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 Cell Biology' source. Tue, 09 Mar 2010 17:34:44 +0100 http://www.medworm.com/index.php?rid=3100642&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-535-4_25 With the entire genome sequence of several animals now available, it is becoming possible to identify in silico all putative peptides and their precursors in an organism. In this chapter we describe a searching algorithm that can be used to scan the genome for predicted proteins with the structural hallmarks of (neuro)peptide precursors. We also describe how to use search strings such as the presence of a glycine residue as a putative amidation site, dibasic cleavage sites, the presence of a signal peptide, and specific peptide motifs to improve a standard BLAST search and make it suitable for searching (neuro)peptides in EST data. We briefly explain how bioinformatic tools and in silico predicted peptide precursor sequences can aid experimental peptide identification with mass spectrometr... Springer protocols feed by Cell Biology info http://www.medworm.com/rss/comments.php?id=3100642 Fri, 18 Dec 2009 16:23:51 +0100 3100642 http://www.medworm.com/index.php?rid=3100641&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-535-4_24 Polypeptide and protein arrays enable high-throughput screening capabilities for studying molecular interactions and profiling of biomarkers, and provide a powerful functional screening tool for peptidomics. To overcome the limitations of conventional arraying methods, we have exploited cell-free systems for generating arrays of polypeptides by direct on-chip biosynthesis from DNA templates. Here we describe two protocols: (i) Protein In Situ Array (PISA), which allows the generation of polypeptide arrays in a single reaction by spotting cell-free lysate together with PCR DNA on a glass surface pre-coated with a capturing reagent, and (ii) DNA Array to Protein Array (DAPA), which is capable of producing multiple copies of a polypeptide array from a single DNA array template. The main advan... Springer protocols feed by Cell Biology info http://www.medworm.com/rss/comments.php?id=3100641 Fri, 18 Dec 2009 16:23:51 +0100 3100641 http://www.medworm.com/index.php?rid=3100640&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-535-4_23 Affinity peptidomics relies on the successfully proven approach used widely in mass-spectrometry-based protein analysis, where protein samples are proteolytically digested prior to the analysis. Unlike traditional proteomic analyses, affinity peptidomics employs affinity detection instead of, or in addition to, the mass-spectrometry detection. Affinity peptidomics, therefore, bridges the gap between protein microarrays and mass spectrometry and can be used for the detection, identification and quantification of endogenous or proteolytic peptides on microarrays and by MALDI-MS. Phage display technology is a widely applicable generic molecular display method suitable for studying protein–protein or protein–peptide interactions and the development of recombinant affinity reagents.... Springer protocols feed by Cell Biology info http://www.medworm.com/rss/comments.php?id=3100640 Fri, 18 Dec 2009 16:23:51 +0100 3100640 http://www.medworm.com/index.php?rid=3100639&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-535-4_22 The tachykinins represent the largest known peptide family and are responsible for a range of pleiotropic functions in both vertebrates and invertebrates. Recent research has shown a diversity of mechanisms such as mRNA splicing, precursor processing and post-translation modification that can lead to a complex and continually expanding repertoire of tachykinin peptides. The peptidomic analysis of the tachykinins has been hindered by the lack of specific methodologies to capture, purify and characterise each tachykinin. This chapter summarises some of the methods that have been developed in order to further purify and characterise individual groups of tachykinin peptides from the peptidome. (Source: Springer protocols feed by Cell Biology) Springer protocols feed by Cell Biology info http://www.medworm.com/rss/comments.php?id=3100639 Fri, 18 Dec 2009 16:23:51 +0100 3100639 http://www.medworm.com/index.php?rid=3100638&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-535-4_21 Little is known about peptides that control developmental processes such as cell differentiation and pattern formation in metazoans. The cnidarian Hydra is one of the most basal metazoans and is a key model system for studying the peptides involved in these processes. We developed a novel peptidomic approach to the isolation and identification of functional signalling peptides from Hydra (the Hydra peptide project). First, peptides extracted from the tissue of Hydra magnipapillata are purified to homogeneity using high-performance liquid chromatography (HPLC). The isolated peptides are then tested for their ability to alter gene expression in Hydra using differential display-PCR (DD-PCR). If gene expression is altered, the peptide is considered as a putative signalling peptide and is subje... Springer protocols feed by Cell Biology info http://www.medworm.com/rss/comments.php?id=3100638 Fri, 18 Dec 2009 16:23:51 +0100 3100638 http://www.medworm.com/index.php?rid=3100637&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-535-4_9 Direct MALDI-TOF mass spectrometric peptide profiling is increasingly used to analyze the peptide complement in the nervous system of a variety of invertebrate animals, from leech to Aplysia and many arthropod species, especially insects and crustaceans. Proper sample preparation is often the most crucial step to obtain the necessary data. Here, we describe protocols for the use of MALDI-TOF mass spectrometry to directly analyze the peptidome of neuroendocrine tissues of insects, particularly Drosophila melanogaster, by MALDI-TOF MS. (Source: Springer protocols feed by Cell Biology) Springer protocols feed by Cell Biology info http://www.medworm.com/rss/comments.php?id=3100637 Fri, 18 Dec 2009 16:23:51 +0100 3100637 http://www.medworm.com/index.php?rid=3100636&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-535-4_8 The diversity of insect neuropeptides coupled with the limitations from the small size of the insects themselves combine to make positive identification through peptide sequencing a highly challenging task. The advent of the “soft-ionisation” techniques of MALDI-TOF and electrospray (ESI)-Q-TOF mass spectrometry, coupled with the additional information from insect genome projects have revolutionised the characterisation of insect neuropeptides, such that sequences can now be obtained from just a few cells, where before thousands of insects had to be laboriously dissected, extracted and purified. Some of the procedures that are now used to identify these peptides are described here. Once the neuropeptides have been identified, it then becomes possible to use this knowledge to de... Springer protocols feed by Cell Biology info http://www.medworm.com/rss/comments.php?id=3100636 Fri, 18 Dec 2009 16:23:51 +0100 3100636 http://www.medworm.com/index.php?rid=3100635&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-535-4_7 Spider venoms represent invaluable sources of biologically active compounds suitable for use in life science research and also having a significant potential for biotechnology and therapeutic applications. The methods reported herewith are based on our long experience of spider venom fractionation and peptides purification. We routinely screen new peptides for antimicrobial and insecticidal activities and our detailed protocols are also reported here. So far these have been tested on species of Central Asian and European spiders from the families Agelenidae, Eresidae, Gnaphosidae, Lycosidae, Miturgidae, Oxyopidae, Philodromidae, Pisauridae, Segestriidae, Theridiidae, Thomisidae, and Zodariidae. The reported protocols should be easily adaptable for use with other arthropod species. (Source:... Springer protocols feed by Cell Biology info http://www.medworm.com/rss/comments.php?id=3100635 Fri, 18 Dec 2009 16:23:51 +0100 3100635 http://www.medworm.com/index.php?rid=3100634&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-535-4_6 Spider venom contains a complex mixture of components with a large range of molecular masses (0.1–60 kDa) exhibiting a diverse array of actions. Most of these components are proteinaceous molecules – biologically active proteins and peptides. Proteomics profiling of spider venoms (the components with MW >10 kDa) could be achieved through conventional 2-DE-based proteomics methods combined with MS or MS/MS detection. Peptidomic profiling (of the components with MW below ~10 kDa) is usually achieved through off-line separation by a combination of ion-exchange and reverse-phase chromatography, and it relies more heavily on de novo sequencing by Edman degradation or MS/MS for peptide identification. (Source: Springer protocols feed by Cell Biology) Springer protocols feed by Cell Biology info http://www.medworm.com/rss/comments.php?id=3100634 Fri, 18 Dec 2009 16:23:51 +0100 3100634 http://www.medworm.com/index.php?rid=3100633&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-535-4_5 Neuropeptides are important signaling molecules that regulate many essential physiological processes. Microdialysis offers a way to sample neuropeptides in vivo. When combined with liquid chromatography–mass spectrometry detection, many known and unknown neuropeptides can be identified from a live organism. This chapter describes sample preparation techniques and general strategies for the mass spectral analysis of neuropeptides collected via microdialysis sampling. Methods for the in vitro microdialysis of a neuropeptide standard as well as the in vivo microdialysis sampling of neuropeptides from a live crab are described. (Source: Springer protocols feed by Cell Biology) Springer protocols feed by Cell Biology info http://www.medworm.com/rss/comments.php?id=3100633 Fri, 18 Dec 2009 16:23:51 +0100 3100633 http://www.medworm.com/index.php?rid=3100632&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-535-4_4 The central nervous systems of molluscan species contain high levels of structurally diverse peptides that function as neurotransmitters, neuromodulators or neurohormones. Peptide diversity is believed to be a way to increase the information handling capacity of neurons in the context of a brain with low cell numbers and neuronal connectivity. Accordingly, much effort has been made to identify peptides from single neurons and tissues of interest. In the past decade a mass spectrometry-based approach has been applied to detect and characterize peptides from single neurons, nerves and tissues of the molluscan brain. Peptides from single neurons are often analysed directly by mass spectrometry without prior sample preparation. Single neurons from the molluscan brain may be identified based on... Springer protocols feed by Cell Biology info http://www.medworm.com/rss/comments.php?id=3100632 Fri, 18 Dec 2009 16:23:51 +0100 3100632 http://www.medworm.com/index.php?rid=3100631&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-535-4_3 The transparent soil nematode Caenorhabditis elegans can be considered an important model organism due to its ease of cultivation, suitability for high-throughput genetic screens, and extremely well-defined anatomy. C. elegans contains exactly 959 cells that are ordered in defined differentiated tissues. Although C. elegans only possesses 302 neurons, a large number of similarities among the neuropeptidergic signaling pathways can be observed with other metazoans. Neuropeptides are important messenger molecules that regulate a wide variety of physiological processes. These peptidergic signaling molecules can therefore be considered important drug targets or biomarkers. Neuropeptide signaling is in the nanomolar range, and biochemical elucidation of individual peptide sequences in the past ... Springer protocols feed by Cell Biology info http://www.medworm.com/rss/comments.php?id=3100631 Fri, 18 Dec 2009 16:23:51 +0100 3100631 http://www.medworm.com/index.php?rid=3100630&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-535-4_2 Host–pathogen interactions are complex competitions during which both the host and the pathogen adapt rapidly to each other in order for one or the other to survive. Salmonella enterica serovar Typhimurium is a pathogen with a broad host range that causes a typhoid fever-like disease in mice and severe food poisoning in humans. The murine typhoid fever is a systemic infection in which S. typhimurium evades part of the immune system by replicating inside macrophages and other cells. The transition from a foodborne contaminant to an intracellular pathogen must occur rapidly in multiple, ordered steps in order for S. typhimurium to thrive within its host environment. Using S. typhimurium isolated from rich culture conditions and from conditions that mimic the hostile intracellular envir... Springer protocols feed by Cell Biology info http://www.medworm.com/rss/comments.php?id=3100630 Fri, 18 Dec 2009 16:23:51 +0100 3100630 http://www.medworm.com/index.php?rid=3100629&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-535-4_20 Recent years have seen great advances in mass spectrometry and proteomics, the science dealing with the analysis of proteins, their structure and function. A branch of proteomics dealing with naturally occurring peptides is often referred to as peptidomics. Direct analysis of peptides produced by processing or degradation of proteins might be useful for example for detecting and identifying pathogenic and/or biomarker peptides in body fluids like blood. In this paper, we introduce one of the standard protocols for comprehensive analysis of serum-derived peptides, which consists of methods for purification of serum peptides, detection of peptides, pattern recognition and clustering (bioinformatics), and identification of peptide sequences. Peptide identification should be followed by the in... Springer protocols feed by Cell Biology info http://www.medworm.com/rss/comments.php?id=3100629 Fri, 18 Dec 2009 16:23:51 +0100 3100629 http://www.medworm.com/index.php?rid=3100628&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-535-4_19 We describe a number of technologies which we developed for the peptidomic profiling of lymphoblastoid cell extracts from patients with leukodystrophies, linked to mutations in the genes encoding the eukaryotic initiation factor 2B (eIF2B; eIF2B-related disorders). (Source: Springer protocols feed by Cell Biology) Springer protocols feed by Cell Biology info http://www.medworm.com/rss/comments.php?id=3100628 Fri, 18 Dec 2009 16:23:51 +0100 3100628 http://www.medworm.com/index.php?rid=3100627&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-535-4_18 An emerging way to study neuropsychiatric or neurodegenerative diseases is by performing proteomic analyses of brain tissues. Here, we describe methods used to isolate and identify the proteins associated with a sample of interest, such as the synapse, as well as to compare the levels of proteins in the sample under different conditions. These techniques, involving subcellular fractionation and modern quantitative proteomics using isotopic labels, can be used to understand the organization of neuronal compartments and the regulation of synaptic function under various conditions. (Source: Springer protocols feed by Cell Biology) Springer protocols feed by Cell Biology info http://www.medworm.com/rss/comments.php?id=3100627 Fri, 18 Dec 2009 16:23:51 +0100 3100627 http://www.medworm.com/index.php?rid=3100626&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-535-4_17 Due to the complexity of the mammalian central nervous system, neuropeptidomic studies in mammals often yield very complicated mass spectra that make data analysis difficult. Careful sample preparation and extraction protocols must be employed in order to minimize spectral complexity and enable extraction of useful information on neuropeptides from a given sample. Controlling post-mortem protease activity is essential to simplifying mass spectra and to identifying low-abundance neuropeptides in tissue samples. Post-mortem microwave-irradiation coupled with cryostat dissection has proven to be effective in arresting protease activity to allow detection of endogenous neuropeptides instead of protein degradation products. (Source: Springer protocols feed by Cell Biology) Springer protocols feed by Cell Biology info http://www.medworm.com/rss/comments.php?id=3100626 Fri, 18 Dec 2009 16:23:51 +0100 3100626 http://www.medworm.com/index.php?rid=3100625&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-535-4_16 Here we report our approach to the peptidomic analysis of mouse liver. We use ultrafiltration for peptide prefractionation, which is followed by size exclusion chromatography. The low molecular weight peptides (MW below ~3 kDa) are analysed directly by nanoLC-MS/MS, and the higher molecular weight peptides (MW above ~3 kDa) are characterized with MALDI-TOF MS first and then proteolytically digested prior to nanoLC-MS/MS analyses. (Source: Springer protocols feed by Cell Biology) Springer protocols feed by Cell Biology info http://www.medworm.com/rss/comments.php?id=3100625 Fri, 18 Dec 2009 16:23:51 +0100 3100625 http://www.medworm.com/index.php?rid=3100624&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-535-4_15 Endocrine tissues like the pituitary, hypothalamus and islets of Langerhans are rich in bioactive peptides. These are used for intercellular signalling and are involved in regulation of almost all physiological processes. Peptidomics is the comprehensive analysis of peptides in tissues, fluids and cells. Peptidomics applied to (neuro-)endocrine tissues aims therefore to identify as many bioactive peptides as possible. Peptidomics of (neuro-)endocrine tissues requires an integrated approach that consists of careful sample handling, peptide separation techniques, mass spectrometry and bioinformatics. Here we describe the methods for isolation and dissection of endocrine tissues, the extraction of bioactive peptides and further sample handling and identification of peptides by mass spectromet... Springer protocols feed by Cell Biology info http://www.medworm.com/rss/comments.php?id=3100624 Fri, 18 Dec 2009 16:23:51 +0100 3100624 http://www.medworm.com/index.php?rid=3100623&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-535-4_14 We describe here a systematic peptidomics approach which we combined with genomics and functional testing. This has proven to be an effective way to identify amphibian antimicrobial peptides, including novel peptide families. Protocols are exemplified for Bombina maxima and Odorrana grahami and can be easily adapted for use with other amphibian species. (Source: Springer protocols feed by Cell Biology) Springer protocols feed by Cell Biology info http://www.medworm.com/rss/comments.php?id=3100623 Fri, 18 Dec 2009 16:23:51 +0100 3100623 http://www.medworm.com/index.php?rid=3100622&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-535-4_13 Antimicrobial peptides (AMPs) play an important role in the host’s innate defence system in many organisms. Amphibian skin is expected to be a particularly rich source of novel AMPs. In amphibians, AMPs are produced from precursor proteins via specific cleavage by processing enzymes. While the nucleotide sequences of the AMP coding region in precursors are hypervariable, those of other regions, including the 5′- and 3′-untranslated regions (UTRs), are highly or relatively conserved in different precursors. Such nucleotide sequence conservation suggests an efficient strategy for molecular cloning of the antimicrobial peptide genes by 3′-rapid amplification of cDNA ends (3′-RACE) and reverse transcriptase polymerase chain reaction (RT-PCR) methods using specific... Springer protocols feed by Cell Biology info http://www.medworm.com/rss/comments.php?id=3100622 Fri, 18 Dec 2009 16:23:51 +0100 3100622 http://www.medworm.com/index.php?rid=3100621&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-535-4_12 Skin secretions from anurans (frogs and toads), particularly those species belonging to the Hylidae and Ranidae families, are a rich source of biologically active peptides. Cytolytic peptides with broad-spectrum antimicrobial activities and highly variable amino acid sequences are often released into these secretions in high concentrations. Identification and characterization of these components can prove to be valuable in species identification, elucidation of evolutionary histories and phylogenetic relationships between species, and may lead to development of agents with potential for therapeutic application. This chapter describes the use of norepinephrine (injection or immersion) to stimulate peptide release in a procedure that does not appear to cause distress to the animals. The pept... Springer protocols feed by Cell Biology info http://www.medworm.com/rss/comments.php?id=3100621 Fri, 18 Dec 2009 16:23:51 +0100 3100621 http://www.medworm.com/index.php?rid=3100620&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-535-4_11 Today, commercially available mass spectrometers increasingly meet all the demands of the proteomics community including high throughput, high sensitivity, and significant fragmentation capability for sequence determinations. Therefore, proper sample preparation is often the most crucial step to obtain the necessary data, particularly when working with biological samples. Depending on the size, sample preparation techniques differ and have to be optimized empirically. This is particularly apparent at the single cell level. In this chapter, we describe protocols for the use of MALDI-TOF mass spectrometry to directly analyse the peptidome of single insect neurons. (Source: Springer protocols feed by Cell Biology) Springer protocols feed by Cell Biology info http://www.medworm.com/rss/comments.php?id=3100620 Fri, 18 Dec 2009 16:23:50 +0100 3100620 http://www.medworm.com/index.php?rid=3100619&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-535-4_10 Direct MALDI-TOF mass spectrometric peptide profiling is increasingly used to analyze the peptide complement in the nervous system of a variety of invertebrate animals from leech to Aplysia and many arthropod species, especially insects and crustaceans. Here, we describe a protocol for direct peptide profiling of defined areas of the central nervous system of insects. With this method, one can routinely and reliably obtain neuropeptide signatures of selected brain areas from various insects. (Source: Springer protocols feed by Cell Biology) Springer protocols feed by Cell Biology info http://www.medworm.com/rss/comments.php?id=3100619 Fri, 18 Dec 2009 16:23:50 +0100 3100619 http://www.medworm.com/index.php?rid=3100618&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-535-4_1 The term “peptidomics” can be defined as the systematic analysis of the peptide content within a cell, organelle, tissue or organism. The science of peptidomics usually refers to the studies of naturally occurring peptides. Another meaning refers to the peptidomics approach to protein analysis. An ancient Roman strategy divide et impera (divide and conquer) reflects the essence of peptidomics. Most effort in this field is spent purifying and dividing the peptidomes, which consist of tens, hundreds or sometimes thousands of functional peptides, followed by their structural and functional characterisation. This chapter introduces the concept of peptidomics, outlines the range of methodologies employed and describes key targets – the peptide groups which are often sought aft... Springer protocols feed by Cell Biology info http://www.medworm.com/rss/comments.php?id=3100618 Fri, 18 Dec 2009 16:23:50 +0100 3100618 http://www.medworm.com/index.php?rid=2988966&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-369-5_9 New challenges will arise as research into human embryonic stem (hES) cell differentiation moves from optimization and overcoming technical hurdles to mechanistic considerations. An immediate need will be to culture hES cells in the absence of contaminating feeder layers and allow for the preparation of purified DNA, RNA, and proteins to analyze changes in microRNA levels, gene expression, protein expression, and signal transduction. Purified, uniform populations of hES cells will allow researchers to better explore the biochemical mechanisms by which differentiation occurs. (Source: Springer protocols feed by Cell Biology) Springer protocols feed by Cell Biology info http://www.medworm.com/rss/comments.php?id=2988966 Tue, 13 Oct 2009 00:00:00 +0100 2988966 http://www.medworm.com/index.php?rid=2988965&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-369-5_8 Human embryonic stem cells (hESC) involve long-term cultures that must remain undifferentiated. The real-time PCR (RT-PCR) technique allows the relative quantification of target genes, including undifferentiation and differentiation markers when referred to a housekeeping control with the addition of a calibrator that serves as an internal control to compare different lots of reactions during the time. The main aspects will include a minimal number of cells to be analyzed, genes to be tested, and how to choose the appropriate calibrator sample and the reference gene. In this chapter, we present how to apply the RT-PCR technique, protocols for its performance, experimental set-up and software analysis, as of the gene expression of hESC lines in consecutive passages for long-term culture sur... Springer protocols feed by Cell Biology info http://www.medworm.com/rss/comments.php?id=2988965 Tue, 13 Oct 2009 00:00:00 +0100 2988965 http://www.medworm.com/index.php?rid=2988964&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-369-5_7 The routine culture and expansion of human embryonic stem (hES) cells has been and is still posing a challenge to researchers wishing to take advantage of the cells' unique potential. In contrast to mouse embryonic stem cells, hES cells usually have to be expanded by tedious mechanical microdissection or by enzymatic dissociation to cell clusters of a very narrow size range. (Source: Springer protocols feed by Cell Biology) Springer protocols feed by Cell Biology info http://www.medworm.com/rss/comments.php?id=2988964 Tue, 13 Oct 2009 00:00:00 +0100 2988964 http://www.medworm.com/index.php?rid=2988963&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-369-5_6 Human embryonic stem cells (hESCs) are pluripotent stem cells derived from the inner cell mass of human blastocysts. hESCs have become a great asset to studying human diseases and genetic functions of healthy organisms. The rate at which hESCs are being used in laboratories is exponentially increasing, and with that, the need for xeno-free hESCs is also increasing. Xeno-free grade hESCs, cells that have not come into contact with any animal-derived components except those of human origin, are critical for eventual drug therapy, cell therapy, and disease treatment in humans. However, advances toward a xeno-free hESC environment are still being developed. Replacement of murine feeder layers with extracellular matrix proteins has advanced the research, and some advances toward a serum-free an... Springer protocols feed by Cell Biology info http://www.medworm.com/rss/comments.php?id=2988963 Tue, 13 Oct 2009 00:00:00 +0100 2988963 http://www.medworm.com/index.php?rid=2988962&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-369-5_5 We describe in this chapter the development of a xenofree molecularly defined medium, SBX, associated with xenofree matrices, to maintain human embryonic stem cell (hESC) pluripotency as determined by phenotypic, functional and TLDA studies. This simple, inexpensive, and more physiological culture condition has been chosen because (1) it is xenofree and molecularly defined; it is devoid of albumin, which is a carrier of undefined molecules; (2) it maintains pluripotency, but very significantly reduces differentiation gene expression during hESC self-renewal, as compared to the widely used culture conditions tested so far; and (3) it can be further improved by replacing high concentrations of expensive additives by physiological concentrations of new factors. Xenofree molecularly defined me... Springer protocols feed by Cell Biology info http://www.medworm.com/rss/comments.php?id=2988962 Tue, 13 Oct 2009 00:00:00 +0100 2988962 http://www.medworm.com/index.php?rid=2988961&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-369-5_4 Human embryonic stem cells (hESCs) have the capacity to self-renew and to differentiate into all components of the embryonic germ layers (ectoderm, mesoderm, endoderm) and subsequently all cell types that comprise human tissues. HESCs can potentially provide an extraordinary source of cells for tissue engineering and great insight into early embryonic development. Much attention has been given to the possibility that hESCs and their derivatives may someday play major roles in the study of the development, disease therapeutics, and repair of injuries to the central and peripheral nervous systems. This tantalizing promise will be realized only when we understand fundamental biological questions about stem cell growth and development into distinct tissue types. In vitro, differentiation of hE... Springer protocols feed by Cell Biology info http://www.medworm.com/rss/comments.php?id=2988961 Tue, 13 Oct 2009 00:00:00 +0100 2988961 http://www.medworm.com/index.php?rid=2988960&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-369-5_3 The majority of human embryonic stem cell (hESC) lines have been derived and grown using mouse or human feeder cells, or using Matrigel®, an animal derivative rich in extracellular matrix (ECM) proteins. However, reliance on feeder layers and animal products limits the manipulation and clinical application of hESC. Alternatively, human fibroblasts produce an ECM which could be employed to coated plates and be easily sterilized. We have shown that hESC grown on this matrix and in the presence of medium conditioned by fibroblast cells maintain markers of pluripotency, including expression of cell surface proteins (SSEA3, SSEA4, TRA-1-60, TRA-1-81), alkaline phosphatase activity, and specific intracellular markers (NANOG, OCT, REX1). Moreover, hESC cultured on this novel human-derived ECM... Springer protocols feed by Cell Biology info http://www.medworm.com/rss/comments.php?id=2988960 Tue, 13 Oct 2009 00:00:00 +0100 2988960 http://www.medworm.com/index.php?rid=2988959&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-369-5_2 We describe here methods required to derive new embryonic stem cell lines starting from the initial cryopreservation of an embryo and finishing with a new cell line. We cover embryo cryopreservation and warming using a highly efficient vitrification method, the production of feeder cells and feeder plates, as well as embryo handling, plating and critical early passages, including earliest possible cryopreservation of putative stem cells using vitrification. (Source: Springer protocols feed by Cell Biology) Springer protocols feed by Cell Biology info http://www.medworm.com/rss/comments.php?id=2988959 Tue, 13 Oct 2009 00:00:00 +0100 2988959 http://www.medworm.com/index.php?rid=2988958&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-369-5_23 The recent discovery of genomic reprogramming of human somatic cells to an embryonic stem (ES) cell-like pluripotent state provides a unique opportunity for stem cell research. The reprogrammed cells, named as induced pluripotent stem (iPS) cells, possess many of the properties of ES cells and represent one of the most promising sources of patient-specific cells for use in disease model, development of pharmacology and toxicology, screening teratogens, and regenerative medicine. Here we describe the detailed methods for the generation of undifferentiated human iPS (hiPS) cells in feeder layer- and serum-free conditions. This system eliminates direct contact of stem cells with MEFs and reduces use of unknown serum factors that may have undesired activities and enables consistency in large-s... Springer protocols feed by Cell Biology info http://www.medworm.com/rss/comments.php?id=2988958 Tue, 13 Oct 2009 00:00:00 +0100 2988958 http://www.medworm.com/index.php?rid=2988957&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-369-5_22 Generic methods for genetic manipulation of human embryonic stem cells (hESCs) are important for both present research and future commercial applications. To date, differences in cell derivation and culture have required independent optimization of transfection and transduction protocols and some lines have remained refractile to all methods. Here we describe a culture protocol that has been extensively tested in 12 different hESC lines (1, 2) and shown to support efficient gene transfer independent of the method of gene delivery or history of the cell line. The system is based on Matrigel monolayer culture and conditioned medium from mouse embryonic feeder cells (MEFs) and entails transient high-density culture followed by rapid adaptation to low density for gene transfer. Under these con... Springer protocols feed by Cell Biology info http://www.medworm.com/rss/comments.php?id=2988957 Tue, 13 Oct 2009 00:00:00 +0100 2988957 http://www.medworm.com/index.php?rid=2988956&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-369-5_21 One of the great advantages of embryonic stem (ES) cells over other cell types is their accessibility to genetic manipulation. They can easily undergo genetic modifications while remaining pluripotent and can be selectively propagated, allowing the clonal expansion of genetically altered cells in culture. Since the first isolation of ES cells in mice, many effective techniques have been developed for gene delivery and manipulation of ES cells. These include transfection, electroporation, and infection protocols, as well as different approaches for inserting, deleting, or changing the expression of genes. These methods proved to be extremely useful in mouse ES cells, for monitoring and directing differentiation, discovering unknown genes and studying their function, and are now being initia... Springer protocols feed by Cell Biology info http://www.medworm.com/rss/comments.php?id=2988956 Tue, 13 Oct 2009 00:00:00 +0100 2988956 http://www.medworm.com/index.php?rid=2988955&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-369-5_20 Traditional methods of studying the differentiation of human embryonic stem cells (hESCs) include generation of embryoid bodies, induced differentiation in vitro, and transplantation to immune-deficient mice. The chick embryo is a well-studied and accessible experimental system that has been used for many years as a xenograft host for mammalian cells. Several years ago, we performed experiments transplanting colonies of hESC into organogenesis-stage chick embryos to establish a novel system for studying the developmental programs and decisions of pluripotent human cells. Fluorescent hESC were used, in order to permit identification of the hESC in living embryos. We transplanted hESC into the trunk of chick embryos, both into and instead of developing somites. Our results showed that hESC s... Springer protocols feed by Cell Biology info http://www.medworm.com/rss/comments.php?id=2988955 Tue, 13 Oct 2009 00:00:00 +0100 2988955 http://www.medworm.com/index.php?rid=2988954&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-369-5_1 The culture and critical assessment of early human embryos during the first week of human development are reviewed for the derivation of ESC. Both normal and abnormal features are assessed by phase contrast microscopy of whole embryos and in serial sections of fixed material by light and electron microscopy (TEM). Normal embryos follow a time table of development and have equal blastomeres with minimal fragmentation and nuclear defects. Abnormal embryos show more fragmentation and nuclear aberrations such as micronucleation and multinucleation, reflected by aneuploidy, polyploidy, and mosaicism. The selection of normal embryos and the hardiest of embryos that survive to blastocysts is recommended for the derivation and culture of ESC. (Source: Springer protocols feed by Cell Biology) Springer protocols feed by Cell Biology info http://www.medworm.com/rss/comments.php?id=2988954 Tue, 13 Oct 2009 00:00:00 +0100 2988954 http://www.medworm.com/index.php?rid=2988953&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-369-5_19 We describe here a reproducible, chemically defined protocol that allows directed differentiation of hESCs to nearly pure neuroectodermal cells and neurons. First, hESC colonies are detached from mouse fibroblast feeder layers and form aggregates to initiate the differentiation procedure. Second, after 4 days of suspension culture, the ESC growth medium is replaced with neural induction medium to guide neuroectodermal specification. Third, the differentiating hESC aggregates are attached onto the culture surface at day 6–7, where columnar neural epithelial cells appear and organize into rosettes. Fourth, the neural rosettes are enriched by detaching rosettes and leaving the peripheral flat cells attached and expanded as neuroepithelial aggregates in the same medium. Finally, the neur... Springer protocols feed by Cell Biology info http://www.medworm.com/rss/comments.php?id=2988953 Tue, 13 Oct 2009 00:00:00 +0100 2988953 http://www.medworm.com/index.php?rid=2988952&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-369-5_18 The vascularization of tissue constructs remains a major challenge in regenerative medicine, as the diffusional supply of oxygen can support only 100–200 μm thick layers of viable tissue. The formation of a mature and functional vascular network requires communication between endothelial cells (ECs) and smooth muscle cells (SMCs). Potential sources of these cells that involve noninvasive methodologies are required for numerous applications including tissue-engineered vascular grafts, myocardial ischemia, wound healing, plastic surgery, and general tissue-engineering applications. Human embryonic stem cells (hESCs) can be an unlimited source of these cells. They can be expanded in vitro in an undifferentiated state without apparent limit, and hES-derived cells can be created i... Springer protocols feed by Cell Biology info http://www.medworm.com/rss/comments.php?id=2988952 Tue, 13 Oct 2009 00:00:00 +0100 2988952 http://www.medworm.com/index.php?rid=2988951&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-369-5_17 Human embryonic stem cells (hESCs) have the ability to self-renew and differentiate into any cell lineage of the three germ layers, therefore holding great promise for regenerative medicine applications. However, directing lineage-restricted differentiation of hESCs and obtaining a homogenous differentiated cell population is still a challenge. We previously described a micromass culture system as a model system to study chondrogenic commitment of the hESCs. Using this system, various growth factors including BMP2 and TGFβ1 direct chondrogenic differentiation and modulate cartilage-specific matrix gene expression in a distinctive manner. Furthermore, a high percentage of differentiated cells exhibit typical morphological characteristics of chondrocytes and express cartilage matrix pro... Springer protocols feed by Cell Biology info http://www.medworm.com/rss/comments.php?id=2988951 Tue, 13 Oct 2009 00:00:00 +0100 2988951 http://www.medworm.com/index.php?rid=2988950&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-369-5_16 Melanocytes are neural crest-derived pigment-producing cells that reside in the inner ear, in the uveal tract, in hair follicles, and in the skin. The main function of melanocytes is to provide pigmentation through melanin production and secretion to the immediate surrounding area. Although much is known about mature melanocyte function and regulation, particularly in the skin, little is known with regard to the signals and gene expression patterns that ensue upon melanocyte development and differentiation from embryonic precursors. The ability to examine these patterns in an in vitro specified setting through the use of embryonic stem cells holds great potential for understanding melanocyte biology. In this chapter, we outline our procedures for the differentiation of human embryonic stem... Springer protocols feed by Cell Biology info http://www.medworm.com/rss/comments.php?id=2988950 Tue, 13 Oct 2009 00:00:00 +0100 2988950 http://www.medworm.com/index.php?rid=2988949&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-369-5_15 Peripheral somatic sensory neurons (PSNs) are responsible for the critical function of transmitting multiple modalities of information from the outside world, including heat, touch, and pain, as well as the position of muscles required for coordinated voluntary movement to the central nervous system. Many peripheral neuropathies exist, including hereditary neurodegeneration in Familial Dysautonomia, infections of PSNs by viruses such as Varicella zoster and damage to PSNs and/or their process resulting from other disease conditions such as diabetes. Understanding of the etiology of these diseases and development of treatments is hampered by the lack of normal and healthy human PSNs for study, which are only available from abortuses or rare surgical procedures. (Source: Springer protocols f... Springer protocols feed by Cell Biology info http://www.medworm.com/rss/comments.php?id=2988949 Tue, 13 Oct 2009 00:00:00 +0100 2988949 http://www.medworm.com/index.php?rid=2988948&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-369-5_14 Human embryonic stem cells' (hESCs) unlimited proliferative potential and differentiation capability to all somatic cell types made them potential cell source in different cell-based tissue engineering strategies as well as various experimental applications in fields such as developmental biology, pharmacokinetics, toxicology, and genetics. Periodontal tissue engineering aims to improve the outcome of regenerative therapies which have variable success rates when contemporary techniques are used. Cell-based therapies may offer potential advantage in overcoming the inherent limitations associated with guided tissue-regeneration procedures, such as dependency on defect type and size and the pool and capacity of progenitor cells resident in the wound area. Elucidation of developmental mechanis... Springer protocols feed by Cell Biology info http://www.medworm.com/rss/comments.php?id=2988948 Tue, 13 Oct 2009 00:00:00 +0100 2988948 http://www.medworm.com/index.php?rid=2988947&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-369-5_13 Bacteriophage recombinases can target specific loci in human embryonic stem cells (hESCs) at high efficiency allowing for long-term expression of transgenes. In this chapter, we describe a retargeting system where phiC31 integrase is used to deliver a chromosomal target for a second integrase, R4. The engineered hESC line can be adapted for complex element assembly using Multisite Gateway technology. Retargeted clones show sustained expression and appropriate regulation of the transgenes over long-term culture and upon differentiation. The system described here represents a method to rapidly assemble complex plasmid-based assay systems, controllably insert them into the hESC genome, and have them actively express in pluripotent as well as in differentiated lineages there from. (Source: Spr... Springer protocols feed by Cell Biology info http://www.medworm.com/rss/comments.php?id=2988947 Tue, 13 Oct 2009 00:00:00 +0100 2988947 http://www.medworm.com/index.php?rid=2988946&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-369-5_12 Gap junctional intercellular communication (GJIC) has been described in different cell types including stem cells and has been involved in different biological events. GJIC is required for mouse embryonic stem cell maintenance and proliferation, and various studies suggest that functional GJIC is a common characteristic of human embryonic stem cells (hESC) maintained in different culture conditions. This chapter introduces methods to study gap junctions in hESC, from expression of gap junction proteins to functional study of GJIC in hESC proliferation, apoptosis, colony growth, and pluripotency. (Source: Springer protocols feed by Cell Biology) Springer protocols feed by Cell Biology info http://www.medworm.com/rss/comments.php?id=2988946 Tue, 13 Oct 2009 00:00:00 +0100 2988946 http://www.medworm.com/index.php?rid=2988945&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-369-5_11 This chapter describes the procedures in order to do immunofluorescence (IF) microscopy and quantitative PCR (qPCR) analysis of human embryonic stem cells (hESCs) grown specifically under feeder-free conditions. A detailed protocol outlining the steps from initially growing the cells, passaging onto 16-well glass chambers, and continuing with the general IF and qPCR steps will be provided. The techniques will be illustrated with new results on cellular localization of transcriptional factors and components of the Hedgehog, Wnt, and PDGF signaling pathways to primary cilia in stem cell maintenance and differentiation. Furthermore, a sample qPCR experiment will be shown illustrating that these techniques can be important tools in answering basic questions about hESC biology. (Source: Springe... Springer protocols feed by Cell Biology info http://www.medworm.com/rss/comments.php?id=2988945 Tue, 13 Oct 2009 00:00:00 +0100 2988945 http://www.medworm.com/index.php?rid=2988944&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-369-5_10 We describe a technique allowing paraffin embedding an entire hESC colony (e.g., ˜ 150 µm thick) and prepare 2-µm thick serial sections. Different staining procedures applied to individual sections produce a 2D survey of the developing hESC colony. Furthermore, a new and useful visualization of this 2D-expression pattern can be created by developing a 3D-model of the culture, based on serial paraffin sections. Individual sections are stained using individual markers. Using 3D image processing software such as Mimics or 3D-Doctor, the actual 3D-rendering of an entire colony can be accomplished. An extended version of this technique even allows for a high-magnification 3D-reconstruction of an area of interest (AOI), e.g., the developing hepatic stem cells. These techniques ... Springer protocols feed by Cell Biology info http://www.medworm.com/rss/comments.php?id=2988944 Tue, 13 Oct 2009 00:00:00 +0100 2988944 http://www.medworm.com/index.php?rid=3044477&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-421-0_2 Dendritic cells (DCs) are key coordinators of the immune response, governing the choice between tolerance and immunity. DCs are professional antigen-presenting cells capable of presenting antigen on MHC molecules and priming CD4 and CD8 T-cell responses. They form a heterogeneous group of cells based on phenotype, location, and function. In this review, murine DCs will be discussed regarding their function with special emphasis on their tissue distribution. Recent findings on DC homeostasis during cancer progression will be presented. Finally, the developmental pathways leading to DC differentiation from their precursors will be summarized. (Source: Springer protocols feed by Cell Biology) Springer protocols feed by Cell Biology info http://www.medworm.com/rss/comments.php?id=3044477 Fri, 09 Oct 2009 00:00:00 +0100 3044477 http://www.medworm.com/index.php?rid=3044476&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-421-0_1 Dendritic cells (DC) are critical to the induction and regulation of the innate and adaptive immune responses. They have been implicated in the pathogenesis of many autoimmune and chronic inflammatory diseases as well as contributing to the development of tumours by their lack of appropriate function. As such, understanding human DC biology provides the insight needed to develop applications for their use in the treatment of diseases. Currently, studies on mouse DC outnumber those on human cells; however, the comparison between mouse and human models has been somewhat misleading due to the basic biological and practical differences between the two models. In this review, we summarise the current understanding of human DC subtypes by describing the phenotype of the populations and how this ... Springer protocols feed by Cell Biology info http://www.medworm.com/rss/comments.php?id=3044476 Fri, 09 Oct 2009 00:00:00 +0100 3044476 http://www.medworm.com/index.php?rid=2988968&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-380-0_11 We describe here our use and refinement of a variant EMSA that can employ multiple and relatively long (up to 1000 bp) probes of promoter sequence in one binding reaction for interaction with nuclear proteins in general and individual transcription factors in particular. We provide labeling and electrophoresis methods suitable for such probes and also highlight the mobility shift differences one can expect with the variant probe method. (Source: Springer protocols feed by Cell Biology) Springer protocols feed by Cell Biology info http://www.medworm.com/rss/comments.php?id=2988968 Wed, 07 Oct 2009 00:00:00 +0100 2988968 http://www.medworm.com/index.php?rid=2988967&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-380-0_10 Gene silencing approaches afford investigators the ability to gain important insight into the normal functional requirements of specific epidermal proteins and promise to yield a powerful therapeutic means to dampen the level of proteins that are mutated or frequently overexpressed in skin disease. The efficient and tractable delivery of siRNAs into epidermal keratinocytes is seminal to this process. Here, we describe techniques for transient and long-term silencing of a representative gene product, namely desmoglein 1, in primary human epidermal keratinocytes maintained as submerged cultures or three-dimensional organotypic raft cultures. As a complement to epidermal-specific gene targeting strategies in mice, these technical approaches permit relatively rapid loss-of-function studies pur... Springer protocols feed by Cell Biology info http://www.medworm.com/rss/comments.php?id=2988967 Wed, 07 Oct 2009 00:00:00 +0100 2988967 http://www.medworm.com/index.php?rid=2875831&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-325-1_1 A gas chromatography-mass spectrometric method was developed that allowed the accurate, highly sensitive and specific quantification of F2-isoprostanes (F2-IsoPs) in different tissues and body fluids. Measurement of F2-IsoPs in isolated rat brain mitochondria, HaCaT keratinocytes, human plasma, and microdialysates of human skin has established the occurrence of oxidative stress in a variety of model systems and disease states. F2-IsoPs correlated with other markers of lipid peroxidation (e.g., TBARS, HETEs) in experimental models of oxidative stress. F2-IsoPs were elevated about 100-fold after iron/ascorbate-induced oxidative stress and 2- to 4-fold after pentylenetetrazol (PTZ)-induced seizures, in hemodialysis patients with end stage renal disease, in psoriasis patients, in HaCaT keratin... Springer protocols feed by Cell Biology info http://www.medworm.com/rss/comments.php?id=2875831 Thu, 01 Oct 2009 23:00:00 +0100 2875831 http://www.medworm.com/index.php?rid=2875830&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-325-1_3 Lysophosphatidylcholine (LPC) is a bioactive lipid implicated to play a functional role in various diseases including atherosclerosis, diabetes, cancer, and inflammation. Conventional methods are of limited value for a systematic evaluation of LPC species concentrations due to complicated, time-consuming procedures. Here we describe a methodology based on electrospray ionization tandem mass spectrometry (ESI-MS/MS) applicable for high-throughput LPC species quantification. This assay provides accuracy and precision sufficient for the analysis of large clinical studies as well as basic biochemical studies in a broad range of biological material including plasma, tissues, and cell culture material. This method may be combined with methods based on the same analytical setup for glycerophospho... Springer protocols feed by Cell Biology info http://www.medworm.com/rss/comments.php?id=2875830 Thu, 01 Oct 2009 23:00:00 +0100 2875830 http://www.medworm.com/index.php?rid=2875829&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-325-1_2 Plants form volatile oxylipins and related compounds under stress. Some of them are important flavor chemicals and give big impact on the flavor quality of food made from plant materials. They are also involved in defense responses of plants against pathogens and herbivores. Furthermore, in some instances, they cause harmful effects on plants themselves. Because of these significances of volatile oxylipins and related compounds, demands to perform comprehensive analyses of these compounds are increasing. In this chapter, we describe the simple but efficient procedures to reveal profiles of volatile oxylipins and related compounds by using HPLC and GC-MS. They are simple and can be performed in biochemical laboratories equipped with common facilities. (Source: Springer protocols feed by Cel... Springer protocols feed by Cell Biology info http://www.medworm.com/rss/comments.php?id=2875829 Thu, 01 Oct 2009 23:00:00 +0100 2875829 http://www.medworm.com/index.php?rid=2875828&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-325-1_4 Various analytical techniques have been adopted for the isolation and identification of the oxolipids and for determining their functionality. Gas chromatography in combination with mass spectrometry (MS) has been specifically utilized in analysis of isoprostanes and other low molecular weight oxolipids, although it requires derivatization of the solutes. In contrast, liquid chromatography (LC) in combination with on-line MS has proven to be well suited for analysis of intact oxolipids without (or minimal) derivatization. LC-MS has also been helpful for the identification of lipidomic changes resulting from covalent binding of lipid ester core aldehydes to amino lipids, amino acids, peptides, and proteins. This chapter reviews the use of the above techniques for lipidomic analysis of the a... Springer protocols feed by Cell Biology info http://www.medworm.com/rss/comments.php?id=2875828 Thu, 01 Oct 2009 23:00:00 +0100 2875828 http://www.medworm.com/index.php?rid=2875827&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-325-1_5 Membrane lipid rafts (LRs) have been demonstrated to be importantly involved in transmembrane signaling in a variety of mammalian cells. Many receptors can be aggregated within the LR clusters to form signaling platforms. Currently, LRs were reported to be clustered to aggregate, recruit, and assemble NADPH oxidase subunits and related proteins in various cells in response to various stimuli, forming redox signaling platforms. These LR signaling platforms may play important roles in the regulation of cellular activity and cell function, and also in the development of cell dysfunction or injury associated with various pathological stimuli. This LRs clustering-mediated mechanism is considered to take a center stage in redox signaling associated with death receptors. In this chapter, some bas... Springer protocols feed by Cell Biology info http://www.medworm.com/rss/comments.php?id=2875827 Thu, 01 Oct 2009 23:00:00 +0100 2875827 http://www.medworm.com/index.php?rid=2875826&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-325-1_6 Direct analysis of polyisoprenoid alcohols by electrospray ionization mass spectrometry (ESI-MS) often produces poor results requiring off-line time- and sample-consuming derivatization techniques. In this chapter, we describe a simple ESI-MS approach for the direct analysis of polyisoprenoid alcohols from biological samples. Lithium iodide is used to promote cationization by intense formation of [M+Li]+ adducts. Detection of polyisoprenoids with mass determination can thus be performed with high sensitivity (LOD near 100 pM), whereas characteristic collision-induced dissociations observed for both dolichols and polyprenols permit investigation of their structure. We also describe a simple ESI-MS approach for the direct analysis of carotenoids in biological samples using lithium iodide to ... Springer protocols feed by Cell Biology info http://www.medworm.com/rss/comments.php?id=2875826 Thu, 01 Oct 2009 23:00:00 +0100 2875826 http://www.medworm.com/index.php?rid=2875825&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-325-1_7 An amide-type adduct, hexanoyl-lysine (HEL) is generated from the reaction between n-6 fatty acid (FA)-derived lipid peroxide and lysine. Immunochemical and chemical methods can be used to detect the formation of HEL. For example, an ELISA kit using the monoclonal antibody to HEL is now commercially available. We recently identified propanoyl-lysine (propionyl-lysine, PRL) from the reaction of an n-3 FA and a lysine residue. The antibody to PRL has been prepared and characterized. Using these monoclonal antibodies, the localization of adducts in tissues has been confirmed. Moreover, both amide-type adducts, HEL and PRL, can be simultaneously measured using liquid chromatography mass spectrometry (LC/MS/MS) with isotope dilution methods. The LC/MS/MS analysis reveals the rigid amounts of th... Springer protocols feed by Cell Biology info http://www.medworm.com/rss/comments.php?id=2875825 Thu, 01 Oct 2009 23:00:00 +0100 2875825 http://www.medworm.com/index.php?rid=2875824&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-325-1_9 We describe application of this methodology in assessments of phospholipid hydroperoxides using as an example their characterization and quantitative determinations in different tissues of mice exposed to total body irradiation (TBI, 10 and 15 Gy). Using ESI-MS, we identified individual molecular species – with particular emphasis on polyunsaturated molecules as preferred peroxidation substrates – in major classes of phospholipids: cardiolipin (CL), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), and phosphatidylinositol (PI) isolated from mouse brain, lung, muscles, small intestine, and bone marrow. We show that the pattern of phospholipid oxidation 24 h after TBI is nonrandom and does not follow the phospholipid abundance in tissues. The anio... Springer protocols feed by Cell Biology info http://www.medworm.com/rss/comments.php?id=2875824 Thu, 01 Oct 2009 23:00:00 +0100 2875824 http://www.medworm.com/index.php?rid=2875823&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-325-1_8 Advances in understanding the neurodegenerative pathologies are creating new opportunities for the development of neuroprotective therapies, such as antioxidant food factors, lifestyle modification, and drugs. However, the biomarker by which to determine the effect of the agent on neurodegeneration is limited. We here address hexanoyl dopamine (HED), one of novel dopamine adducts derived from brain polyunsaturated acid, referring to its in vitro formation, potent toxicity to SH-SY5Y cells, and application to assess the neuroprotective effect of antioxidative food factors. Dopamine is a neurotransmitter and its deficiency is a characterized feature in Parkinson’s disease (PD), thereby HED represents a new addition to understanding of dopamine biology and pathophysiology of PD and a no... Springer protocols feed by Cell Biology info http://www.medworm.com/rss/comments.php?id=2875823 Thu, 01 Oct 2009 23:00:00 +0100 2875823 http://www.medworm.com/index.php?rid=2875822&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-325-1_10 Quantitative real-time reverse-transcription (RT)-PCR is a precise and sensitive method to measure mRNA levels over a broad dynamic range. This chapter describes the quantitative transcript analysis of 41 selected lipid-related transcripts in macrophages and microglia using a novel “Lipidomic” Taqman Array. The Taqman Array results show that (1) stimulation with the liver-X-receptor and retinoid-X-receptor ligands T0901317 and 9-cis retinoic acid induces several genes of lipid metabolism, (2) lipopolysaccharide (LPS) and interferon-g (Ifn-g) strongly repress lipid-related genes, and (3) coincubation with docosahexaenoic acid dampens the repressing effect of LPS. The method described in this chapter can be used to monitor the transcriptional response of 41 dynamic “lipid&r... Springer protocols feed by Cell Biology info http://www.medworm.com/rss/comments.php?id=2875822 Thu, 01 Oct 2009 23:00:00 +0100 2875822 http://www.medworm.com/index.php?rid=2875821&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-325-1_12 Sterols are essential lipid components of eukaryotic membranes. They are synthesized in the endoplasmatic reticulum (ER) from where they are efficiently transported to the plasma membrane, which harbors ~90% of the free sterol pool of the cell. The molecular mechanisms that govern this lipid transport, however, are not well characterized and are challenging to analyze. Saccharomyces cerevisiae offers the opportunity to circumvent some of the technical limitations associated with studying this forward transport of sterols from the ER to the plasma membrane, because the organism can also take up sterols from the environment, incorporate them into the plasma membrane and transport them back to the ER, where the free sterol is converted to steryl esters. This reverse sterol transport, however,... Springer protocols feed by Cell Biology info http://www.medworm.com/rss/comments.php?id=2875821 Thu, 01 Oct 2009 23:00:00 +0100 2875821 http://www.medworm.com/index.php?rid=2875820&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-325-1_11 Imaging membrane lipid domains to characterize their organization and function has been hindered by the lack of reliable lipid-specific probes. In this paper, we provide detailed methods to investigate, mainly by confocal microscopy, the distribution and dynamics of two components of the “lipid rafts,” sphingomyelin (SM) and cholesterol, using two specific lipid probes that have been extensively studied in the laboratory: lysenin, a SM-binding toxin and the fluorescent esters of poly(ethylene glycol) cholesteryl ether (PEG-Chol) that label cholesterol-rich domains. The production of nontoxic forms of lysenin as well as its specific binding behavior have allowed monitoring the distribution and the dynamics of SM-rich domains in living cell membranes. Because of its water-solubil... Springer protocols feed by Cell Biology info http://www.medworm.com/rss/comments.php?id=2875820 Thu, 01 Oct 2009 23:00:00 +0100 2875820 http://www.medworm.com/index.php?rid=2875819&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-325-1_13 The process of fatty acid transport across the plasma membrane occurs by several mechanisms that involve distinct membrane-bound and membrane-associated proteins and enzymes. Among these are the fatty acid transport proteins (FATP) and long-chain acyl CoA synthetases (Acsl). Previous studies in yeast and adipocytes have shown FATP and Acsl form a physical complex at the plasma membrane and are required for fatty acid transport, which proceeds through a coupled process-linking transport with metabolic activation termed vectorial acylation. At present, six isoforms of FATP and five isoforms of ACSL have been identified in mice and man. In addition, there are a number of splice variants of different FATP and Acsl isoforms. The different FATP and Acsl isoforms have distinct tissue expression p... Springer protocols feed by Cell Biology info http://www.medworm.com/rss/comments.php?id=2875819 Thu, 01 Oct 2009 23:00:00 +0100 2875819 http://www.medworm.com/index.php?rid=2875818&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-325-1_14 The ultimate goal of proteomics is to characterize the function of all proteins in parallel and in the most physiologically relevant settings possible. A step toward this goal has been the introduction of activity-based proteomics. The simultaneous detection of individual protein activities can be facilitated directly in the proteome using specific activity-based probes consisting of a recognition site targeting a certain enzyme species, a properly positioned reactive site which forms a covalent bond with the target and a reporter tag for visualization and/or purification of the covalently bound target. As properties like polarity, size, charge, structure, and chemical reactivity of the reporter tag have a large impact on the reactivity of the probes toward the target enzymes probes suitab... Springer protocols feed by Cell Biology info http://www.medworm.com/rss/comments.php?id=2875818 Thu, 01 Oct 2009 23:00:00 +0100 2875818 http://www.medworm.com/index.php?rid=2875817&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-325-1_15 Aging is the major risk factor for age-related maculopathy (ARM), the biggest cause of vision loss among the elderly in industrialized societies, and a major change in the affected tissues is the age-related accumulation of neutral lipid in Bruch’s membrane (BrM) of the eye throughout adulthood. Here we show that esterified cholesterol (EC) is the major neutral lipid species in this tissue, which has implications for potential sources of this material. The combination of filipin histochemistry and comprehensive lipid profiling made possible this insight on a complex tissue. (Source: Springer protocols feed by Cell Biology) Springer protocols feed by Cell Biology info http://www.medworm.com/rss/comments.php?id=2875817 Thu, 01 Oct 2009 23:00:00 +0100 2875817 http://www.medworm.com/index.php?rid=2875816&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-325-1_16 Mass spectrometry (MS), particularly electrospray-MS, is the key tool in modern lipidomics. However, as even a modest scale experiment produces a great amount of data, data processing often becomes limiting. Notably, the software provided with MS instruments are not well suited for quantitative analysis of lipidomes because of the great variety of species present and complexities in response calibration. Here we describe the use of two recently introduced software tools: lipid mass spectrum analysis (LIMSA) and spectrum extraction from chromatographic data (SECD), which significantly increase the speed and reliability of mass spectrometric analysis of complex lipidomes. LIMSA is a Microsoft Excel add-on that (1) finds and integrates the peaks in an imported spectrum, (2) identifies the pea... Springer protocols feed by Cell Biology info http://www.medworm.com/rss/comments.php?id=2875816 Thu, 01 Oct 2009 23:00:00 +0100 2875816 http://www.medworm.com/index.php?rid=2875815&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-325-1_17 Triacylglycerols (TGs) are principal components of vegetable oils and animal fats. Natural TGs form extremely complex mixtures composed of tens or hundreds of molecular species. HPLC/MS suits well for their analyses, but manual data processing is laborious and time-consuming. Specialized software algorithms are needed to accelerate the interpretation process. Here we present software named TriglyAPCI for interpreting APCI, APPI, or ESI MS/MS spectra of TGs. The chapter shows how to build and use the software, what are its advantages and limitations. The algorithm uses diacylglycerol fragments and molecular adducts for determining TG structure. Each ion in a spectrum is tested whether it might be a fragment or a molecular adduct. If so, the number of carbons and double bonds is assigned to ... Springer protocols feed by Cell Biology info http://www.medworm.com/rss/comments.php?id=2875815 Thu, 01 Oct 2009 23:00:00 +0100 2875815 http://www.medworm.com/index.php?rid=2875814&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-325-1_18 Computer simulation has become an increasingly popular tool in the study of lipid membranes, complementing experimental techniques by providing information on structure and dynamics at high spatial and temporal resolution. Molecular visualization is the most powerful way to represent the results of molecular simulations, and can be used to illustrate complex transformations of lipid aggregates more easily and more effectively than written text. In this chapter, we review some basic aspects of simulation methodologies commonly employed in the study of lipid membranes and we describe a few examples of complex phenomena that have been recently investigated using molecular simulations. We then explain how molecular visualization provides added value to computational work in the field of biolog... Springer protocols feed by Cell Biology info http://www.medworm.com/rss/comments.php?id=2875814 Thu, 01 Oct 2009 23:00:00 +0100 2875814 http://www.medworm.com/index.php?rid=2875813&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-325-1_19 Owing to their importance in cellular physiology and pathology as well as to recent technological advances, the study of lipids has reemerged as a major research target. However, the structural diversity of lipids presents a number of analytical and informatics challenges. The field of lipidomics is a new postgenome discipline that aims to develop comprehensive methods for lipid analysis, necessitating concomitant developments in bioinformatics. The evolving research paradigm requires that new bioinformatics approaches accommodate genomic as well as high-level perspectives, integrating genome, protein, chemical and network information. The incorporation of lipidomics information into these data structures will provide mechanistic understanding of lipid functions and interactions in the con... Springer protocols feed by Cell Biology info http://www.medworm.com/rss/comments.php?id=2875813 Thu, 01 Oct 2009 23:00:00 +0100 2875813 http://www.medworm.com/index.php?rid=2875812&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-325-1_20 Past literature on exposure to lipophilic agents such as organochlorines (OCs) is conflicting, posing challenges for the interpretation of their potential human health risks. Since blood is often used as a proxy for adipose tissue, it is necessary to model serum lipids when assessing health risks of OCs. Using a simulation study, we evaluated four statistical models (unadjusted, standardized, adjusted, and two-stage) for the analysis of polychlorinated biphenyls (PCBs) exposure, serum lipids, and health outcome risk. Eight candidate true causal scenarios, depicted by directed acyclic graphs, were used to illustrate the ramifications of misspecification of underlying assumptions when interpreting results. Biased results were produced when statistical models that deviated from the underlying... Springer protocols feed by Cell Biology info http://www.medworm.com/rss/comments.php?id=2875812 Thu, 01 Oct 2009 23:00:00 +0100 2875812 http://www.medworm.com/index.php?rid=2819937&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-322-0_25 Lipases are responsible for the hydrolysis of acylglycerols and cholesteryl esters in animals, plants, and microorganisms. In this chapter we describe a tool for the concomitant analysis of lipases in complex proteomes. For this purpose, the target enzymes are selectively and covalently labelled with fluorescent suicide inhibitors. Stable lipid–protein complexes are formed that are separated by gel electrophoresis and identified by mass spectrometry. (Source: Springer protocols feed by Cell Biology) Springer protocols feed by Cell Biology info http://www.medworm.com/rss/comments.php?id=2819937 Tue, 22 Sep 2009 18:18:54 +0100 2819937 http://www.medworm.com/index.php?rid=2819936&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-322-0_24 Plant oil has become an important component in the search for a replacement for non-renewable energy sources, as well as being used for a wide range of industrial purposes, all in addition to its vital importance for human diet. Detailed knowledge of the lipid distribution in plants is fundamental for the understanding of local regulatory networks covering storage metabolism, and for the development of new approaches for plant breeding and transgenic research. We here review a measurement protocol or “tool” based on magnetic resonance imaging (MRI), which allows the non-invasive detection and quantitative visualization of lipid in living plant tissue. The method provides quantitative lipid maps with a resolution close to the cellular level and can be used on a wide range of pla... Springer protocols feed by Cell Biology info http://www.medworm.com/rss/comments.php?id=2819936 Tue, 22 Sep 2009 18:18:54 +0100 2819936 http://www.medworm.com/index.php?rid=2819935&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-322-0_9 Matrix-assisted laser desorption and ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) can provide rapid, sensitive determinations of lipids from small tissue samples in both single determinations and automated high-throughput assays. MALDI-TOF MS is a sensitive, high-throughput technique for the determination of lipids such as the phosphoinositides, PtdIns (phosphatidylinositol), PIP (phosphatidylinositol-4-phosphate, and PIP2 (phosphatidylinositol-4,5-bisphosphate), but in crude extracts the signals are weak or not observed due in large part to ion suppression by phosphatidylcholine and other cationic lipids. A rapid separation step using a small column of a strong cation exchange (SCX) gel can be utilized easily and effectively to adsorb or capture cationic lipids from chloro... Springer protocols feed by Cell Biology info http://www.medworm.com/rss/comments.php?id=2819935 Tue, 22 Sep 2009 18:18:54 +0100 2819935 http://www.medworm.com/index.php?rid=2819934&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-322-0_8 Eicosanoids are oxygenated, endogenous, unsaturated fatty acids derived from arachidonic acid. Detection and quantification of these compounds are of great interest because they play important roles in a number of significant diseases, including asthma, chronic obstructive pulmonary disease (COPD), cardiovascular disease, and cancer. Because the endogenous levels of eicosanoids are quite low, sensitive and specific analytical methods are required to reliably quantify these compounds. High-performance liquid chromatography mass spectrometry (HPLC/MS) has emerged as one of the main techniques used in eicosanoid profiling. Herein, we describe the main LC/MS techniques and principles as well as their application in eicosanoid analysis. In addition, a protocol is given for extracting eicosanoid... Springer protocols feed by Cell Biology info http://www.medworm.com/rss/comments.php?id=2819934 Tue, 22 Sep 2009 18:18:53 +0100 2819934 http://www.medworm.com/index.php?rid=2819933&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-322-0_7 Applications of tandem mass spectrometry in the field of lipid clinical chemistry are considered. Haemato­logical and biochemical advantages are presented favoring the choice of red blood cell membranes as a starting material in a wide variety of biomedical fields. Practical considerations are discussed with respect to methods of sampling, storage, and lipid extraction of red blood cells. The chapter describes the capabilities of a direct infusion of raw lipid extracts in the electro-spray ionization source compared with the more sophisticated method of high-performance liquid chromatography coupled with hybrid tandem mass spectrometry. Both methods have been evaluated and have been shown to be suitable for diagnosis and/or monitoring for a variety of human disorders. (Source: Springer... Springer protocols feed by Cell Biology info http://www.medworm.com/rss/comments.php?id=2819933 Tue, 22 Sep 2009 18:18:53 +0100 2819933 http://www.medworm.com/index.php?rid=2819932&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-322-0_6 The important roles of lipids particularly certain phospholipids in signal transduction processes and as important disease markers are becoming increasingly evident. Unfortunately, however, sensitive methods of lipid analysis are established to a much lesser extent than, e.g., methods of protein analysis. Mass spectrometry (MS) is an increasingly used technique of lipid analysis and electrospray ionization (ESI) MS is the so far most established ionization method. Although matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS) was so far primarily used for protein analysis, however, this method has itself proven to be very useful in the field of lipid analysis, too. (Source: Springer protocols feed by Cell Biology) Springer protocols feed by Cell Biology info http://www.medworm.com/rss/comments.php?id=2819932 Tue, 22 Sep 2009 18:18:53 +0100 2819932 http://www.medworm.com/index.php?rid=2819931&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-322-0_5 The use of NMR spectroscopy in lipid research has been traditionally reserved for the analysis and structural elucidation of discrete lipid molecules. Although NMR analysis of organic molecules provides a plethora of structural information that is normally unattainable by most other techniques, its use for global analysis of mixed lipid pools has been hampered by its relatively low sensitivity and overlapping of signals in the spectrum. However, the last few decades have witnessed great advancements in NMR spectroscopy that generally resulted in greater sensitivity and offered more flexibility in sampling techniques. The method discussed in this chapter describes the use of NMR for global lipidome analysis. This methodology benefits from the quantitative nature of this technique together w... Springer protocols feed by Cell Biology info http://www.medworm.com/rss/comments.php?id=2819931 Tue, 22 Sep 2009 18:18:53 +0100 2819931 http://www.medworm.com/index.php?rid=2819930&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-322-0_23 A gradient HPLC-charged aerosol detection method was applied to the measurement of different lipids including: free fatty acids, fatty alcohols, glycerides, steroids, phospholipids, and fat-soluble vitamins. An algal oil sample is used as an example. Charged aerosol detection offers a new approach to the routine analysis of any nonvolatile lipid. It shows low ng (on column) sensitivity, a dynamic range of over four orders of magnitude, good reproducibility, gradient compatibility, and similar analyte response for nonvolatile species, independent of chemical structure. Furthermore, charged aerosol detection uses mobile phases that are fully compatible with LC–MS – enabling both detectors to be used in a “lipidomics platform.” (Source: Springer protocols feed by Cell ... Springer protocols feed by Cell Biology info http://www.medworm.com/rss/comments.php?id=2819930 Tue, 22 Sep 2009 18:18:53 +0100 2819930 http://www.medworm.com/index.php?rid=2819929&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-322-0_22 There has been a recent explosion in research concerning novel bioactive sphingolipids (SPLs) such as ceramide (Cer), sphingosine (Sph), and sphingosine 1-phosphate (Sph-1P) and this has necessitated the development of accurate and user-friendly methodology for analyzing and quantitating the endogenous levels of these molecules. ESI/MS/MS methodology provides a universal tool used for detecting and monitoring changes in SPL levels and composition from biological materials. Simultaneous ESI/MS/MS analysis of sphingoid bases (SBs), sphingoid base 1-phosphates (SB-1Ps), ceramides (Cers), ceramide 1-phosphates (Cer-1P), glucosyl/galactosyl-ceramides (Glu-Cers), and sphingomyelins (SMs) is performed on a Thermo Fisher Scientific triple quadrupole mass spectrometer operating in a multiple reacti... Springer protocols feed by Cell Biology info http://www.medworm.com/rss/comments.php?id=2819929 Tue, 22 Sep 2009 18:18:53 +0100 2819929 http://www.medworm.com/index.php?rid=2819928&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-322-0_21 Modern lipidomics relies heavily on mass spectrometry for the structural characterization and quantification of lipids of biological origins. Structural information is gained by tandem mass spectrometry (MS/MS) whereby lipid ions are fragmented to elucidate lipid class, fatty acid chain length, and degree of unsaturation. Unfortunately, however, in most cases double bond position cannot be assigned based on MS/MS data alone and thus significant structural diversity is hidden from such analyses. For this reason, we have developed two online methods for determining double bond position within unsaturated lipids; ozone electrospray ionization mass spectrometry (OzESI–MS) and ozone-induced dissociation (OzID). Both techniques utilize ozone to cleave C–C double bonds that result in ... Springer protocols feed by Cell Biology info http://www.medworm.com/rss/comments.php?id=2819928 Tue, 22 Sep 2009 18:18:53 +0100 2819928 http://www.medworm.com/index.php?rid=2819927&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-322-0_20 In the last decades, free radical processes delineated an interdisciplinary field linking chemistry to biology and medicine. Free radical mechanisms became of importance as molecular basis of physiological and pathological conditions. Lipids, in particular, unsaturated fatty acids, are susceptible of free radical attack. The reactivity of the double bond toward free radicals is well known, in particular the reversible addition of radical species to this functionality determines the cis–trans double bond isomerization. Since the prevalent geometry displayed by unsaturated fatty acids in eukaryotes is cis, the occurrence of the cis–trans isomerization by free radicals corresponds to the loss of an important structural information linked to biological activity. The formation of tr... Springer protocols feed by Cell Biology info http://www.medworm.com/rss/comments.php?id=2819927 Tue, 22 Sep 2009 18:18:53 +0100 2819927 http://www.medworm.com/index.php?rid=2819926&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-322-0_19 Very long chain fatty acids confer functional diversity on cells by variations in their chain length and degree of unsaturation. Microsomal fatty acid elongation represents the major pathway for determining the chain length of saturated, monounsaturated, and polyunsaturated fatty acids in cellular lipids. The overall reaction for fatty acid elongation involves four enzymes and utilizes malonyl CoA, NADPH, and fatty acyl CoA as substrates. While the fundamental pathway and its requirements have been known for many years, recent advances have revealed a family of enzymes involved in the first step of the reaction, i.e., the condensation reaction. Seven fatty acid elongase subtypes (Elovl #1–7) have been identified in the mouse, rat, and human genomes. These enzymes determine the rate o... Springer protocols feed by Cell Biology info http://www.medworm.com/rss/comments.php?id=2819926 Tue, 22 Sep 2009 18:18:53 +0100 2819926 http://www.medworm.com/index.php?rid=2819925&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-322-0_18 Quantitative analysis of components from different subcellular fractions is a key to the understanding of metabolic function as well as to the origin, the biogenesis, and the crosstalk of organelles. The yeast is an excellent model organism to address such questions from the biochemical, molecular biological, and cell biological viewpoints. A yeast organelle which gained much interest during the last decade is the lipid particle/droplet (LP), a storage compartment for nonpolar lipids but at the same time an organelle actively contributing to cellular metabolism. In this chapter, we describe methods and techniques that are commonly used to analyze lipids from LP at the molecular level by thin-layer chromatography, gas–liquid chromatography, and mass spectrometry. We provide an easy to... Springer protocols feed by Cell Biology info http://www.medworm.com/rss/comments.php?id=2819925 Tue, 22 Sep 2009 18:18:53 +0100 2819925 http://www.medworm.com/index.php?rid=2819924&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-322-0_17 We describe the procedure to extract, isolate, and purify the adipose components (TG-glycerol, TG-palmitate, and genomic DNA) and the derivation procedure to analyze the isotopic 2H-enrichment of these components by gas chromatography/mass spectrometry. The calculation principles are described to obtain the fractional and absolute synthesis rates of TG, of DNL, and of DNA measured in the adipose tissues. The method is nonradioactive, nonhazardous, accurate, reproducible, and very sensitive. We present recent in vivo data on the ontogeny of adipose tissue growth dynamics in young and adult obese Zucker rats compared with lean Zucker rats. (Source: Springer protocols feed by Cell Biology) Springer protocols feed by Cell Biology info http://www.medworm.com/rss/comments.php?id=2819924 Tue, 22 Sep 2009 18:18:53 +0100 2819924 http://www.medworm.com/index.php?rid=2819923&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-322-0_16 This study depicts the potential of MALDI-TOF/MS as an easy, fast, and reliable technique to characterize the TAG and FAPs in vegetable oils. (Source: Springer protocols feed by Cell Biology) Springer protocols feed by Cell Biology info http://www.medworm.com/rss/comments.php?id=2819923 Tue, 22 Sep 2009 18:18:53 +0100 2819923 http://www.medworm.com/index.php?rid=2819922&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-322-0_15 In this chapter we are going to mention about three different approaches in lipidomics and how to effectively profile or calculate the amounts of phospholipids from major molecular species up to minor ones. 1) Precise identification and profiling of individual molecular species of phospholipids by data-dependent LC–ESIMS/MS combination with “Lipid Search”. We have been using this method as a global analysis of phospholipid. We usually applied this method at least once for new biological samples. We constructed an automated search engine, “Lipid Search”, for identification and profiling of phospholipids. Once after applying this analysis, a specified retention time can be obtained for each elution peak of i... Springer protocols feed by Cell Biology info http://www.medworm.com/rss/comments.php?id=2819922 Tue, 22 Sep 2009 18:18:53 +0100 2819922 http://www.medworm.com/index.php?rid=2819921&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-322-0_14 Lipidomics aim to generate qualitative and quantitative information on different classes of lipids and their species, and when applied in conjunction with proteomic and genomic assays, facilitate the comprehensive study of lipid metabolism in cellular, organ, or body systems. Advances in mass spectrometry have underpinned the expansion of lipidomic methodologies. Prostanoids are potent autacoids present in a plethora of cellular systems, known best for their intimate role in inflammation. Electrospray ionisation (ESI) allows the efficient ionisation of prostanoids in aqueous systems. ESI can be readily coupled to liquid chromatography (LC) followed by tandem mass spectrometry (MS/MS)-based detection, thus allowing the development of a potent and selective LC/ESI–MS/MS quantitative as... Springer protocols feed by Cell Biology info http://www.medworm.com/rss/comments.php?id=2819921 Tue, 22 Sep 2009 18:18:53 +0100 2819921 http://www.medworm.com/index.php?rid=2819920&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-322-0_13 Membrane rafts are ordered microdomains of the plasma membrane consisting of cholesterol, sphingolipids, and saturated fatty acids which appear to regulate many cellular signaling pathways. One such type of membrane raft is caveolae, which are cave-like invaginations of the plasma membrane. Interestingly, changes in the acyl composition of cellular membranes have been shown to alter the specific localization of membrane raft associated proteins and their function. This is noteworthy because modification of membrane acyl composition is readily accomplished through changes in dietary fat composition. Here we describe a common approach used to fractionate cell membranes to obtain an enriched preparation of caveolae and gas chromatographic techniques to determine fatty acyl composition. In add... Springer protocols feed by Cell Biology info http://www.medworm.com/rss/comments.php?id=2819920 Tue, 22 Sep 2009 18:18:53 +0100 2819920 http://www.medworm.com/index.php?rid=2819919&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-322-0_12 Imaging mass spectrometry (IMS) provides a unique method to probe for chemical distributions within tissue sections with high chemical specificity. The direct analysis of tissue sections by mass spectrometry, which is the field of IMS, is relatively young, 10 year old; however, the techniques for mass spectrometric analysis are well known. Critical aspects of IMS then are the preparation of tissue specimens for insertion into a vacuum chamber and the interpretation of results with respect to disease studies. Here, we describe the methodologies for geographic localization of phospholipids in flat-mounted eye segments from rhesus monkey using IMS with matrix-assisted laser desorption/ionization (MALDI) and tandem mass spectrometry. (Source: Springer protocols feed by Cell Biology) Springer protocols feed by Cell Biology info http://www.medworm.com/rss/comments.php?id=2819919 Tue, 22 Sep 2009 18:18:53 +0100 2819919 http://www.medworm.com/index.php?rid=2819918&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-322-0_11 High-pressure liquid chromatography–mass spectrometry (HPLC–MS) has become a de facto standard analytical tool in lipidomic analyses of complex biological samples. This technique offers the best combination of selectivity and sensitivity among the currently available analytical methods, and provides not only the retention times of analytes, but also their m/z values, from which molecular masses of the compounds can be deduced. Further enhancement of the technique comes from the fact that some of the MS instruments (also known as ion traps, or MS n instruments) are capable of multistage fragmenting of the analytes, thus enabling the researcher to perform their structural elucidation. These capabilities make HPLC–MS n an ideal tool for an... Springer protocols feed by Cell Biology info http://www.medworm.com/rss/comments.php?id=2819918 Tue, 22 Sep 2009 18:18:53 +0100 2819918 http://www.medworm.com/index.php?rid=2819917&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-322-0_10 Lipidomics studies the large-scale changes in nonwater-soluble metabolites (lipids) accompanying perturbations of biological systems. Because lipids are involved in crucial biological mechanisms, there is a growing scientific interest in using lipidomic approaches to understand the regulation of the lipid meta-bolism in all eukaryotic and prokaryotic organisms. Lipidomics is a powerful tool in system biology that can be used together with genomics, transcriptomics, and proteomics to answer biological questions arising from various scientific areas such as environmental sciences, pharmacology, nutrition, biophysics, cell biology, physiology, pathology, and disease diagnostics. One of the main challenges for lipidomic analysis is the range of concentrations and chemical complexity of differe... Springer protocols feed by Cell Biology info http://www.medworm.com/rss/comments.php?id=2819917 Tue, 22 Sep 2009 18:18:53 +0100 2819917 http://www.medworm.com/index.php?rid=2819916&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-322-0_4 Lipids from dietary sources or from de novo synthesis are transported while bound to proteins to other tissues where they are used for cell membrane synthesis or stored for energy generation. In cell membranes or in plasma, lipids can undergo several modifications that are important in cell function. Several proteins orchestrate the transport, biosynthesis, and modification of lipids. Thus, the intersection of lipids and proteins is important in human metabolic pathways. Recent advances in mass spectrometry and bioinformatics have made it possible to obtain compositional (structural and functional) data of lipid molecular species and proteins in biological samples. This combination of lipidomics and proteomics is advantageous because it allows us to better define biochemical pathways, disc... Springer protocols feed by Cell Biology info http://www.medworm.com/rss/comments.php?id=2819916 Tue, 22 Sep 2009 18:18:52 +0100 2819916 http://www.medworm.com/index.php?rid=2819915&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-322-0_3 Despite an increasing recognition of the causative and diagnostic role of lipids in the onset and progression of retinal disease, information on the global lipid profile of the normal retina is quite limited. Here, a “shotgun” tandem mass spectrometry approach involving the use of multiple lipid class-specific precursor ion and neutral loss scan mode experiments has been employed to analyze lipid extracts from normal rat retina, obtained with minimal sample handling prior to analysis. Redundant information for the identification and characterization of molecular species in each lipid class was obtained by complementary analysis of their protonated or deprotonated precursor ions, or by analysis of their various ionic adducts (e.g., Na+, NH4 +, Cl–, CH3OCO2 ... Springer protocols feed by Cell Biology info http://www.medworm.com/rss/comments.php?id=2819915 Tue, 22 Sep 2009 18:18:52 +0100 2819915 http://www.medworm.com/index.php?rid=2819914&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-322-0_2 Docosahexaenoic acid (DHA, 22:6 n-3) is an omega-3 fatty acid with a 22 carbon acyl chain containing six cis double bonds and is predominantly found in membrane glycerophospholipids. Dietary consumption of DHA has been positively linked with the prevention of numerous pathologies and consequently, it has been the focus of extensive research over the last four decades. Nevertheless, our understanding of its molecular mode of action is not well understood. One likely mechanism is through DHA’s influence on cell membranes and the proteins embedded within them. This influence may be altered depending on the glycerophospholipid head group DHA is esterified to and its fatty acid partner, i.e., the specific glycerophospholipid molecule. Accordingly, an understanding of the exact glycerophos... Springer protocols feed by Cell Biology info http://www.medworm.com/rss/comments.php?id=2819914 Tue, 22 Sep 2009 18:18:52 +0100 2819914 http://www.medworm.com/index.php?rid=2819913&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-322-0_1 Contamination from subcellular organelles and myelin has hindered attempts to characterize the lipidome of brain mitochondria. A high degree of mitochondrial purity is required for accurate measurements of the content and molecular species composition of mitochondrial lipids. We devised a discontinuous Ficoll and sucrose gradient procedure for the isolation and purification of brain mitochondria free from any detectable contamination. Shotgun lipidomics was used to analyze the lipid composition of the brain mitochondria. These procedures can be used to determine whether intrinsic lipid abnormalities underlie mitochondrial dysfunction associated with neurological and neurodegenerative diseases. (Source: Springer protocols feed by Cell Biology) Springer protocols feed by Cell Biology info http://www.medworm.com/rss/comments.php?id=2819913 Tue, 22 Sep 2009 18:18:52 +0100 2819913 http://www.medworm.com/index.php?rid=2778281&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-103-5_1 Visualization of meiotic chromosomes in the model organism S. cerevisiae has become an integral part of the study of wild-type meiosis and the characterization of mutant phenotypes. This chapter describes a simple method for chromosome spreading, which is a variation on a protocol originally developed by Dresser and Giroux (1). This method uses osmotic pressure to spread the nuclear contents of spheroplasted meiotic cells over a glass slide enabling unobstructed inspection of the chromosomal morphology. Chromosomes from all meiotic stages can be analyzed using indirect immunofluorescence to visualize meiotic proteins involved in different processes of meiosis, including recombination, synapsis, sister chromatid cohesion, and chromosome disjunction. (Source: Springer protocols feed by Cell ... Springer protocols feed by Cell Biology info http://www.medworm.com/rss/comments.php?id=2778281 Fri, 31 Jul 2009 23:00:00 +0100 2778281 http://www.medworm.com/index.php?rid=2778280&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-103-5_3 Fluorescent in situ hybridization (FISH) provides a powerful tool to study the localization of DNA sequences in relationship to one another. FISH has the advantage over other methods, notably use of GFP-tagged repressor/operator arrays, that an almost unlimited number of probes can be utilized without having to make new strains for each new locus one wants to study. Also, the number of sites that can be visualized at the same time is limited only by the number of fluorophores that are available and can be distinguished by the available microscope. Described here is a method for FISH analysis and its application to analysis of chromosome pairing during meiosis in S. cerevisiae. (Source: Springer protocols feed by Cell Biology) Springer protocols feed by Cell Biology info http://www.medworm.com/rss/comments.php?id=2778280 Fri, 31 Jul 2009 23:00:00 +0100 2778280 http://www.medworm.com/index.php?rid=2778279&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-103-5_2 The fission yeast, Schizosaccharomyces pombe, much like the budding yeast, is a particularly well-suited model organism for genetic research. However, the miniscule size of both yeasts’ nuclei has hindered their success as research models for cytologists. A solution to this problem is provided by the spreading of nuclei, which increases their volume and allows for a better spatial resolution of nuclear contents. Here we describe nuclear spreading in fission yeast. Spreading of meiotic nuclei is particularly helpful in exposing the linear elements (LinEs), which are the fission yeasts’ rudimentary version of the synaptonemal complex. Although the LinEs’ role is still not fully understood, they serve as important meiotic hallmarks and their presence and morphology can be us... Springer protocols feed by Cell Biology info http://www.medworm.com/rss/comments.php?id=2778279 Fri, 31 Jul 2009 23:00:00 +0100 2778279 http://www.medworm.com/index.php?rid=2778278&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-103-5_4 The fission yeast Schizosaccharomyces pombe has provided a useful experimental system to study nuclear structures during meiosis. Unlike many higher animals in which meiosis takes place only in specialized tissues deep inside their bodies, S. pombe is a unicellular eukaryote and its meiosis can be induced simply by depleting nitrogen sources from the culture medium. The entire process of meiosis is completed within several hours, and thus can be followed in individual living cells. These features provide ease of microscopic observation. A more trivial merit is its rod-like cell shape, which aids microscopic observation, as the long axis of cells is kept in the microscope image plane. Here we describe methods for induction of meiosis and fluorescence microscopy observation in living cells o... Springer protocols feed by Cell Biology info http://www.medworm.com/rss/comments.php?id=2778278 Fri, 31 Jul 2009 23:00:00 +0100 2778278 http://www.medworm.com/index.php?rid=2778277&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-103-5_5 Movements are implicit in the chromosome behaviors of bouquet formation, pairing and synapsis during meiotic prophase. In S. cerevisiae, the positions of chromosomes, specific structures, and individual chromosomal loci marked by fluorescent fusion proteins are easily visualized in living cells. Time-lapse analyses have revealed rapid and varied chromosome movements throughout meiotic prophase. To facilitate the analysis of these movements, we have developed a simple, inexpensive, and efficient method to prepare sporulating cells for fluorescence microscopy. This method produces a monolayer of cells that progress from meiosis through spore formation, allows visualization of hundreds of cells in a single high-resolution frame and is suitable for most methods of fluorescence microscopy. (Sou... Springer protocols feed by Cell Biology info http://www.medworm.com/rss/comments.php?id=2778277 Fri, 31 Jul 2009 23:00:00 +0100 2778277 http://www.medworm.com/index.php?rid=2778276&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-103-5_6 Important information on cellular physiology can be obtained by directly observing living cells. The nucleus, and the chromatin within, is of particular interest to many researchers. Monitoring the behavior of specific DNA loci in the living cell is now commonly achieved through the insertion of binding sites for fluorescently tagged proteins at the sequence of interest (e.g. Ref 1). However, visualizing the behavior of full length chromosomes can only be achieved when they constitute discrete, relatively well individualized units. During meiotic mid-prophase, chromosomes of budding yeast are well-organized structures that present such characteristics, making them remarkably suited for visualization. Here we describe the optimized protocols and techniques that allow monitoring of chromosom... Springer protocols feed by Cell Biology info http://www.medworm.com/rss/comments.php?id=2778276 Fri, 31 Jul 2009 23:00:00 +0100 2778276 http://www.medworm.com/index.php?rid=2778275&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-103-5_7 The filamentous fungi Neurospora crassa and Sordaria macrospora are materials of choice for recombination studies because each of the DNA strands involved in meiosis can be visually analyzed using spore-color mutants. Well-advanced molecular genetic methodologies have been developed for each of these fungi, and several mutants defective in recombination and/or pairing are available. Moreover, the complete genome sequence of N. crassa has made it possible to clone virtually any gene involved in their life cycle. Both fungi provide also a particularly attractive experimental system for cytological analysis of meiosis: stages can be determined independently of chromosomal morphology and their seven chromosomes are easily identified. The techniques for light, immunofluorescence and electron mi... Springer protocols feed by Cell Biology info http://www.medworm.com/rss/comments.php?id=2778275 Fri, 31 Jul 2009 23:00:00 +0100 2778275 http://www.medworm.com/index.php?rid=2778274&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-103-5_8 We describe iron-hematoxylin staining of intact gill segments for the brightfield examination of meiotic progression, and the use of surface spreads and fluorescence in situ hybridization (FISH) to investigate meiotic chromosome pairing. Gill segments can alternatively be stained with DAPI for the determination of meiotic stage, or propidium iodide for the quantitation of nuclear DNA content, and the chromosome fixation and spreading techniques used for FISH are also suitable for immunolocalization studies of chromosomal proteins. (Source: Springer protocols feed by Cell Biology) Springer protocols feed by Cell Biology info http://www.medworm.com/rss/comments.php?id=2778274 Fri, 31 Jul 2009 23:00:00 +0100 2778274 http://www.medworm.com/index.php?rid=2778273&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-103-5_11 The nematode Caenorhabditis elegans has emerged as an informative experimental system for analysis of meiosis, in large part because of the advantageous physical organization of meiotic nuclei as a gradient of stages within the germline. Here we provide tools for detailed observational studies of cells within the worm gonad, including techniques for light and electron microscopy. (Source: Springer protocols feed by Cell Biology) Springer protocols feed by Cell Biology info http://www.medworm.com/rss/comments.php?id=2778273 Fri, 31 Jul 2009 23:00:00 +0100 2778273 http://www.medworm.com/index.php?rid=2778272&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-103-5_10 Many of the structures involved in meiotic synapsis and recombination such as synaptonemal complexes (SCs) and recombination nodules (RNs) can be resolved only by electron microscopy. Therefore, electron microscopic (EM) immunolocalization using gold-conjugated antibodies is the best way to verify whether certain proteins are components of SCs or RNs. Here, we describe (1) preparing tomato primary microsporocyte protoplasts in leptotene, zygotene, and pachytene stages; (2) hypotonically bursting the protoplasts on glow-discharged glass and plastic-coated slides to make spreads of SCs; (3) immunolabeling proteins in SCs and RNs with colloidal gold; (4) staining SC spreads for EM; and (5) transferring SC spreads on plastic films to grids for EM. (Source: Springer protocols feed by Cell Biolo... Springer protocols feed by Cell Biology info http://www.medworm.com/rss/comments.php?id=2778272 Fri, 31 Jul 2009 23:00:00 +0100 2778272 http://www.medworm.com/index.php?rid=2778271&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-103-5_13 A wide variety of techniques have been utilized to determine the localization of various proteins from premeiotic through meiotic stages in Drosophila males. Live imaging has been instrumental in monitoring chromosome pairing and the localization of fusion proteins. Immunofluorescence has been a widely utilized technique to examine the localization and colocalization of the many proteins involved in meiosis. Recently, an immuno-FISH protocol was developed to observe the co-localization of DNA probes and proteins. In this chapter, detailed protocols outlining these three types of experiments are presented. (Source: Springer protocols feed by Cell Biology) Springer protocols feed by Cell Biology info http://www.medworm.com/rss/comments.php?id=2778271 Fri, 31 Jul 2009 23:00:00 +0100 2778271 http://www.medworm.com/index.php?rid=2778270&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-103-5_12 Methods are described to analyze two different parts of the Drosophila ovary, which correspond to early stages (pachytene) and late stages (metaphase I and beyond) of meiosis. In addition to taking into account morphology, the techniques differ by fixation conditions and the method to isolate the tissue. Most of these methods are whole mounts, which preserve the three-dimensional structure. (Source: Springer protocols feed by Cell Biology) Springer protocols feed by Cell Biology info http://www.medworm.com/rss/comments.php?id=2778270 Fri, 31 Jul 2009 23:00:00 +0100 2778270 http://www.medworm.com/index.php?rid=2778269&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-103-5_14 The genus Daphnia has an intriguing reproductive mode of cyclical parthenogenesis. This reproductive mode has been studied for centuries, but cytogenetic information is lacking due to technical limitations of classical methods. We have developed methods for the preparation and examination of meiotic chromosomes of Daphnia pulex from oocytes and spermatocytes. Oocyte chromosome preparations are obtained by isolating individual oocytes after the release of yolk granules from the ovary using pressure and capillary action. Spermatocyte chromosomes are prepared using a conventional squash method. Cryosectioning is an easy and fast way to prepare sections. We also illustrate the application of immunofluorescence staining against α tubulin, as well as fluorescence in situ hybridization (FIS... Springer protocols feed by Cell Biology info http://www.medworm.com/rss/comments.php?id=2778269 Fri, 31 Jul 2009 23:00:00 +0100 2778269 http://www.medworm.com/index.php?rid=2778268&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-103-5_9 Advances in molecular biology and in the genetics of Arabidopsis thaliana have led to this organism becoming an important model for the analysis of meiosis in plants. Cytogenetic investigations are pivotal to meiotic studies and a number of technological improvements for Arabidopsis cytology have provided a range of tools to investigate chromosome behaviour during meiosis. This chapter includes protocols on basic cytology, FISH analysis, immunocytology, a procedure for a meiotic time course and electron microscopy. (Source: Springer protocols feed by Cell Biology) Springer protocols feed by Cell Biology info http://www.medworm.com/rss/comments.php?id=2778268 Fri, 31 Jul 2009 23:00:00 +0100 2778268 http://www.medworm.com/index.php?rid=2778267&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-103-5_15 In recent years, zebrafish (Danio rerio) has been used as a model vertebrate organism for studies of human disease, development, and genetics. This chapter describes detailed methods for the preparation of whole-mount meiotic oocytes and spermatocytes as well as cryostat sectioning of ovaries and testes for immunofluorescence microscopy studies of zebrafish. (Source: Springer protocols feed by Cell Biology) Springer protocols feed by Cell Biology info http://www.medworm.com/rss/comments.php?id=2778267 Fri, 31 Jul 2009 23:00:00 +0100 2778267 http://www.medworm.com/index.php?rid=2778266&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-103-5_16 Spermatogenesis is a cyclic process during which, within each epithelial area, various generations of germ cells undergo a series of developmental steps according to a fixed time schedule. The cycle of the seminiferous epithelium can be subdivided into stages. In the mouse, 12 such stages have been described that can be distinguished from one another by steps in spermatid development. The best way to recognize the stages in seminiferous tubule cross-sections is to use Bouin’s-fixed testes of normal mice and sections stained with the Periodic acid Schiff (PAS) technique and hematoxylin. Unfortunately, this is not always possible. Sometimes PAS staining cannot be used, such as when immunohistochemistry is carried out. Moreover, not all germ cell types may be present in some instances, ... Springer protocols feed by Cell Biology info http://www.medworm.com/rss/comments.php?id=2778266 Fri, 31 Jul 2009 23:00:00 +0100 2778266 http://www.medworm.com/index.php?rid=2778265&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-103-5_18 The studies of molecular events that occur in single cell types within a tissue often require the disaggregation of the tissue into a single cell suspension, followed by isolation of distinct cell populations. The germinal epithelium of mammals is composed of several cell types, which divide mitotically, before entering meiosis. In this chapter, we describe the isolation of five mouse germ-cell fractions by centrifugal elutriation, and characterize them by their DNA content (flow cytometry), cell morphology (DAPI staining of nuclei, Giemsa staining of squashed cells) and deposition of stage-specific meiotic markers (SYCP3, H1t, SAM68) on chromosome spreads and whole cells. Within 2 h it is possible to obtain enriched populations of elongated spermatids (up to ∼50% of the fraction)... Springer protocols feed by Cell Biology info http://www.medworm.com/rss/comments.php?id=2778265 Fri, 31 Jul 2009 23:00:00 +0100 2778265 http://www.medworm.com/index.php?rid=2778264&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-103-5_17 Understanding meiosis is facilitated by in vitro experimental approaches, but this has not been easily applicable to mammalian meiocytes. Available methods for in vitro analysis of mammalian oocytes are generally limited to experimental analysis of the late prophase period. Short-term cultures of male germ cells have been useful for analysis of earlier meiotic prophase pathways, as well as onset of the meiotic division phase, but no studies have achieved reliable spermatogenesis in vitro. Here we describe a method for preparing highly enriched pachytene spermatocytes from mouse testicular cell suspensions using cell-size fractionation by sedimentation through a bovine serum albumin gradient at unit gravity. We also provide a procedure for short-term culture of spermatocytes and the pharmac... Springer protocols feed by Cell Biology info http://www.medworm.com/rss/comments.php?id=2778264 Fri, 31 Jul 2009 23:00:00 +0100 2778264 http://www.medworm.com/index.php?rid=2778263&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-103-5_20 The goal of this chapter is twofold: First, to acquaint the reader with the problems inherent in analyzing mammalian female meiosis and, second, to provide a step-by-step approach to mastering the necessary techniques. Although the methods presented are for use in the human and mouse, with subtle alterations the same techniques should be applicable to most mammalian species. (Source: Springer protocols feed by Cell Biology) Springer protocols feed by Cell Biology info http://www.medworm.com/rss/comments.php?id=2778263 Fri, 31 Jul 2009 23:00:00 +0100 2778263 http://www.medworm.com/index.php?rid=2778262&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-103-5_19 This chapter describes methods of preparation of meiotic nuclei for their analysis with electron microscopy. Procedures include surface spreading as well as fixation, staining, and sectioning of tissue and subsequent 3-D reconstruction analysis. Immunostaining protocols for electron microscopy are provided. The rationale for using electron microscopy in place of light or fluorescent microscopy for specific cytological analyses is presented. (Source: Springer protocols feed by Cell Biology) Springer protocols feed by Cell Biology info http://www.medworm.com/rss/comments.php?id=2778262 Fri, 31 Jul 2009 23:00:00 +0100 2778262 http://www.medworm.com/index.php?rid=2778261&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-103-5_21 We describe how to make cytological preparations that are suitable for the analysis of interference among these foci, and how to estimate the strength of interference among foci, using the gamma distribution as a mathematical model for focus/CO positioning. (Source: Springer protocols feed by Cell Biology) Springer protocols feed by Cell Biology info http://www.medworm.com/rss/comments.php?id=2778261 Fri, 31 Jul 2009 23:00:00 +0100 2778261 http://www.medworm.com/index.php?rid=2778260&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-103-5_22 A complex meiotic differentiation program generates genetically diverse haploid cells (gametes or spores) to compensate for the genome doubling that occurs at fertilization. To this end, homologous chromosomes must undergo pairing and recombination before they become partitioned in haploid sets by two consecutive meiotic divisions. Chromosome ends (telomeres) contain a protective complex that is crucial for genomic stability. In meiosis, telomeres become key players in the chromosome pairing process during prophase to the first meiotic division. At the onset of prophase I, telomeres attach to the nuclear envelope, about which they move and transiently cluster in a limited sector of the nuclear periphery. The dynamic clustering of telomeres (bouquet formation) occurs at the onset of the zyg... Springer protocols feed by Cell Biology info http://www.medworm.com/rss/comments.php?id=2778260 Fri, 31 Jul 2009 23:00:00 +0100 2778260 http://www.medworm.com/index.php?rid=2778259&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-103-5_23 New immunofluorescence techniques allow visual identification of human cells in various stages of meiotic prophase. Antibodies to the synaptonemal complex, the centromere and sites of recombination allow these stages of meiotic prophase to be identified. The progress of chromosome synapsis, recombination and associated phenomena such as interference can be studied in normal men, translocation heterozygotes and men with infertility problems. This has greatly stimulated research in human meiosis, leading to many exciting studies on the mechanisms underlying recombination and the generation of chromosome abnormalities. (Source: Springer protocols feed by Cell Biology) Springer protocols feed by Cell Biology info http://www.medworm.com/rss/comments.php?id=2778259 Fri, 31 Jul 2009 23:00:00 +0100 2778259 http://www.medworm.com/index.php?rid=2778258&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-103-5_24 Most of the human aneuploidies have a maternal origin. This feature makes the study of human female meiosis a fundamental topic to understand the reasons leading to this important social problem. Unfortunately, due to sample collection difficulties, not many studies have been performed on human female meiotic prophase. In this chapter we present a comprehensive collection of protocols that allows the study of human female meiotic prophase through different technical approaches using both spread and structurally preserved oocytes. (Source: Springer protocols feed by Cell Biology) Springer protocols feed by Cell Biology info http://www.medworm.com/rss/comments.php?id=2778258 Fri, 31 Jul 2009 23:00:00 +0100 2778258 http://www.medworm.com/index.php?rid=2778257&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-103-5_25 During mouse meiosis, gene expression and homologous synapsis are intimately linked. Chromosomes that fail to synapse at the zygotene–pachytene transition become transcriptionally silenced by a process called Meiotic Silencing of Unsynapsed Chromatin (MSUC), and this silencing (or defects in it) may in turn cause germ cell losses and infertility. Gene transcription at the chromosomal level can be readily observed using RNA fluorescence in-situ hybridisation (FISH), and this approach allows one to directly compare expression at a specific locus with the synaptic status of the chromosome domain on which it resides. Here we describe a protocol for carrying out RNA FISH on male meiotic cells, together with detail on the important controls and common problems associated with this techniqu... Springer protocols feed by Cell Biology info http://www.medworm.com/rss/comments.php?id=2778257 Fri, 31 Jul 2009 23:00:00 +0100 2778257 http://www.medworm.com/index.php?rid=2715811&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-993-2_9 Progress from our present understanding of the mechanisms behind mitosis has been compromised by the fact that model systems that were ideal for molecular and genetic studies (such as yeasts, C. elegans, or Drosophila) were not suitable for intracellular micromanipulation. Unfortunately, those systems that were appropriate for micromanipulation (such as newt lung cells, PtK1 cells, or insect spermatocytes) are not amenable for molecular studies. We believe that we can significantly broaden this scenario by developing high-resolution live cell microscopy tools in a system where micromanipulation studies could be combined with modern gene-interference techniques. Here we describe a series of methodologies for the functional dissection of mitosis by the use of simultaneous live cell microscop... Springer protocols feed by Cell Biology info http://www.medworm.com/rss/comments.php?id=2715811 Fri, 31 Jul 2009 23:00:00 +0100 2715811 http://www.medworm.com/index.php?rid=2715810&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-993-2_8 In this chapter we describe the preparation of early mitotic C. elegans embryos for the tomographic reconstruction of end-morphologies of spindle microtubules. Early embryos are prepared by high-pressure freezing and freeze-substitution for thin-layer embedding in Epon/Araldite. We further describe data acquisition, tomographic reconstruction, and 3-D modeling of microtubules in serially sectioned mitotic spindles. The presented techniques are applicable to other model systems. (Source: Springer protocols feed by Cell Biology) Springer protocols feed by Cell Biology info http://www.medworm.com/rss/comments.php?id=2715810 Fri, 31 Jul 2009 23:00:00 +0100 2715810 http://www.medworm.com/index.php?rid=2715809&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-993-2_7 Fluorescence live microscopy is a powerful technique to study complex cellular dynamics such as cell division. The availability of fluorescent markers based on GFP fusion proteins for virtually any cellular structure allows efficient visualization of specific processes, and the combination of different fluorophores can be used to study their coordination. In this chapter, we present methods for automated live cell microscopy to study mitotic gene function systematically and in high throughput. In particular, we provide protocols for efficient generation of fluorescent reporter cell lines stably expressing combinations of cellular markers, and provide detailed guidelines for optimizing imaging protocols for automated long-term live microscopy. (Source: Springer protocols feed by Cell Biolog... Springer protocols feed by Cell Biology info http://www.medworm.com/rss/comments.php?id=2715809 Fri, 31 Jul 2009 23:00:00 +0100 2715809 http://www.medworm.com/index.php?rid=2715808&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-993-2_6 The identification of all the individual components that constitute the plethora of complexes in each cell type represents perhaps the most exciting challenge of postgenomic biology. This is particularly important in the study of events such as mitosis and cytokinesis, in which rapid and precise protein–protein interactions regulate both the direction and accuracy of these intricate processes. Here we describe an experimental strategy to isolate protein complexes involved in mitosis and cytokinesis in cultured Drosophila cells. This method involves the tagging of the bait protein with two IgG binding domains of Protein A and the isolation of the tagged bait along with its interacting partners by a single affinity purification step. These isolated complexes can then be analysed by sev... Springer protocols feed by Cell Biology info http://www.medworm.com/rss/comments.php?id=2715808 Fri, 31 Jul 2009 23:00:00 +0100 2715808 http://www.medworm.com/index.php?rid=2715807&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-993-2_5 Hydrodynamic analysis is a powerful tool to dissect the molecular architecture of macromolecular protein assemblies. These techniques have been successfully used in yeast systems but are also well suited to the analysis of protein complexes from human cells. Furthermore, the combination of hydrodynamic analysis with siRNA mediated protein depletion provides an excellent system to probe the composition of protein complexes isolated from human cells. In this chapter we describe the use of these approaches in the analysis of macromolecular protein complexes during mitosis in human cells, using the kinetochore as an example. (Source: Springer protocols feed by Cell Biology) Springer protocols feed by Cell Biology info http://www.medworm.com/rss/comments.php?id=2715807 Fri, 31 Jul 2009 23:00:00 +0100 2715807 http://www.medworm.com/index.php?rid=2715806&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-993-2_4 A novel protein expression system in budding yeast is described which has been used to express many yeast mitotic regulators as well as a wide range of other recombinant proteins from several different species. The expression system relies on autoselection with essential genes to maintain high copy numbers of expression plasmids. Autoselection permits expression cells to be grown in rich medium with no need for plasmid selection with drugs or nutritional conditions. This optimizes growth and expression of recombinant proteins. The use of the expression system is illustrated by purifying budding yeast mitotic regulators, Cdc14 and Net1, and recapitulating their activities in vitro. (Source: Springer protocols feed by Cell Biology) Springer protocols feed by Cell Biology info http://www.medworm.com/rss/comments.php?id=2715806 Fri, 31 Jul 2009 23:00:00 +0100 2715806 http://www.medworm.com/index.php?rid=2715805&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-993-2_3 Genetic studies on model organisms, particularly yeasts and Drosophila melanogaster, have proven powerful in identifying the cell cycle machinery and its regulatory mechanisms. In more recent years RNAi has been used in a variety of genome-wide screens and single molecule studies to elucidate the mechanisms of cell cycle progression. In Drosophila cultured cells, RNAi is extremely simple, and a strong effect can be observed by adding the dsRNA to the cultured cells, with few complications of off-target effects. Functions in cell cycle progression can be followed by a variety of assays. One of the advantages of these cells is that they allow high-resolution spatiotemporal observations to be made by microscopy, with no particular complexity in terms of media and temperature. Here we discuss ... Springer protocols feed by Cell Biology info http://www.medworm.com/rss/comments.php?id=2715805 Fri, 31 Jul 2009 23:00:00 +0100 2715805 http://www.medworm.com/index.php?rid=2715804&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-993-2_2 With the human genome fully sequenced (1, 2), biologists continue to face the challenging task of evaluating the function of each of the ∼25,000 genes contained within it. Gene targeting in human cells provides a powerful and unique experimental tool in this regard (3–8). Although somewhat more involved than RNAi or pharmacological approaches, somatic cell gene targeting is a precise technique that avoids both incomplete knockdown and off-target effects, but is still much quicker than analogous manipulations in the mouse. Moreover, immortal knockout cell lines provide excellent platforms for both complementation analysis and biochemical purification of multiprotein complexes in native form. Here we present a detailed gene-targeting protocol that was recently applied to the mitoti... Springer protocols feed by Cell Biology info http://www.medworm.com/rss/comments.php?id=2715804 Fri, 31 Jul 2009 23:00:00 +0100 2715804 http://www.medworm.com/index.php?rid=2715803&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-993-2_20 The Anaphase Promoting Complex (APC) ubiquitin ligase is critical for multiple processes including cell cycle, development, meiosis, and senescence. The importance of regulation of the APC by substrate competitive (pseudosubstrate) inhibitors, such as Emi1 and BubR1, has recently been demonstrated. Substrate competitive inhibitors typically bind to enzymes via the same site as substrates, but by having any combination of increased enzyme affinity and low turnover numbers, are able to “clog” the ability of the enzyme to bind and turnover substrates. For the APC, these pseudosubstrates can both position and block the APC and have been well validated as critical regulators for the APC enzymes. (Source: Springer protocols feed by Cell Biology) Springer protocols feed by Cell Biology info http://www.medworm.com/rss/comments.php?id=2715803 Fri, 31 Jul 2009 23:00:00 +0100 2715803 http://www.medworm.com/index.php?rid=2715802&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-993-2_1 Reverse genetic methods, such as homologous gene targeting, have greatly contributed to our understanding of molecular pathways in mitosis, especially in yeast. The chicken B-lymphocyte line, DT40, represents a unique example among vertebrate somatic cells where homologous gene targeting occurs at very high frequency. DT40 cells therefore provide a useful and accessible somatic genetic system for wide-ranging biochemical and cell biological assays. In this chapter, we describe the main principles of homologous gene targeting, the concept of targeting construct design and the detailed experimental protocol of how to achieve successful knockouts. We also mention methods for conditional disruption of essential genes and conclude with specific procedures for the study of mitosis in DT40 cells.... Springer protocols feed by Cell Biology info http://www.medworm.com/rss/comments.php?id=2715802 Fri, 31 Jul 2009 23:00:00 +0100 2715802 http://www.medworm.com/index.php?rid=2715801&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-993-2_19 Ubiquitination and protein degradation regulate cell cycle progression in all eukaryotes. During mitosis, ubiquitination by the Anaphase-Promoting Complex/Cyclosome (APC/C) triggers sister chromatid separation and mitotic exit. The APC/C is tightly regulated by phosphorylation, ubiquitination, association of activators or inhibitors, and competitive binding of substrates. Much of our understanding of the mechanism of APC/C-dependent ubiquitination has been obtained from studies using extracts of Xenopus laevis eggs or synchronized human tissue culture cells. Here, we describe protocols to prepare extracts of synchronized human cells, and discuss experiments to use extracts for the biochemical analysis of APC/C-dependent ubiquitination. (Source: Springer protocols feed by Cell Biology) Springer protocols feed by Cell Biology info http://www.medworm.com/rss/comments.php?id=2715801 Fri, 31 Jul 2009 23:00:00 +0100 2715801 http://www.medworm.com/index.php?rid=2715800&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-993-2_18 The anaphase-promoting complex/cyclosome (APC/C), a large (20S) multisubunit E3 ligase, has an essential role to ubiquitylate numerous substrates at specific times during mitosis and G1 phase as well as in meiosis. The deregulation of the APC/C causes cell death or genomic instability, which is a hallmark of cancers. Although 13 years have passed since its discovery, the molecular mechanisms of the APC/C-dependent ubiquitylation and proteolysis are still poorly understood. The development of in vitro systems enables the identification of new substrates and investigation of the molecular mechanisms by which the APC/C recognizes its substrates. This chapter describes in vitro assays reconstituted in Xenopus egg extracts. (Source: Springer protocols feed by Cell Biology) Springer protocols feed by Cell Biology info http://www.medworm.com/rss/comments.php?id=2715800 Fri, 31 Jul 2009 23:00:00 +0100 2715800 http://www.medworm.com/index.php?rid=2715799&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-993-2_17 Cell cycle transitions are controlled, in part, by ubiquitin-dependent proteolysis. In mitosis, the metaphase to anaphase transition is governed by an E3 ubiquitin ligase called the cyclosome or Anaphase-Promoting Complex (APC), and a WD40-repeat protein co-factor called Cdc20. In vitro Cdc20-dependent APC (APCCdc20) assays have been useful in the identification and validation of target substrates, and in the study of APC enzymology and regulation. Many aspects of the regulation of cell cycle progression have been discovered in the budding yeast Saccharomyces cerevisiae, and proteins purified from this model organism have been employed in a wide variety of in vitro assays. Here we outline a quantitative in vitro mitotic APCCdc20 assay that makes use of a highly active form of the APC that ... Springer protocols feed by Cell Biology info http://www.medworm.com/rss/comments.php?id=2715799 Fri, 31 Jul 2009 23:00:00 +0100 2715799 http://www.medworm.com/index.php?rid=2715798&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-993-2_16 The targeted destruction of key regulators helps to drive the cell cycle. Here we describe a quantitative assay to measure destruction of different regulators in mitotic cells. This assay uses GFP-tagged substrates and time-lapse fluorescence microscopy of single cells to pinpoint the timing of destruction of different substrates at different stages in mitosis. (Source: Springer protocols feed by Cell Biology) Springer protocols feed by Cell Biology info http://www.medworm.com/rss/comments.php?id=2715798 Fri, 31 Jul 2009 23:00:00 +0100 2715798 http://www.medworm.com/index.php?rid=2715797&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-993-2_15 The spindle checkpoint is a surveillance mechanism that ensures the fidelity of chromosome segregation by inhibiting anaphase onset until all chromosomes have established stable bipolar attachments. Here we describe a number of protocols that can be used to assay the ability of budding and fission yeast cells to (1) establish and maintain a spindle checkpoint arrest, and (2) segregate chromosomes efficiently upon recovery from mitotic arrest. We focus on experimental detail of the budding yeast protocols, but also point out important differences between budding and fission yeast assays. (Source: Springer protocols feed by Cell Biology) Springer protocols feed by Cell Biology info http://www.medworm.com/rss/comments.php?id=2715797 Fri, 31 Jul 2009 23:00:00 +0100 2715797 http://www.medworm.com/index.php?rid=2715796&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-993-2_14 For high-fidelity chromosome segregation, kinetochores must be properly captured by spindle microtubules, but the mechanisms of initial kinetochore capture have remained elusive. Observation of individual kinetochore–microtubule interaction has been difficult, because multiple kinetochores are captured by microtubules during a short period and within a small space. By isolating one of the kinetochores from others through regulation of the activity of a centromere, we could visualize individual kinetochore–microtubule interactions in Saccharomyces cerevisiae. This technique, which we have called the ‘centromere reactivation system’, allowed us to dissect the process of kinetochore capture and transport on the mitotic spindle into several steps, thus enabling us to id... Springer protocols feed by Cell Biology info http://www.medworm.com/rss/comments.php?id=2715796 Fri, 31 Jul 2009 23:00:00 +0100 2715796 http://www.medworm.com/index.php?rid=2715795&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-993-2_13 Kinetochores are multiprotein machines that initiate mitotic checkpoint signaling and control chromosome movement through interactions with microtubules. Our lab has utilized Xenopus laevis frog egg extracts to investigate the requirements for kinetochore assembly and disassembly in vertebrates. Egg extracts support the assembly of functional kinetochores that are capable of binding microtubules, aligning and segregating chromosomes, and sending spindle checkpoint signals. This is the only in vitro system that assembles functional kinetochores, making it particularly well suited for these types of studies. Probing kinetochore assembly using the biochemically tractable egg extract system has elucidated the intricate assembly requirements for numerous vertebrate kinetochore proteins. ... Springer protocols feed by Cell Biology info http://www.medworm.com/rss/comments.php?id=2715795 Fri, 31 Jul 2009 23:00:00 +0100 2715795 http://www.medworm.com/index.php?rid=2715794&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-993-2_12 During cell division microtubules of the mitotic spindle segregate the duplicated chromosomes into the two daughter cells. Chromosome–microtubule attachment is mediated by kinetochores, multiprotein complexes assembled on specialized regions of the DNA. Kinetochores modulate microtubule dynamics to generate the forces necessary to power chromosome movement and regulate the spindle checkpoint. Errors in kinetochore function can cause aneuploidy, a hallmark of 80% of solid tumors in humans, suggesting a fundamental link to tumorigenesis. Human kinetochores are complex protein machines with over 100 different proteins. Here we present fixed- and live-cell-based assays used to functionally categorize kinetochore proteins with regard to spindle checkpoint activity and kinetochore–mi... Springer protocols feed by Cell Biology info http://www.medworm.com/rss/comments.php?id=2715794 Fri, 31 Jul 2009 23:00:00 +0100 2715794 http://www.medworm.com/index.php?rid=2715793&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-993-2_11 Mitotic spindle microtubules pull chromosomes toward each pole to generate two daughter cells. Proper spindle formation and function are required to prevent tumorigenesis and cell death. The fission yeast Schizosaccharomyces pombe has been widely used as a model organism to understand the molecular mechanism of mitosis due to its convenience in genetics, molecular biology, and cell biology. The development of fluorescent protein systems and microscopy enables us to investigate the “true” behavior of proteins in living fission yeast cells using a strain with a fluorescence-tagged gene under its native promoter. In this way the level of expression of tagged protein is similar to the level of wild-type nontagged protein. In this chapter we illustrate standard methods to generate s... Springer protocols feed by Cell Biology info http://www.medworm.com/rss/comments.php?id=2715793 Fri, 31 Jul 2009 23:00:00 +0100 2715793 http://www.medworm.com/index.php?rid=2715792&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-993-2_10 The formation of a bipolar spindle is essential for the equal segregation of duplicated DNA into two daughter cells during mitosis. Spindle bipolarity is largely dependent on the mitotic cell possessing two centrosomes that can each establish one spindle pole. The centrosome is also now known to regulate many other aspects of cell cycle progression, including G1/S progression, spindle orientation and symmetry, cytokinesis, and checkpoint signalling. As a result, defects in centrosome arrangement or number can lead to loss of cell polarity, defective cell division, and abnormal chromosome segregation, all events that are typical of cancer cells. Indeed, cancer cells often exhibit overduplicated centrosomes and multipolar spindles. Here, we outline a number of fluorescence imaging methodolog... Springer protocols feed by Cell Biology info http://www.medworm.com/rss/comments.php?id=2715792 Fri, 31 Jul 2009 23:00:00 +0100 2715792 http://www.medworm.com/index.php?rid=2126049&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-310-7_9 This chapter describes a procedure for isolation and analysis of fractions enriched in plasma membranes from minute amounts of tissue. It consists of a method for extraction and fractionation of membranes and a method for enzymatic digestion of membrane proteins without use of detergents. The method for isolation of membranes comprises of a stepwise depletion of nonintegral membrane molecules from entire tissue homogenate by high-salt, carbonate, and urea washes followed by a treatment of the membranes with sublytic concentrations of a detergent and enrichment of the plasma membranes by a density gradient fractionation. Fluorometric assays for protein content and plasma membrane marker activity allow calculation of the yield and extent of plasma membrane enrichment. Reduction, carboxymethy... Springer protocols feed by Cell Biology info http://www.medworm.com/rss/comments.php?id=2126049 Fri, 23 Jan 2009 12:13:16 +0100 2126049 http://www.medworm.com/index.php?rid=2126048&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-310-7_8 Plasma membranes encompass a complex and varying set of proteins essential to life. In addition, plasma membrane proteins represent the majority of all known drug targets. The characterization of plasma membrane proteomes is, therefore, of eminent importance. A current bottleneck is the lack of efficient protocols to isolate plasma membranes from tissues or entire organs. To this end, we recently established a simple and effective isolation procedure which is based on aqueous polymer two-phase systems. In this chapter, we provide a detailed protocol for the isolation of plasma membranes from brain tissue, which could easily be adapted to other sources. (Source: Springer protocols feed by Cell Biology) Springer protocols feed by Cell Biology info http://www.medworm.com/rss/comments.php?id=2126048 Fri, 23 Jan 2009 12:13:16 +0100 2126048 http://www.medworm.com/index.php?rid=2126047&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-310-7_7 Sequential detergent extraction of proteins from eukaryotic cells has been used to increase proteome coverage of 2D-PAGE. We have adapted sequential detergent extraction for use with the high-throughput non-electrophoretic proteomics method of liquid chromatography and electrospray ionisation tandem mass spectrometry. This method of extraction yields comprehensive proteomes that include up to twice as many membrane proteins as other published methods. Two thirds of these membrane proteins have more than one transmembrane domain and many of these have multiple transmembrane domains. Since sequential detergent extraction (SDE) separates proteins based upon their physicochemistry and sub-cellular localisation, this method also provides useful data about cellular localisation. (Source: Springe... Springer protocols feed by Cell Biology info http://www.medworm.com/rss/comments.php?id=2126047 Fri, 23 Jan 2009 12:13:15 +0100 2126047 http://www.medworm.com/index.php?rid=2126046&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-310-7_6 Mitochondria are essential organelles in cellular metabolism. These organelles are bounded by two membranes, the outer and inner membrane. Especially the inner membrane comprises a high content of proteins, for example, the protein complexes of the respiratory chain. High-resolution separation and analysis of such membrane proteins, for example, by two-dimensional gel electrophoresis (2-DE), is hampered by their hydrophobicity and tendency for aggregation. Here, we describe the separation of mitochondrial membrane proteins of Saccharomyces cerevisiae by 16-benzyldimethyl-n-hexadecylammonium chloride/sodium dodecyl sulfate polyacrylamide gel electrophoresis (16-BAC/SDS-PAGE). This method enables the separation of membrane proteins owing to the solubilizing power of the ionic detergents 16-B... Springer protocols feed by Cell Biology info http://www.medworm.com/rss/comments.php?id=2126046 Fri, 23 Jan 2009 12:13:14 +0100 2126046 http://www.medworm.com/index.php?rid=2126045&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-310-7_5 Identification and characterization of membrane proteins is of increasing importance in modern proteomic studies. It is of central interest to have access to methods that combine efficient solubilization with enrichment of proteins and intact protein complexes. Separation methods have been developed based on nondenaturing detergent extraction of yeast mitochondrial membrane proteins followed by enrichment of hydrophobic proteins in aqueous two-phase system. Combining the zwitterionic detergent Zwittergent 3-10 and the nonionic detergent Triton X-114 results in a complementary solubilization of proteins, which is similar to that of the anionic detergent sodium dodecyl sulfate (SDS) but with the important advantage of being nondenaturing. Detergent/polymer two-phase system partitioning offer... Springer protocols feed by Cell Biology info http://www.medworm.com/rss/comments.php?id=2126045 Fri, 23 Jan 2009 12:13:11 +0100 2126045 http://www.medworm.com/index.php?rid=2126044&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-310-7_4 Generally, a combination of two or more chromatographic and/or electrophoretic methods is conducted to separate membrane protein complexes. Here we describe how thylakoid membrane protein complexes from the photosynthetic apparatus can be successfully separated by two main steps: preparative methods that enable purification of membrane protein complexes in the native (intact) form, and analytical methods that allow resolution of each membrane protein. Thus, separation of intact supercomplexes was achieved by solubilisation of the sample using mild detergents followed either by sucrose gradient ultracentrifugation or by blue native gel (BNG) electrophoresis. Complexes, thus, recovered were then resolved further using either reversed phase liquid chromatography or SDS-PAGE respectively. (Sou... Springer protocols feed by Cell Biology info http://www.medworm.com/rss/comments.php?id=2126044 Fri, 23 Jan 2009 12:13:09 +0100 2126044 http://www.medworm.com/index.php?rid=2126043&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-310-7_3 A full understanding of leukocyte responses to external stimuli requires knowledge of the full complement of proteins found on their surfaces. Systematic examination of the mammalian cell surfaces at the protein level is hampered by technical difficulties associated with proteomic analysis of so many membrane proteins and the large amounts of starting material required. The use of transcriptomic analyses avoids challenges associated with protein stability and separation and enables the inclusion of an amplification step; thus allowing the use of cell numbers applicable to the study of sub populations of, for example, primary lymphocytes. Here we present a transcriptomic methodology based on Serial Analysis of Gene Expression (SAGE) to recover an essentially complete and quantitative profil... Springer protocols feed by Cell Biology info http://www.medworm.com/rss/comments.php?id=2126043 Fri, 23 Jan 2009 12:13:06 +0100 2126043 http://www.medworm.com/index.php?rid=2126042&cid=s_37121_171_f&fid=37121&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-310-7_23 Pancreatic zymogen granules (ZG) are specialized for digestive enzyme storage and regulated secretion in the exocrine pancreas and are a classical model for studying secretory granule function. To understand the function of this organelle, we have conducted a proteomic study to identify the ZG membrane proteins from ZGs purified by Percoll gradient centrifugation. By combining multiple separation strategies including two-dimensional gel electrophoresis and two-dimensional liquid chromatography with tandem mass spectrometry (TMS), we identified 101 proteins from purified ZG membranes including a large number of proteins previously unknown on ZG membranes. To distinguish intrinsic membrane proteins from soluble and peripheral membrane proteins, a quantitative proteomics strategy was develope... Springer protocols feed by Cell Biology info http://www.medworm.com/rss/comments.php?id=2126042 Fri, 23 Jan 2009 12:13:05 +0100 2126042