288 http://www.medworm.com/index.php?rid=1539287&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-242-7_15 Chronic consumption of ethanol induces hepatic steatosis and inflammation, which can eventually lead to more severe liver injury, characterized by fibrosis and cirrhosis. Recruitment of neutrophils to the liver, as well as activation of Kupffer cells, mediates the inflammatory responses observed after chronic ethanol exposure. Kupffer cells, the resident macrophages of the liver, are critical to the onset of ethanol-induced liver injury. Activation of Kupffer cells leads to an increased production of proinflammatory cytokines, such as tumor necrosis factor-α and also reactive oxygen species, a process mediated in part by changes in lipopolysaccharide-induced TLR4-dependent signal transduction. The isolation and culture of Kupffer cells is an important technique with which one can elu... Springer protocols feed by Biochemistry info http://www.medworm.com/rss/comments.php?id=1539287 Fri, 02 May 2008 04:00:00 +0100 1539287 http://www.medworm.com/index.php?rid=1539286&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-242-7_23 Chronic ethanol consumption dysregulates glucose and lipid homeostasis, is associated with insulin resistance, and alters serum levels of adipokines including adiponectin and tumor necrosis factor-α. However, the mechanisms involved in these chronic ethanol-induced pathologies are not fully understood. Adipose tissue has been implicated as an important contributor to chronic ethanol-induced disease states and, therefore, the effects of chronic ethanol feeding in rats on adipocytes has been investigated. Three major functions of the adipocyte include glucose transport, adipokine secretion, and triglyceride breakdown via lipolysis. Included in this chapter are protocols for studying the effect of chronic ethanol feeding on these adipocyte functions. (Source: Springer protocols feed by ... Springer protocols feed by Biochemistry info http://www.medworm.com/rss/comments.php?id=1539286 Fri, 02 May 2008 04:00:00 +0100 1539286 http://www.medworm.com/index.php?rid=1539285&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-242-7_9 Both acute and chronic alcohol consumption have significant immunomodulatory effects of which alterations in innate immune functions contribute to impaired antimicrobial defense and inflammatory responses. Blood monocytes, macrophages, and dendritic cells play a central role in innate immune recognition as these cells recognize pathogens, respond with inflammatory cytokine production, and induce antigen-specific T-lymphocyte activation. All of these innate immune cell functions are affected in humans by alcohol intake. Here, we summarize the different effects of acute and chronic alcohol on monocyte, macrophage, and dendritic cell functions in humans and describe methods for separation and functional evaluation of these cell types. (Source: Springer protocols feed by Biochemistry) Springer protocols feed by Biochemistry info http://www.medworm.com/rss/comments.php?id=1539285 Fri, 02 May 2008 04:00:00 +0100 1539285 http://www.medworm.com/index.php?rid=1539284&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-242-7_10 In vivo studies are ideal for identifying the phenomenology of ethanol toxicity and teratology. They are limited in being able to explore cellular and molecular mechanisms of action. Two types of culture models have proven to be very instructive: monolayer primary cultures of dissociated cells and organotypic slice cultures. Dissociated cell preparations have the advantage of being enriched populations of cells, whereas the organotypic cultures have the advantage of providing normal cell associations. Details for the methods used to generate these preparations are described. As ethanol is a volatile liquid, the success of a culture model depends upon stabilizing the ethanol content in the culture medium. A method to maintain the ethanol concentration is described. (Source: Springer protoco... Springer protocols feed by Biochemistry info http://www.medworm.com/rss/comments.php?id=1539284 Fri, 02 May 2008 04:00:00 +0100 1539284 http://www.medworm.com/index.php?rid=1539283&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-242-7_3 Alcohol-associated life-style disease, as exemplified by alcoholic liver disease (ALD), is multifactorial with intricate interactions among genetic and environmental factors predicating individual predisposition. To experimentally dissect the interfaces of these interactions for better understanding of the pathogenesis, it is essential to have an animal model that provides maximal control over ethanol and dietary intake and that enables a precise addition or deletion analysis for a risk or protective factor of interest. Rodent intragastric ethanol infusion (IEI) model was developed two decades ago to meet this requirement. Work conducted with the model to date demonstrates the importance of both maximal ethanol intake and secondary risk factors in ALD. Mouse IEI model proved to be particul... Springer protocols feed by Biochemistry info http://www.medworm.com/rss/comments.php?id=1539283 Fri, 02 May 2008 04:00:00 +0100 1539283 http://www.medworm.com/index.php?rid=1539282&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-242-7_12 During the second trimester period, neuroepithelial stem cells give birth to millions of new neuroblasts, which migrate away from their germinal zones to populate the developing brain and terminally differentiate into neurons. During this period, large numbers of cells are also eliminated by programmed cell death. Therefore, the second trimester constitutes an important critical period for neuronal proliferation, migration, differentiation and apoptosis. Substantial evidence indicates that teratogens like ethanol can interfere with neuronal maturation. However, there is a paucity of good model systems to study early, second trimester events. In vivo models are inherently interpretatively complex because cell proliferation, migration, differentiation, and death mechanisms occur concurrently... Springer protocols feed by Biochemistry info http://www.medworm.com/rss/comments.php?id=1539282 Fri, 02 May 2008 04:00:00 +0100 1539282 http://www.medworm.com/index.php?rid=1539281&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-242-7_8 Animal models of fetal alcohol spectrum disorder (FASD) have been instrumental in isolating alcohol as a teratogen and demonstrating behavioral and neural effects. There are a number of different models for rodents with various strengths and weaknesses. A three-trimester model of FASD is described here; the model uses intragastric intubation of both pregnant dams and pups to mimic alcohol exposure across all three trimesters in humans. The model does not use expensive equipment and is relatively easy to accomplish. The model allows excellent control of alcohol dose and uses an oral route of administration. There are no undernutrition effects with the doses used here. A drawback of the model is the stress of the intubation procedures and ways in which to minimize this stress are discussed. ... Springer protocols feed by Biochemistry info http://www.medworm.com/rss/comments.php?id=1539281 Fri, 02 May 2008 04:00:00 +0100 1539281 http://www.medworm.com/index.php?rid=1539280&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-242-7_20 Chronic alcohol abuse leads to multiple defects in the immune system, leading to an increased risk of infectious disease and malignancy. Immune lesions encompass both the innate and adaptive arms and include deficiencies in the B-cell compartment. Long-term alcoholics exhibit loss of B cells in the periphery and diminished ability to generate protective antibodies. To better mimic the chronic alcoholic patient, our group has used an ethanol-in-drinking-water mouse model. Mice consuming alcohol in this manner progressively develop a range of immune abnormalities, including defects in humoral immunity. To document and explore B-cell lesions in ethanol-consuming mice, our laboratory has used a broad panel of technologies. These include protocols to define the physical state of B cells in the ... Springer protocols feed by Biochemistry info http://www.medworm.com/rss/comments.php?id=1539280 Fri, 02 May 2008 04:00:00 +0100 1539280 http://www.medworm.com/index.php?rid=1539279&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-242-7_17 Most ingested ethanol is eliminated from the body through oxidative metabolism in the liver. Alcohol dehydrogenase is the enzyme that is most important in the oxidation of ethanol to acetaldehyde. However, it has also been demonstrated that cytochrome P4502E1 also can contribute to this process. However, this is not the only aldehyde that is produced after chronic ethanol consumption because oxidative stress and lipid peroxidation can be induced in the liver, which results in the production of malondialdehyde and 4-hydroxy-2-nonenal. These aldehydes are highly reactive and have the ability to react with (adduct) many macromolecules to alter their structure and play a major role in the derangements of hepatic function. Therefore, the formation of these types of adducts in the liver has been... Springer protocols feed by Biochemistry info http://www.medworm.com/rss/comments.php?id=1539279 Fri, 02 May 2008 04:00:00 +0100 1539279 http://www.medworm.com/index.php?rid=1539278&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-242-7_14 Oxidative stress is increasingly suspected to contribute to the initiation and progression of many disease, including those caused by alcohol exposure. Two major products of reactive oxygen and nitrogen species formation are 4OH-nonenal and 3-nitrotyrosine protein adducts, both of which can be detected by immunohistochemistry. In the past, immunohistochemical techniques have served largely as qualitative measures of changes. However, coupled with digital capture and analysis of photomicrographs, one can now quantitate treatment-related changes with immunohistochemistry. This chapter summarizes techniques for immunohistochemical detection of these products of reactive oxygen and nitrogen species and subsequent image-analysis. Although the methods described herein are based on liver, these t... Springer protocols feed by Biochemistry info http://www.medworm.com/rss/comments.php?id=1539278 Fri, 02 May 2008 04:00:00 +0100 1539278 http://www.medworm.com/index.php?rid=1539277&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-242-7_2 The variety of animal models used in the study of alcoholic liver disease reflects the formidable task of developing a model that replicates the human disease. We show that oral feeding of fatty acids derived from fish oil and ethanol induces fatty liver, necrosis, inflammation, and fibrosis. Together with the study of oxidative and nitrosative stress markers, cytokines, proteasome function, and protein studies, this model has provided an inexpensive and technically simple method of establishing pathological alcoholic liver injury. (Source: Springer protocols feed by Biochemistry) Springer protocols feed by Biochemistry info http://www.medworm.com/rss/comments.php?id=1539277 Fri, 02 May 2008 04:00:00 +0100 1539277 http://www.medworm.com/index.php?rid=1539276&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-242-7_19 Evaluation of the functional responses of T cells is of importance in determining the mechanism(s) of immunodeficiency resulting from chronic alcohol abuse and other conditions that lead to immune dysfunction. Mice that are chronically exposed to 20% (w/v) ethanol in water develop immunodeficiency and have T cells with abnormal activation profiles, reduced total numbers, increased CD4/CD8 ratios, and an increased memory/naïve phenotype ratio. These cells also have abnormal antigen-specific responses after inoculation of the ethanol mice with model infectious organisms. Study of the functional abnormalities of these cells requires a reliable system that can present appropriate activation stimuli in vitro for the generation of polyclonal or antigen-specific responses in enriched or puri... Springer protocols feed by Biochemistry info http://www.medworm.com/rss/comments.php?id=1539276 Fri, 02 May 2008 04:00:00 +0100 1539276 http://www.medworm.com/index.php?rid=1539275&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-242-7_5 It is largely accepted that vertebrates are more susceptible to chemical insult during the early life stage. It is implied that if a chemical such as ethanol is developmentally toxic, it must interfere with, or modulate, critical signaling pathways. The probable molecular explanation for increased embryonic susceptibility is that collectively there is no other period of an animal's lifespan when the full repertoire of molecular signaling is active. Understanding the mechanism by which ethanol exposure disrupts vertebrate embryonic development is enormously challenging; it requires a thorough understanding of the normal molecular program to understand how transient ethanol exposure disrupts signaling and results in detrimental long-lasting effects. During the past several years, investigato... Springer protocols feed by Biochemistry info http://www.medworm.com/rss/comments.php?id=1539275 Fri, 02 May 2008 04:00:00 +0100 1539275 http://www.medworm.com/index.php?rid=1539274&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-242-7_16 Dendritic cells (DCs) play a key role in the initiation of effective immune responses against infectious agents because they are unique in their ability to provide antigen-specific activation of naïve T cells. To do this, they must acquire antigen and migrate to spleen or lymph node to present the antigen to T cells in association with costimulatory molecules and cytokines. Murine models of chronic EtOH exposure have been developed for dissecting the mechanisms by which EtOH alters immune cell functions. This chapter details methods for assessing DC functions in such models. Methods are presented for 1) the identification and isolation of various DC subsets from spleen, epidermis, and lung, 2) measurement of LC migration out of epidermis and DC migration into peripheral and peribronch... Springer protocols feed by Biochemistry info http://www.medworm.com/rss/comments.php?id=1539274 Fri, 02 May 2008 04:00:00 +0100 1539274 http://www.medworm.com/index.php?rid=1539273&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-242-7_13 A significant body of evidence indicates that endotoxemia plays a crucial role in the pathogenesis of alcoholic liver disease. There are several possible factors that may be involved in inducing alcoholic endotoxemia, but increased intestinal permeability to enteric endotoxins appears to be the major contributing factor. In the normal gut, the epithelial barrier function prevents diffusion of toxins across the epithelium. However, the barrier is disrupted in patients with alcoholic liver disease. We showed that acetaldehyde disrupts intestinal epithelial tight junctions and increases paracellular permeability to endotoxins in Caco-2 cell monolayer, the extensively studied model of the differentiated intestinal epithelium. The mechanisms involved in acetaldehyde-induced increase in intestin... Springer protocols feed by Biochemistry info http://www.medworm.com/rss/comments.php?id=1539273 Fri, 02 May 2008 04:00:00 +0100 1539273 http://www.medworm.com/index.php?rid=1539272&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-242-7_25 Chronic alcohol consumption has been shown to severely compromise mitochondrial protein synthesis. Hepatic mitochondria isolated from alcoholic animals contain decreased levels of respiratory complexes and display depressed respiration rates when compared to pair-fed controls. One underlying mechanism for this involves ethanol-elicited alterations in the structural and functional integrity of the mitochondrial ribosome. Ethanol feeding results in ribosomal changes that include decreased sedimentation rates, larger hydrodynamic volumes, increased levels of unassociated subunits and changes in the levels of specific ribosomal proteins. The methods presented in this chapter detail how to isolate mitochondrial ribosomes, determine ribosomal activity, separate ribosomes into nucleic acid and pr... Springer protocols feed by Biochemistry info http://www.medworm.com/rss/comments.php?id=1539272 Fri, 02 May 2008 04:00:00 +0100 1539272 http://www.medworm.com/index.php?rid=1539271&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-242-7_22 The development of alcoholic muscle disease, which affects both cardiac and skeletal muscle, leads to increased morbidity and mortality in patients who abuse alcohol. The disease pathology includes myocyte degeneration, loss of striations, and myofilament dissolution, which is consistent with alterations in structural and myofibrillar proteins. One explanation for the changes in myofibrillar architecture is that the expression of cellular proteins may be compromised by ethanol consumption. The dynamic balance of proteins in striated muscle is dependent upon rates of protein synthesis and protein degradation. We have shown that protein synthesis is depressed in striated muscle after either acute alcohol intoxication or chronic alcohol ingestion. The loss of myofibrillar proteins occurs prio... Springer protocols feed by Biochemistry info http://www.medworm.com/rss/comments.php?id=1539271 Fri, 02 May 2008 04:00:00 +0100 1539271 http://www.medworm.com/index.php?rid=1539270&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-242-7_7 Prenatal alcohol exposure disrupts development, leading to a range of effects referred to as fetal alcohol spectrum disorders (FASD). FASDs include physical, central nervous system, and behavioral alterations. Animal model systems are used to study the relationship between alcohol-related central nervous system damage and behavioral alterations, risk factors for FASD, mechanisms of alcohol-induced damage, as well as treatments and interventions. When using a rodent model, it is important to recognize that the timing of brain development relative to birth differs between humans and rodents. Thus, to model alcohol exposure during the third trimester equivalent, rats must be exposed during early postnatal development (postnatal days 4-9). Artificial rearing is one experimental paradigm that i... Springer protocols feed by Biochemistry info http://www.medworm.com/rss/comments.php?id=1539270 Fri, 02 May 2008 04:00:00 +0100 1539270 http://www.medworm.com/index.php?rid=1539269&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-242-7_1 Acute alcohol administration has minimal effects on basal immune function. However, when the immune system is challenged, acute alcohol administration alters the immune system's response. In the first 3 h after infection or traumatic injury, the presence of alcohol is associated with a decreased inflammatory response. This defect lasts long after the alcohol is cleared. Conversely, by 48 h after traumatic injury, the presence of alcohol is associated with a heightened inflammatory response. Aside from its in vivo actions, systemic administration of alcohol also alters the ex vivo response of immune cells, resulting in a decreased production of multiple cytokines after stimulation by lipopolysaccharide, concanavilin A, zymosan, and CpG DNA. Here, we describe a standardized model of acute ad... Springer protocols feed by Biochemistry info http://www.medworm.com/rss/comments.php?id=1539269 Fri, 02 May 2008 04:00:00 +0100 1539269 http://www.medworm.com/index.php?rid=1539268&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-242-7_24 Mitochondrial dysfunction is recognized as a contributing factor to a number of diseases, including chronic alcohol-induced hepatotoxicity. Although there is a detailed understanding of the metabolic pathways and proteins of the liver mitochondrion, little is known of how changes in the mitochondrial proteome contribute to the development of hepatic pathologies. In this short overview the insights gained from study of changes in the mitochondrial proteome in alcoholic liver disease will be described. Profiling the liver mitochondrial proteome has the potential to shed light on the alcohol-mediated molecular defects responsible for mitochondrial and cellular dysfunction. The methods presented herein demonstrate the power of using complementary proteomics approaches, that is, 2-D IEF/SDS-PAG... Springer protocols feed by Biochemistry info http://www.medworm.com/rss/comments.php?id=1539268 Fri, 02 May 2008 04:00:00 +0100 1539268 http://www.medworm.com/index.php?rid=1539267&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-242-7_4 Mice provide a useful model for the study of immune deficiency caused by chronic alcohol abuse. Their suitability is related to several factors, including in particular the extensive knowledge base in the immunology of mice already existing in the literature. Specific modeling of the immunodeficiency of the chronic human alcoholic requires that ethanol must be administered to the model for a significant portion of its life span. In mice, it has proven to be necessary to administer ethanol daily for up to 32 wk or longer to observe all the immune abnormalities that occur in middle-aged alcoholic humans. Such time spans are problematic with many of the common protocols for ethanol administration. It has been shown by others and confirmed by our group that the most practical way of accomplish... Springer protocols feed by Biochemistry info http://www.medworm.com/rss/comments.php?id=1539267 Fri, 02 May 2008 04:00:00 +0100 1539267 http://www.medworm.com/index.php?rid=1539266&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-242-7_21 Bone is an important target tissue for alcohol. Moderate alcohol consumption may slow bone loss during aging, but alcohol consumption inhibits bone growth during adolescence, and alcohol abuse in adults is an important risk factor for osteoporosis. Various techniques have been applied for evaluating the impact of alcohol on bone, including densitometry for assessment of bone mass and density, computed tomography for evaluation of bone microarchitecture, serum biochemistry for measurement of markers of global bone resorption and formation, and histomorphometry for assessment of cellular activity. Of these methods, histomorphometry is the gold standard for assessing bone because it is the only method for the direct in situ analysis of bone cells and their activities. The procedures described... Springer protocols feed by Biochemistry info http://www.medworm.com/rss/comments.php?id=1539266 Fri, 02 May 2008 04:00:00 +0100 1539266 http://www.medworm.com/index.php?rid=1539265&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-242-7_11 CYP2E1, a member of the cytochrome P450 family, is induced by ethanol. CYP2E1 activates many hepatotoxins to their reactive toxic intermediate form, and generates reactive oxygen species (ROS) during its catalytic cycle. Induction of CYP2E1 plays an important role in ethanol-induced oxidant stress and ethanol toxicity. To study the biochemical and toxicological properties of CYP2E1, our laboratory developed a HepG2 cell line which constitutively expresses the human CYP2E1 form. These cells displayed elevated oxidative stress, loss of mitochondrial function and loss of viability when challenged with prooxidants such as ethanol, polyunsaturated fatty acids (PUFA) such as arachidonic acid, iron, or when depleted of the critical antioxidant glutathione, as compared with control HepG2 cells whi... Springer protocols feed by Biochemistry info http://www.medworm.com/rss/comments.php?id=1539265 Fri, 02 May 2008 04:00:00 +0100 1539265 http://www.medworm.com/index.php?rid=1539264&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-242-7_18 Natural killer (NK) cells part of innate immunity. NK cells have been assigned numerous functions, including the ability to serve as a bridge between innate and adaptive immunity. In evaluating NK cell function, two pathways need to be examined: their ability to kill certain tumors spontaneously and their ability to secrete cytokines, interferon-gamma (IFN-γ), in particular. Although NK cells are distinct from T lymphocytes, a new lymphocyte subset, termed NKT cell, has been described. NKT cells express surface markers that are unique to NK cells (e.g., NK1.1) as well as markers that are unique to T cells (e.g., CD3). Most NKT cells recognize glycolipids and are thought to play an important immunoregulatory role. This chapter will detail the methodology needed for examination of NK a... Springer protocols feed by Biochemistry info http://www.medworm.com/rss/comments.php?id=1539264 Fri, 02 May 2008 04:00:00 +0100 1539264