MedWorm.com provides a medical RSS filtering service. Over 6000 RSS medical sources are combined and output via different filters. This feed contains the latest items from the 'Springer protocols feed by Biochemistry' source. Thu, 09 Feb 2012 13:38:43 +0100 http://www.medworm.com/index.php?rid=5473430&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-382-0_10 Immortalised cultured cells are powerful tools to assess the function of ectopically expressed proteins. However, it must be ensured that the protein of interest is functional in the host system and display native behaviour. In particular, mitochondrial uncoupling proteins (UCPs) displayed (non-native) artefactual uncoupling when expressed in yeast or can possess functions upon reconstitution in proteoliposomes that cannot be reproduced in isolated mitochondria. In the light of newly discovered UCP1 orthologues and paralogues (UCP2, UCP3, plant UCP), comparative functional studies require a system with identical mitochondrial, cellular, and genetic backgrounds. In this chapter, the protocols for the ectopic expression of mouse UCP1 in the human embryonic kidney (HEK293) cell line are intro... Springer protocols feed by Biochemistry news http://www.medworm.com/rss/comments.php?id=5473430 Mon, 05 Dec 2011 07:28:53 +0100 5473430 http://www.medworm.com/index.php?rid=5473429&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-382-0_11 Oxidative phosphorylation is an important energy-conserving mechanism coupling mitochondrial electron transfer to ATP synthesis. Coupling between respiration and phosphorylation is not fully efficient due to proton and electron leaks. In this chapter, methods are presented to measure proton and electron leak activities in isolated mitochondria. The relative strength of a modular kinetic approach to probe oxidative phosphorylation is emphasised. (Source: Springer protocols feed by Biochemistry) Springer protocols feed by Biochemistry news http://www.medworm.com/rss/comments.php?id=5473429 Mon, 05 Dec 2011 07:28:53 +0100 5473429 http://www.medworm.com/index.php?rid=5473428&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-382-0_12 We present different methods applicable for isolated mitochondria or intact cells. We also present experiments demonstrating that a magnitude and a direction (increase or decrease) of a change in mitochondrial ROS production depends on the metabolic state of this organelle. (Source: Springer protocols feed by Biochemistry) Springer protocols feed by Biochemistry news http://www.medworm.com/rss/comments.php?id=5473428 Mon, 05 Dec 2011 07:28:53 +0100 5473428 http://www.medworm.com/index.php?rid=5473427&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-382-0_13 Ca2+-sensitive electrode as a practical approach is used to follow Ca2+ changes in the medium and particularly useful to study mitochondrial Ca2+ uptake (or release); this method permits the continuous recording of Ca2+ movements through the mitochondrial inner membrane. In this chapter, it is described how to prepare a Ca2+-sensitive electrode, and its application on mitochondrial studies with emphasis on the mitochondrial permeability transition. (Source: Springer protocols feed by Biochemistry) Springer protocols feed by Biochemistry news http://www.medworm.com/rss/comments.php?id=5473427 Mon, 05 Dec 2011 07:28:53 +0100 5473427 http://www.medworm.com/index.php?rid=5473426&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-382-0_14 The concentration of calcium ions in the mitochondria has a profound impact on mitochondrial function, modulating respiratory activity at physiological concentrations, while causing lethal damage during calcium overload. The “rhod” series of calcium sensitive fluorescent dyes tend to accumulate preferentially in mitochondria, although the reliability of mitochondrial calcium measurements depends critically on the partitioning of dye within the mitochondria which can vary between preparations. Methods are described to aid verification and quantification of the mitochondrial calcium concentration using single or two photon confocal microscopy and combining the imaging with another cytosolic calcium sensing dye. The method of linear unmixing to separate fluorescent signals based o... Springer protocols feed by Biochemistry news http://www.medworm.com/rss/comments.php?id=5473426 Mon, 05 Dec 2011 07:28:53 +0100 5473426 http://www.medworm.com/index.php?rid=5473425&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-382-0_15 Opening of a large conductance channel in the inner mitochondrial membrane, known as the mitochondrial permeability transition (MPT) pore, has been shown to be a primary mediator of cell death in the heart subjected to ischemia-reperfusion injury. Inhibitors of the MPT have been shown to reduce cardiac ischemia-reperfusion injury. Furthermore, most cardioprotective strategies appear to reduce ischemic cell death either by reducing the triggers for the opening of the MPT, such as reducing calcium overload or reactive oxygen species, or by more direct inhibition of the MPT. This chapter focuses on key issues in the study of the MPT and provides some methods for measuring MPT opening in isolated mitochondria. (Source: Springer protocols feed by Biochemistry) Springer protocols feed by Biochemistry news http://www.medworm.com/rss/comments.php?id=5473425 Mon, 05 Dec 2011 07:28:53 +0100 5473425 http://www.medworm.com/index.php?rid=5473424&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-382-0_17 Mitochondrial energy metabolism depends upon high-flux and low-flux electron transfer pathways. The former provide the energy to support chemiosmotic coupling for oxidative phosphorylation. The latter provide mechanisms for signaling and control of mitochondrial functions. Few practical methods are available to measure rates of individual mitochondrial electron transfer reactions; however, a number of approaches are available to measure steady-state redox potentials (E h) of donor/acceptor couples, and these can be used to gain insight into rate-controlling reactions as well as mitochondrial bioenergetics. Redox changes within the respiratory electron transfer pathway are quantified by optical spectroscopy and measurement of changes in autofluorescence. Low-flux pathways involving t... Springer protocols feed by Biochemistry news http://www.medworm.com/rss/comments.php?id=5473424 Mon, 05 Dec 2011 07:28:53 +0100 5473424 http://www.medworm.com/index.php?rid=5473423&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-382-0_16 Laser scanning confocal microscopy provides the ability to image submicron sections in living cells and tissues. In conjunction with pH-indicating fluorescent probes, confocal microscopy can be used to visualize the distribution of pH inside living cells. Here, we describe a confocal microscopic technique to image intracellular pH in living cells using SNARF-1, a ratiometric pH-indicating fluorescent probe. SNARF-1 is ester-loaded into the cytosol and mitochondria of adult cardiac myocytes. Using 568-nm excitation, emitted fluorescence longer and shorter than 595-nm are imaged and then ratioed after background subtraction. Ratio values for each pixel are converted to values of pH using a standard curve (lookup table). Images of the intracellular distribution of pH show cytosolic and nuclea... Springer protocols feed by Biochemistry news http://www.medworm.com/rss/comments.php?id=5473423 Mon, 05 Dec 2011 07:28:53 +0100 5473423 http://www.medworm.com/index.php?rid=5473422&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-382-0_18 Nuclear magnetic resonance (NMR) spectroscopy is a technique with an increasing importance in the study of metabolic diseases. Its initial important role in the determination of chemical structures (1, 2) has been considerably overcome by its potential for the in vivo study of metabolism (3–5). The main characteristic that makes this technique so attractive is its noninvasiveness. Only nuclei capable of transitioning between energy states, in the presence of an intense and constant magnetic field, are studied. This includes abundant nuclei such as proton (1H) and phosphorous (31P), as well as stable isotopes such as deuterium (2H) and carbon 13 (13C). This allows a wide range of applications that vary from the determination of water distribution in tissues (as obtained in a magnetic ... Springer protocols feed by Biochemistry news http://www.medworm.com/rss/comments.php?id=5473422 Mon, 05 Dec 2011 07:28:53 +0100 5473422 http://www.medworm.com/index.php?rid=5473421&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-382-0_1 Bioenergetic Science started in seventh century with the pioneer works by Joseph Priestley and Antoine Lavoisier on photosynthesis and respiration, respectively. New developments were implemented by Pasteur in 1860s with the description of fermentations associated to microorganisms, further documented by Buchner brothers who discovered that fermentations also occurred in cell extracts in the absence of living cells. In the beginning of twentieth century, Harden and Young demonstrated that orthophosphate and other heat-resistant compounds (cozymase), later identified as NAD, ADP, and metal ions, were mandatory in the fermentation of glucose. The full glycolysis pathway has been detailed in 1940s with the contributions of Embden, Meyeroff, Parnas, Warburg, among others. Studies on the citric... Springer protocols feed by Biochemistry news http://www.medworm.com/rss/comments.php?id=5473421 Mon, 05 Dec 2011 07:28:53 +0100 5473421 http://www.medworm.com/index.php?rid=5473420&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-382-0_19 The advent of techniques with the ability to scan massive changes in cellular makeup (genomics, proteomics, etc.) has revealed the compelling need for analytical methods to interpret and make sense of those changes. Computational models built on sound physico-chemical mechanistic basis are unavoidable at the time of integrating, interpreting, and simulating high-throughput experimental data. Another powerful role of computational models is predicting new behavior provided they are adequately validated. (Source: Springer protocols feed by Biochemistry) Springer protocols feed by Biochemistry news http://www.medworm.com/rss/comments.php?id=5473420 Mon, 05 Dec 2011 07:28:53 +0100 5473420 http://www.medworm.com/index.php?rid=5473419&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-382-0_2 In many studies, the evaluation of mitochondrial function is critical to understand how disease conditions or xenobiotics alter mitochondrial function. One of the classic end-points that can be assessed is oxygen consumption, which can be performed under controlled, yet artificial conditions. Oxygen is the terminal acceptor in the mitochondrial respiratory chain, namely at an enzyme called cytochrome oxidase, which produces water in the process and pumps protons from the matrix to the intermembrane space. Several techniques are available to measure oxygen consumption, including polarography with oxygen electrodes or fluorescent/luminescent probes. The present chapter will deal with the determination of mitochondrial oxygen consumption by means of the Clark-type electrode, which has been wi... Springer protocols feed by Biochemistry news http://www.medworm.com/rss/comments.php?id=5473419 Mon, 05 Dec 2011 07:28:53 +0100 5473419 http://www.medworm.com/index.php?rid=5473418&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-382-0_3 Protocols for high-resolution respirometry (HRR) of intact cells, permeabilized cells, and permeabilized muscle fibers offer sensitive diagnostic tests of integrated mitochondrial function using standard cell culture techniques and small needle biopsies of muscle. Multiple substrate–uncoupler–inhibitor titration (SUIT) protocols for analysis of oxidative phosphorylation improve our understanding of mitochondrial respiratory control and the pathophysiology of mitochondrial diseases. Respiratory states are defined in functional terms to account for the network of metabolic interactions in complex SUIT protocols with stepwise modulation of coupling and substrate control. A regulated degree of intrinsic uncoupling is a hallmark of oxidative phosphorylation, whereas pathological and... Springer protocols feed by Biochemistry news http://www.medworm.com/rss/comments.php?id=5473418 Mon, 05 Dec 2011 07:28:53 +0100 5473418 http://www.medworm.com/index.php?rid=5473417&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-382-0_4 Interest in the investigation of mitochondrial dysfunction has seen a resurgence over recent years due to the implication of such dysfunction in both drug-induced toxicity and a variety of disease states. Here, we describe a methodology to assist in such investigations whereby the oxygen consumption of isolated mitochondria is assessed in a high-throughput fashion using a phosphorescent oxygen-sensitive probe, standard microtitre plates, and plate reader detection. The protocols provided describe the required isolation procedures, initial assay optimization, and subsequent compound screening. Typical data is also provided illustrating the expected activity levels as well as recommended plate maps and data analysis approaches. (Source: Springer protocols feed by Biochemistry) Springer protocols feed by Biochemistry news http://www.medworm.com/rss/comments.php?id=5473417 Mon, 05 Dec 2011 07:28:53 +0100 5473417 http://www.medworm.com/index.php?rid=5473416&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-382-0_5 Nitric oxide (•NO) is a ubiquitous signaling molecule that participates in neuromolecular phenomena associated with memory formation as well as in excitotoxicity. In the hippocampus, neuronal •NO production is coupled to the activation of the NMDA-type of glutamate receptor. More recently, Cytochrome c oxidase has emerged as a novel target for •NO, which competes with O2 for binding to this mitochondrial complex. This reaction establishes •NO not only as a regulator of cellular metabolism but possibly also as a regulator of mitochondrial production of reactive oxygen species which participate in cellular signaling. A major gap in the understanding of •NO bioactivity, namely, in the hippocampus, has been the lack of knowledge of its concentration dynamics. Here, we ... Springer protocols feed by Biochemistry news http://www.medworm.com/rss/comments.php?id=5473416 Mon, 05 Dec 2011 07:28:53 +0100 5473416 http://www.medworm.com/index.php?rid=5473415&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-382-0_6 The study of the mitochondrial membrane potential (ΔΨ) is essential for an integrated appraisal of mitochondrial function, since it reflects differences in electrical potential and represents the main component of the proton electrochemical gradient accounting for more than 90% of the total available respiratory energy. Numerous methods have been used to estimate mitochondrial membrane potential (ΔΨ), including fluorescent methods and electrochemical probes. In this chapter, we describe several practical approaches that allow mitochondrial membrane potential (ΔΨ) evaluation, by using a tetraphenylphosphonium (TPP+)-selective electrode. The main focus is given to the evaluation of ΔΨ in isolated mitochondria. (Source: Springer protocols feed by Bioche... Springer protocols feed by Biochemistry news http://www.medworm.com/rss/comments.php?id=5473415 Mon, 05 Dec 2011 07:28:53 +0100 5473415 http://www.medworm.com/index.php?rid=5473414&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-382-0_7 We present suitable conditions to employ safranine as a Δψ indicator. (Source: Springer protocols feed by Biochemistry) Springer protocols feed by Biochemistry news http://www.medworm.com/rss/comments.php?id=5473414 Mon, 05 Dec 2011 07:28:53 +0100 5473414 http://www.medworm.com/index.php?rid=5473413&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-382-0_8 The mitochondrial membrane potential is the dominant component of the proton-motive force that is the potential term in the proton circuit linking electron transport to ATP synthesis and other energy-dependent mitochondrial processes. Cationic fluorescent probes have been used for many years to detect gross qualitative changes in mitochondrial membrane potentials in intact cell culture. In this chapter, I describe how these fluorescence signals may be used to obtain a semiquantitative measure of changes in mitochondrial membrane potential. (Source: Springer protocols feed by Biochemistry) Springer protocols feed by Biochemistry news http://www.medworm.com/rss/comments.php?id=5473413 Mon, 05 Dec 2011 07:28:53 +0100 5473413 http://www.medworm.com/index.php?rid=5473412&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-382-0_9 Metabolic control analysis provides a quantitative framework for analyzing regulatory properties of enzymes in various metabolic pathways. It has been used for estimation of control parameters of the enzymatic pathways at isolated enzyme, cellular, or whole organism levels. This chapter describes how control and elasticities analysis can be experimentally applied to measure control properties of the components of the oxidative phosphorylation system and how a variant of such analysis – phenomenological kinetic analysis – can be used to investigate the effects of various factors (physiological or pathological) on the system of oxidative phosphorylation in isolated mitochondria. (Source: Springer protocols feed by Biochemistry) Springer protocols feed by Biochemistry news http://www.medworm.com/rss/comments.php?id=5473412 Mon, 05 Dec 2011 07:28:53 +0100 5473412 http://www.medworm.com/index.php?rid=5447424&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-376-9_25 Packaging DNA into compact chromatin enables eukaryotic cells to organize and regulate their genome. Packaging is achieved by wrapping ∼146–147 bp of DNA around a histone octamer to form a nucleosome, the basic unit of chromatin. Chromatin is a barrier of the bound DNA to factors involved in DNA-dependent processes such as transcription, replication, recombination, and repair. Several multisubunit protein complexes can move nucleosome to different positions on DNA utilizing energy derived from ATP hydrolysis and thereby facilitate access to DNA. Several methods are described for measuring nucleosome movement both in vivo and in vitro which provide important insights into the remodeling process. (Source: Springer protocols feed by Biochemistry) Springer protocols feed by Biochemistry news http://www.medworm.com/rss/comments.php?id=5447424 Sat, 26 Nov 2011 07:20:45 +0100 5447424 http://www.medworm.com/index.php?rid=5447423&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-376-9_24 Chromatin immunoprecipitation (ChIP) has been developed for studying protein–DNA interactions and has been extensively used for mapping the localization of posttranslationally modified histones, histone variants, transcription factors, or chromatin modifying enzymes at a given locus or on a genome-wide scale. ChIP methods have been modified and improved over the years to fit a variety of different cell types and tissues. Here, we present a detailed protocol for hippocampal ChIP, of both minced tissue and enzyme-separated hippocampal cells. This protocol enables to study chromatin–protein interactions in a specified population of hippocampal cells, allowing to study chromatin regulation in the central nervous system in a variety of conditions and disorders. Our assay has been de... Springer protocols feed by Biochemistry news http://www.medworm.com/rss/comments.php?id=5447423 Sat, 26 Nov 2011 07:20:45 +0100 5447423 http://www.medworm.com/index.php?rid=5447422&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-376-9_23 During prenatal development, a large number of different cell types are formed, the vast majority of which contain identical genetic material. The basis of the great variety in cell phenotype and function is the differential expression of the approximately 25,000 genes in the mammalian genome. Transcriptional activity is regulated at many levels by proteins, including members of the basal transcriptional apparatus, DNA-binding transcription factors, and chromatin-binding proteins. Importantly, chromatin structure dictates the availability of a specific genomic locus for transcriptional activation as well as the efficiency, with which transcription can occur. Chromatin immunoprecipitation (ChIP) is a method to assess if chromatin modifications or proteins are present at a specific locus. Ch... Springer protocols feed by Biochemistry news http://www.medworm.com/rss/comments.php?id=5447422 Sat, 26 Nov 2011 07:20:45 +0100 5447422 http://www.medworm.com/index.php?rid=5447421&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-376-9_22 Chromatin remodeling is a key mode of transcriptional regulation, and studying the nucleosome positioning at promoters is an important means to understand how genes are regulated. Nucleosome scanning is a convenient method to study nucleosome positioning. Yeast cells are converted to spheroplasts and nuclei are isolated. The nuclei are then digested by micrococcal nuclease to yield mononucleosome-sized DNA. Using a set of overlapping primers that cover the entire promoter, quantitative real-time PCR is performed using the mononucleosome DNA as the template. The nucleosome enrichment for each primer is calculated to yield a map of nucleosome occupancy across the promoter. (Source: Springer protocols feed by Biochemistry) Springer protocols feed by Biochemistry news http://www.medworm.com/rss/comments.php?id=5447421 Sat, 26 Nov 2011 07:20:45 +0100 5447421 http://www.medworm.com/index.php?rid=5447420&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-376-9_21 Cyclobutane pyrimidine dimers (CPDs) and (6,4) pyrimidine–pyrimidone dimers are the major DNA lesions (or photoproducts) induced by ultraviolet light and are removed by the nucleotide excision repair (NER) pathway. If not repaired, DNA damage can lead to genome instability. The genome is organized into nuclear domains with distinct functions and chromatin structures. Although studies on NER in all chromosomal contexts are important to understand the mechanisms of genome maintenance, we focused on NER in the nucleolus. The attractive feature of the rDNA locus is its chromatin structure; not all rRNA genes are transcribed and both active (no nucleosomes) and inactive (nucleosomes) rRNA genes coexist in the nucleolus. These characteristics allow for direct comparison of NER in two very ... Springer protocols feed by Biochemistry news http://www.medworm.com/rss/comments.php?id=5447420 Sat, 26 Nov 2011 07:20:45 +0100 5447420 http://www.medworm.com/index.php?rid=5447419&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-376-9_20 In eukaryotes, multiple copies of ribosomal RNA (rRNA) genes co-exist in two different chromatin states: actively transcribed (nucleosome depleted) chromatin, and nontranscribed (nucleosomal) chromatin. The presence of two rRNA gene populations compromises the interpretation of analyses obtained by the standard biochemical methods that are used to study chromatin structure (e.g., nuclease digestion and chromatin immunoprecipitation). Here, we provide a protocol to investigate the specific association of proteins with the two rRNA gene chromatin populations in vivo, using Saccharomyces cerevisiae as a model eukaryote. (Source: Springer protocols feed by Biochemistry) Springer protocols feed by Biochemistry news http://www.medworm.com/rss/comments.php?id=5447419 Sat, 26 Nov 2011 07:20:45 +0100 5447419 http://www.medworm.com/index.php?rid=5447418&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-376-9_19 Investigation of DNA–protein interactions is a key approach in understanding mechanisms of gene regulation. The method described allows detection of dynamic DNA–protein interactions occurring at gene promoters in living cells during the time scale of seconds and minutes. The combination of chromatin immunoprecipitation with real-time PCR allows for detection of changes in activator and co-activator content of any promoter during transcriptional activation. The described method is most applicable to investigation of processes resulting in nucleosome loss at gene promoters during the induction of transcription. The approach is also applicable to any dynamic process involving DNA–protein interactions. (Source: Springer protocols feed by Biochemistry) Springer protocols feed by Biochemistry news http://www.medworm.com/rss/comments.php?id=5447418 Sat, 26 Nov 2011 07:20:45 +0100 5447418 http://www.medworm.com/index.php?rid=5447417&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-376-9_18 Gene transcription is a complex process that involves a large number of proteins. These proteins can be brought to their target genes by a variety of different mechanisms: many transcription factors interact with specific DNA sequences in promoters or enhancers, several epigenetic regulators bind histones bearing specific modifications, elongation factors and some RNA processing factors bind to the transcribing RNA polymerase, and other factors interact directly with nascent transcripts or noncoding RNA. Immunostaining of Drosophila polytene chromosomes allows the genome-wide localization of factors involved at different stages of transcriptional regulation. In this chapter, we present protocols that adapt the general technique to probe different recruitment mechanisms employed by these fa... Springer protocols feed by Biochemistry news http://www.medworm.com/rss/comments.php?id=5447417 Sat, 26 Nov 2011 07:20:45 +0100 5447417 http://www.medworm.com/index.php?rid=5447416&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-376-9_17 Histone acetylation is the most studied posttranslation modification of nucleosomes. Understanding the mechanisms involved in global and promoter-specific histone acetylation will shed light on the control of transcriptional regulation. Chromatin immunoprecipitation is a powerful technique to study protein–DNA interactions in vivo. Proteins and DNA are cross-linked with formaldehyde, cells are lysed, and DNA is sheared by sonication. Protein–DNA complexes are immunoprecipitated with antibodies specific for total and acetylated histones and the relative occupancy of acetylated and total histones at selected loci is assessed by real-time PCR of the purified DNA. (Source: Springer protocols feed by Biochemistry) Springer protocols feed by Biochemistry news http://www.medworm.com/rss/comments.php?id=5447416 Sat, 26 Nov 2011 07:20:45 +0100 5447416 http://www.medworm.com/index.php?rid=5447415&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-376-9_16 We describe the procedure to generate cells stably expressing recombinant tagged-proteins at physiological level and then to purify the associated chromatin by affinity purification. Targets identified in this manner were validated in independent ChAP assays as well as in ChIP assays using antibodies against the endogenous protein. (Source: Springer protocols feed by Biochemistry) Springer protocols feed by Biochemistry news http://www.medworm.com/rss/comments.php?id=5447415 Sat, 26 Nov 2011 07:20:45 +0100 5447415 http://www.medworm.com/index.php?rid=5447414&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-376-9_15 Regulatory elements in promoter sequences typically function as binding sites for transcription factor proteins and thus are critical determinants of gene transcription. There is growing evidence that chromatin features, such as histone modifications or nucleosome positions, also have important roles in transcriptional regulation. Recent functional genomics and computational studies have yielded extensive datasets cataloging transcription factor binding sites (TFBS) and chromatin features, such as nucleosome positions, throughout the yeast genome. However, much of this data can be difficult to navigate or analyze efficiently. This chapter describes practical methods for the visualization, data mining, and statistical analysis of yeast promoter elements and chromatin features using two Web-... Springer protocols feed by Biochemistry news http://www.medworm.com/rss/comments.php?id=5447414 Sat, 26 Nov 2011 07:20:45 +0100 5447414 http://www.medworm.com/index.php?rid=5447413&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-376-9_9 The development of chromatin immunoprecipitation assays (ChIP) as a tool to examine the interactions between nuclear proteins and DNA has enhanced essentially our understanding of the dynamic association of transcription factors and chromatin modifiers with target DNA sequences. Still in vivo ChIP experiments of the central nervous system continue to represent a challenge given the considerable cellular and functional diversity, which makes the dissection of discrete circumscribed structures highly desirable. Tiny amounts of tissue can result, however, in insufficient quantities of starting material incompatible with many ChIP applications and lead to variable results. Here, we discuss the suitability of currently available ChIP protocols for in vivo ChIP experiments and present a new stre... Springer protocols feed by Biochemistry news http://www.medworm.com/rss/comments.php?id=5447413 Sat, 26 Nov 2011 07:20:45 +0100 5447413 http://www.medworm.com/index.php?rid=5447412&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-376-9_10 Chromatin immunoprecipitation (ChIP) is the most widely used method to measure the interaction of proteins with their target DNA sequences in the living cell. The use of ChIP can address many of the fundamental processes underlying transcription, such as the positioning and modification of nucleosomes, the binding of specific transcription factors to regulatory sequences, the secondary recruitment of chromatin-modifying complexes, and other signalling molecules to chromosomal DNA, and the occupancy of RNA polymerase complexes. ChIP is especially useful to define the dynamic nature of these processes. The basis for ChIP in most applications is the determination of the immunoprecipitation (IP) efficiency of individual genomic regions by comparing the amounts of DNA in the IP sample and the i... Springer protocols feed by Biochemistry news http://www.medworm.com/rss/comments.php?id=5447412 Sat, 26 Nov 2011 07:20:45 +0100 5447412 http://www.medworm.com/index.php?rid=5447411&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-376-9_11 Chromatin immunoprecipitation (ChIP) allows enrichment of genomic regions which are associated with specific transcription factors, histone modifications, and indeed any other epitopes which are present on chromatin. The original ChIP methods used site-specific PCR and Southern blotting to confirm which regions of the genome were enriched, on a candidate basis. The combination of ChIP with genomic tiling arrays (ChIP-chip) allowed a more unbiased approach to map ChIP-enriched sites. However, limitations of microarray probe design and probe number have a detrimental impact on the coverage, resolution, sensitivity, and cost of whole-genome tiling microarray sets for higher eukaryotes with large genomes. The combination of ChIP with high-throughput sequencing technology has allowed more compr... Springer protocols feed by Biochemistry news http://www.medworm.com/rss/comments.php?id=5447411 Sat, 26 Nov 2011 07:20:45 +0100 5447411 http://www.medworm.com/index.php?rid=5447410&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-376-9_12 Chromatin immunoprecipitation (ChIP) assays were developed in order to comprehensively describe physiological interactions between DNA sequences, transcriptional regulators, and the modification status of associated chromatin. In ChIP assays, living cells are treated with chemical cross-linkers to covalently bind proteins to each other and to their DNA targets. Once cross-linked to associated proteins, chromatin is extracted and fragmented by sonication and protein–DNA complexes are isolated using specific antibodies against a target protein. The cross-links that bind proteins to DNA are then reversed, and purified DNA fragments are analyzed by qPCR to determine if a specific sequence is present. As DNA regulatory elements frequently rely on the interaction of multiple transcription ... Springer protocols feed by Biochemistry news http://www.medworm.com/rss/comments.php?id=5447410 Sat, 26 Nov 2011 07:20:45 +0100 5447410 http://www.medworm.com/index.php?rid=5447409&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-376-9_13 Hypoxia-inducible factor (HIF) is the principal transcription factor that regulates adaptive physiologic responses to compromised oxygen tension. von Hippel–Lindau (VHL) tumor-suppressor protein binds and ubiquitylates the catalytic α subunit of HIF in an oxygen-dependent manner for rapid destruction via the 26S proteasome, thereby establishing VHL as a critical negative regulator of HIF. Mutations in VHL cause VHL disease, which is frequently characterized by the overexpression of HIFα and the development of tumors in multiple organ systems, including the central nervous system and the kidney. Here, we describe classical experimental approaches to demonstrate and validate HIF-responsive transcriptional regulation of genes. (Source: Springer protocols feed by Biochemistry... Springer protocols feed by Biochemistry news http://www.medworm.com/rss/comments.php?id=5447409 Sat, 26 Nov 2011 07:20:45 +0100 5447409 http://www.medworm.com/index.php?rid=5447408&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-376-9_14 The ability of prototypical second messenger cyclic AMP (cAMP) to positively control transcription of the somatostatin gene was pivotal to the original identification of the transcription factor cAMP response element-binding protein. However, it is now clear that alternative intracellular cAMP sensors, of which the exchange protein directly activated by cAMP (Epac) proteins have been studied most intensively, also initiate transcription of key genes in response to cAMP elevation. For example, we have demonstrated in vascular endothelial cells that activation of Epac1 is necessary for cAMP-mobilizing agents to trigger the induction of the gene-encoding suppressor of cytokine signaling-3 (SOCS-3), a potent inhibitor of interleukin (IL)-6 signaling. This is achieved through the recruitment of... Springer protocols feed by Biochemistry news http://www.medworm.com/rss/comments.php?id=5447408 Sat, 26 Nov 2011 07:20:45 +0100 5447408 http://www.medworm.com/index.php?rid=5447407&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-376-9_1 Immunoprecipitation of cross-linked chromatin in combination with microarrays (ChIP-chip) or ultra high-throughput sequencing (ChIP-seq) is widely used to map genome-wide in vivo transcription factor binding. Both methods employ initial steps of in vivo cross-linking, chromatin isolation, DNA fragmentation, and immunoprecipitation. For ChIP-chip, the immunoprecipitated DNA samples are then amplified, labeled, and hybridized to DNA microarrays. For ChIP-seq, the immunoprecipitated DNA is prepared for a sequencing library, and then the library DNA fragments are sequenced using ultra high-throughput sequencing platform. The protocols described here have been developed for ChIP-chip and ChIP-seq analysis of sequence-specific transcription factor binding in Drosophila embryos. A series of contr... Springer protocols feed by Biochemistry news http://www.medworm.com/rss/comments.php?id=5447407 Sat, 26 Nov 2011 07:20:45 +0100 5447407 http://www.medworm.com/index.php?rid=5447406&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-376-9_2 Transcription is the first step in the flow of biological information from genome to proteome and its tight regulation is a crucial checkpoint in most biological processes occurring in all living organisms. In eukaryotes, one of the most important mechanisms of transcriptional regulation relies on the activity of transcription factors which, upon binding to specific nucleotide motifs (consensus) present in the promoter region of target genes, modulate the activity of RNA polymerase II activating and/or repressing gene transcription. The identification of binding sites for these transcription factors is crucial to the understanding of transcriptional regulation at the molecular level and to the prediction of putative target genes for each transcription factor. However, transcription regulat... Springer protocols feed by Biochemistry news http://www.medworm.com/rss/comments.php?id=5447406 Sat, 26 Nov 2011 07:20:45 +0100 5447406 http://www.medworm.com/index.php?rid=5447405&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-376-9_3 Transcription factor NFκB is a key regulator of genes involved in immune and inflammatory responses, as well as genes regulating cell proliferation and survival. In addition to many inflammatory disorders, NFκB is constitutively activated in a variety of human cancers and leukemia. Thus, inhibition of NFκB DNA binding activity represents an important therapeutic approach for disorders characterized by high levels of constitutive NFκB activity. We have previously shown that NFκB DNA binding activity is suppressed by the nuclear translocation and accumulation of IκBα, which is induced by inhibition of the 26S proteasome. In this chapter, we describe a protocol that uses small inhibitory RNA (si RNA) interference followed by electrophoretic mobility s... Springer protocols feed by Biochemistry news http://www.medworm.com/rss/comments.php?id=5447405 Sat, 26 Nov 2011 07:20:45 +0100 5447405 http://www.medworm.com/index.php?rid=5447404&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-376-9_4 RNA polymerase II (Pol II) plays a crucial role in eukaryotic biology since it is necessary for the expression of all protein-coding genes as well as most microRNAs and several small nuclear RNAs. Pol II is specifically recruited to core promoter DNA via its association with general transcription factors (GTFs) that possess DNA binding activity such as TFIID, TFIIA, and TFIIB. The large multi-protein assemblies of Pol II together with the GTFs required for productive transcription are termed pre-initiation complexes (PICs). To date, studies of the interaction of PICs with promoter DNA have relied on the use of purified or recombinant GTFs. Recent findings have demonstrated an astonishing diversity in the function of core promoters as well as in the protein composition of PICs. The currentl... Springer protocols feed by Biochemistry news http://www.medworm.com/rss/comments.php?id=5447404 Sat, 26 Nov 2011 07:20:45 +0100 5447404 http://www.medworm.com/index.php?rid=5447403&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-376-9_5 Transcription factors recruit a wide variety of associated co-factors to regulate gene expression. These co-factors include protein kinases, phosphatases, deacetylases, methylases, and ubiquitin ligases, etc. To identify novel protein kinases associated with transcription factor NFAT, we took advantage of the increased ability of DNA binding and used an oligonucleotide affinity-binding approach. Coupling with in-gel kinase assays to detect phosphotransferase activity, we were able to identify p90 ribosomal S6 kinase (RSK) and p70 S6 kinase (S6K) that are present in the NFAT:DNA complex. We further demonstrated that RSK and S6K binds to and physically interacts with NFATc4. Similar oligonucleotide affinity-binding approach can be coupled with other enzymatic reactions, such as dephosphoryla... Springer protocols feed by Biochemistry news http://www.medworm.com/rss/comments.php?id=5447403 Sat, 26 Nov 2011 07:20:45 +0100 5447403 http://www.medworm.com/index.php?rid=5447402&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-376-9_6 This article outlines the general strategies and protocols to study ChIP assays in differential recruitment of transcriptional factors (TFs) and also global analysis of transcription factor recruitment is discussed. Further, the applications of ChIP assays for discovering novel genes that are dependent on specific transcription factors were addressed. (Source: Springer protocols feed by Biochemistry) Springer protocols feed by Biochemistry news http://www.medworm.com/rss/comments.php?id=5447402 Sat, 26 Nov 2011 07:20:45 +0100 5447402 http://www.medworm.com/index.php?rid=5447401&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-376-9_7 Eukaryotic gene regulation is controlled, in part, by inducible transcription factor-binding regulatory sequences in a tissue-specific and hormone-responsive manner. The development of methods for the analysis of transcription factor interaction within native chromatin has been a significant advance for the systematic analyses of the timing of gene regulation and studies on the effects of chromatin modifying enzymes on promoter accessibility. Chromatin immunoprecipitation (ChIP) is a specific method involving formaldehyde mediated protein–chromatin fixation to preserve the interaction for subsequent target identification. However, the conventional single-step cross-linking technique does not preserve all protein–DNA interactions, especially for transcription factors in hyper-dy... Springer protocols feed by Biochemistry news http://www.medworm.com/rss/comments.php?id=5447401 Sat, 26 Nov 2011 07:20:45 +0100 5447401 http://www.medworm.com/index.php?rid=5447400&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-376-9_8 Transcription factor NFκB comprises a family of proteins that serve as crucial regulators of genes involved in host immune and inflammatory responses, cell survival, proliferation, and differentiation. Since transcription of NFκB-dependent genes is increased in numerous inflammatory disorders as well as in many types of cancer and leukemia, inhibition of NFκB-dependent transcription thus represents an important therapeutic target. We have previously shown that in human leukocytes, transcription of NFκB-dependent genes is inhibited by the nuclear translocation and accumulation of IκBα, which can be induced by an inhibitor of CRM1-dependent nuclear export, leptomycin B (LMB). In this chapter, we describe a protocol that uses chromatin immunoprecipitation (... Springer protocols feed by Biochemistry news http://www.medworm.com/rss/comments.php?id=5447400 Sat, 26 Nov 2011 07:20:45 +0100 5447400 http://www.medworm.com/index.php?rid=5281205&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-331-8_25 This article describes a method used to measure amino acid racemase activity by high-performance liquid chromatography (HPLC). The assay involves fluorogenic chiral derivatization of amino acids with a newly developed reagent, and enantioseparation of d- and l-amino acid derivatives by HPLC. The method is accurate and reliable, and can be automated using a programmable autosampling injector. (Source: Springer protocols feed by Biochemistry) Springer protocols feed by Biochemistry news http://www.medworm.com/rss/comments.php?id=5281205 Tue, 04 Oct 2011 06:15:15 +0100 5281205 http://www.medworm.com/index.php?rid=5281204&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-331-8_24 Serine racemase is a glial and neuronal enzyme that reversibly converts l-serine to d-serine, an endogenous co-agonist of N-methyl-d-aspartate receptor type glutamate receptors (NMDARs). Here we present methods to recombinantly express and purify serine racemase in bacteria and two complementary ways to determine d-serine levels in unknown samples. Furthermore, a detailed protocol of serine racemase activity assays is described that can be used to screen for activators and inhibitors in vitro. (Source: Springer protocols feed by Biochemistry) Springer protocols feed by Biochemistry news http://www.medworm.com/rss/comments.php?id=5281204 Tue, 04 Oct 2011 06:15:15 +0100 5281204 http://www.medworm.com/index.php?rid=5281203&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-331-8_23 This paper describes a method for determining the nutritional value of d-amino acids, d-peptides, and amino acid derivatives using a growth assay in mice fed a synthetic all-amino acid diet. A large number of experiments were carried out in which a molar equivalent of the test compound replaced a nutritionally essential amino acid such as l-lysine (l-Lys), l-methionine (l-Met), l-phenylalanine (l-Phe), and l-tryptophan (l-Trp) as well as the semi-essential amino acids l-cysteine (l-Cys) and l-tyrosine (l-Tyr). The results show wide-ranging variations in the biological utilization of test substances. The method is generally applicable to the determination of the biological utilization and safety of any amino acid derivative as a potential nutritional source of the corresponding l-amino acid... Springer protocols feed by Biochemistry news http://www.medworm.com/rss/comments.php?id=5281203 Tue, 04 Oct 2011 06:15:15 +0100 5281203 http://www.medworm.com/index.php?rid=5281202&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-331-8_22 Recent studies have shown that biologically uncommon d-β-aspartic acid residues accumulate in specific proteins during the aging process. However, aspartyl residues are not racemized uniformly because d-Asp appears to be confined to particular sites in these proteins. We here describe the method to identify the specific sites of d-β-aspartic acids inversion in proteins. (Source: Springer protocols feed by Biochemistry) Springer protocols feed by Biochemistry news http://www.medworm.com/rss/comments.php?id=5281202 Tue, 04 Oct 2011 06:15:15 +0100 5281202 http://www.medworm.com/index.php?rid=5281201&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-331-8_21 The presence of d-amino acids in foods is promoted by harsh technological processes (e.g., high temperature or extreme pH values) or can be the consequence of adulteration or microbial contamination (d-amino acids are major components of the bacterial cell wall). For this reason, quality control is becoming more and more important both for the industry (as a cost factor) and for consumer protection. For routine food analysis and quality control, simple and easily applicable analytical methods are needed: biosensors can often satisfy these requirements. The use of an enzymatic, stereospecific reaction could confer selectivity to a biosensor for detecting and quantifying d-amino acids in foodstuffs. The flavoenzyme d-amino acid oxidase from the yeast Rhodotorula gracilis is an ideal biocatal... Springer protocols feed by Biochemistry news http://www.medworm.com/rss/comments.php?id=5281201 Tue, 04 Oct 2011 06:15:15 +0100 5281201 http://www.medworm.com/index.php?rid=5281200&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-331-8_20 The NMDA subtypes of glutamatergic receptors (NMDARs) are unusual in that their activation requires the binding of both glutamate and a co-agonist glycine or d-serine. Whereas glycine was first suggested to play such a role, it was later established that d-serine could serve as an endogenous co-agonist at different central synapses. We still do not know the exact nature of the endogenous co-agonist(s) of NMDARs and the function of the so-called glycine B site in many brain structures. We introduced few years ago the use of enzymes that specifically degrade either d-serine or glycine to decipher the influence of these amino acids on NMDA receptors function. The use of these enzymatic scavengers represents an invaluable technique for neurophysiologists investigating the neuromodulation of th... Springer protocols feed by Biochemistry news http://www.medworm.com/rss/comments.php?id=5281200 Tue, 04 Oct 2011 06:15:15 +0100 5281200 http://www.medworm.com/index.php?rid=5281199&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-331-8_19 d-Serine is a transmitter-like molecule that physiologically activates NMDA receptors in the brain. Although d-serine was thought to be exclusively released by astrocytes, we recently demonstrated endogenous d-serine release from neurons in cultures and slices. So far high-performance liquid chromatography (HPLC) has been the standard technique to monitor d-serine and other amino acids. This method employs pre-column derivatization with a chiral reagent to produce fluorescence derivatives that can be further separated on a reversed-phase column. Due to the close retention times of l-serine, l-glutamine, and d-serine, the quantification of low levels of endogenous d-serine synthesis and release from cell cultures and tissues can be challenging. We here describe an enzymatic treatment method... Springer protocols feed by Biochemistry news http://www.medworm.com/rss/comments.php?id=5281199 Tue, 04 Oct 2011 06:15:15 +0100 5281199 http://www.medworm.com/index.php?rid=5281198&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-331-8_18 d-Amino acids play several key roles and are widely diffused in living organisms, from bacteria (in which d-alanine is a component of the cell wall) to mammals (where d-serine is involved in glutamatergic neurotransmission in the central nervous system). The study of the biological processes involving d-amino acids and their use as clinical or biotechnological biomarkers requires reliable methods of quantifying them. Although “traditional” analytical techniques have been (and still are) employed for such tasks, enzymatic assays based on enzymes which possess a strict stereospecificity (i.e., that are only active on the d-enantiomers of amino acids) allowed the set-up of low-cost protocols with a high sensitivity and selectivity and suitable for determining the d-amino acid cont... Springer protocols feed by Biochemistry news http://www.medworm.com/rss/comments.php?id=5281198 Tue, 04 Oct 2011 06:15:15 +0100 5281198 http://www.medworm.com/index.php?rid=5281197&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-331-8_17 We describe the detailed procedures and the critical points for obtaining reliable estimated ages using the racemization method. (Source: Springer protocols feed by Biochemistry) Springer protocols feed by Biochemistry news http://www.medworm.com/rss/comments.php?id=5281197 Tue, 04 Oct 2011 06:15:15 +0100 5281197 http://www.medworm.com/index.php?rid=5281196&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-331-8_16 We report here improved HPLC methods for the specific determination of d-Asp and NMDA in biological tissues. (Source: Springer protocols feed by Biochemistry) Springer protocols feed by Biochemistry news http://www.medworm.com/rss/comments.php?id=5281196 Tue, 04 Oct 2011 06:15:15 +0100 5281196 http://www.medworm.com/index.php?rid=5281195&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-331-8_15 Chemoselective reactions are important tools for the modification of peptides and proteins. Thereby the modification is desired to be site specific and bioorthogonal. Here we describe the site-specific modification of azido-proteins via a Staudinger-type phosphite ligation. The reaction was carried out in aqueous system on proteins containing p-azido-phenylalanine in a single position introduced by the amber codon technique. A selective introduction of branched polyethylene scaffolds can be achieved with the application of the methodology reported herein. (Source: Springer protocols feed by Biochemistry) Springer protocols feed by Biochemistry news http://www.medworm.com/rss/comments.php?id=5281195 Tue, 04 Oct 2011 06:15:15 +0100 5281195 http://www.medworm.com/index.php?rid=5281194&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-331-8_14 Unnatural amino acid mutagenesis allows us to introduce unnatural α-amino acids into internal positions of proteins in response to expanded codons such as amber and four-base codons. To improve the unnatural amino acid mutagenesis, the incorporation of unnatural α-amino acids and non-α-amino acids into the N-terminus of proteins has been achieved using expanded initiation codons. Here, we describe the method for the incorporation of fluorescent-labeled non-α-amino acids into the N-terminus of proteins in a cell-free translation system. (Source: Springer protocols feed by Biochemistry) Springer protocols feed by Biochemistry news http://www.medworm.com/rss/comments.php?id=5281194 Tue, 04 Oct 2011 06:15:15 +0100 5281194 http://www.medworm.com/index.php?rid=5281193&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-331-8_13 Expanding the repertoire of genetically encoded amino acids in cultured mammalian cells requires the expression of the bacterial or archaeal pair of a tRNA and an aminoacyl-tRNA synthetase variant engineered to be specific to the amino acid, along with the supplementation of an unnatural amino acid in the growth medium. The expression of the pair is generally achieved by transfecting the cultured cells with the plasmids bearing the genes encoding the exogenous pair of translation molecules. Here, we provide a description of some of these plasmids and protocols for transfecting cells with the plasmids and preparing growth media supplemented with unnatural amino acids, to facilitate their incorporation into proteins at specific sites. (Source: Springer protocols feed by Biochemistry) Springer protocols feed by Biochemistry news http://www.medworm.com/rss/comments.php?id=5281193 Tue, 04 Oct 2011 06:15:15 +0100 5281193 http://www.medworm.com/index.php?rid=5281192&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-331-8_12 Unnatural amino acids can be genetically incorporated into proteins in live cells by using an orthogonal tRNA/aminoacyl-tRNA synthetase pair. Here we describe a method to efficiently express the orthogonal tRNA and synthetase in Saccharomyces cerevisiae, which enables unnatural amino acids to be genetically incorporated into target proteins in yeast with high efficiency. We also describe the use of a yeast strain deficient in the nonsense-mediated mRNA decay, which further increases the unnatural amino acid incorporation efficiency when a stop codon is used to encode the unnatural amino acid. These strategies will facilitate the investigation of proteins and their related biological processes in yeast by exploiting the novel properties afforded by unnatural amino acids. (Source: Springer p... Springer protocols feed by Biochemistry news http://www.medworm.com/rss/comments.php?id=5281192 Tue, 04 Oct 2011 06:15:15 +0100 5281192 http://www.medworm.com/index.php?rid=5281191&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-331-8_11 Site-specific incorporation of unnatural amino acids into proteins in vivo relies on the genetic reassignment of nonsense or quadruplet codons. Here, we describe a general procedure for the random introduction of these codons into open reading frames resulting in protein libraries that are scanned with unnatural amino acid residues. These libraries can enable large-scale mutagenesis experiments aimed at understanding and improving protein function. (Source: Springer protocols feed by Biochemistry) Springer protocols feed by Biochemistry news http://www.medworm.com/rss/comments.php?id=5281191 Tue, 04 Oct 2011 06:15:15 +0100 5281191 http://www.medworm.com/index.php?rid=5281190&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-331-8_9 Because of their unique mechanism of cytotoxicity against bacteria and other microorganisms, antimicrobial peptides have received a great deal of attention as possible therapeutic agents. Incorporation of unnatural amino acids into the peptide sequences has the potential to improve the organism selectivity and potency of these peptides as well as increase their metabolic stability. This protocol outlines the logic used to selectively incorporate unnatural amino acid into a peptide sequence in an attempt to obtain peptides with increased therapeutic potential as antibiotic agents. (Source: Springer protocols feed by Biochemistry) Springer protocols feed by Biochemistry news http://www.medworm.com/rss/comments.php?id=5281190 Tue, 04 Oct 2011 06:15:15 +0100 5281190 http://www.medworm.com/index.php?rid=5281189&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-331-8_8 Site-specific in vivo incorporation of unnatural amino acids provides powerful tools for the study of protein interaction and dynamics. Here, we provide a protocol for the incorporation of six such UAA probes into a GFP reporter system, expressed in Escherichia coli from both arabinose and lactose-inducible expression plasmids using an autoinduction media. (Source: Springer protocols feed by Biochemistry) Springer protocols feed by Biochemistry news http://www.medworm.com/rss/comments.php?id=5281189 Tue, 04 Oct 2011 06:15:15 +0100 5281189 http://www.medworm.com/index.php?rid=5281188&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-331-8_7 A large number of modified amino acids other than the canonical amino acid residues can be found in natural products, especially antibiotics. The structure of these peptide-based compounds is investigated using modern two-dimensional NMR techniques. The automatic assignment of the 2D NMR proton spectra and consequent determination of the primary and 3D structure of peptides or small size proteins containing natural amino acids is nowadays routine. However, a deficiency in the ability to readily sequence peptides containing unnatural amino acids still remains and a great human effort and time is required. The experimental methods and the protocols of manual analysis of the data are described in the following sections. (Source: Springer protocols feed by Biochemistry) Springer protocols feed by Biochemistry news http://www.medworm.com/rss/comments.php?id=5281188 Tue, 04 Oct 2011 06:15:15 +0100 5281188 http://www.medworm.com/index.php?rid=5281187&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-331-8_10 Endogenous antimicrobial peptides (AMPs) can have multimodal mechanisms of bacterial inactivation, such as membrane lysis, interference with cell wall biosynthesis or membrane-based protein machineries, or translocation through the membrane to intracellular targets. The controlled variation of side-chain characteristics in their amino acid residues can provide much useful information on structure–activity relationships and mode-of-action, and also lead to improved activities. The small size and relatively low complexity of AMPs make them amenable to solid-phase peptide synthesis, facilitating the use of nonproteinogenic amino acids and vastly increasing the accessible molecular diversity of side chains. Here, we describe how such residues can be used to modulate such key parameters a... Springer protocols feed by Biochemistry news http://www.medworm.com/rss/comments.php?id=5281187 Tue, 04 Oct 2011 06:15:15 +0100 5281187 http://www.medworm.com/index.php?rid=5281186&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-331-8_6 Hydantoinases/dihydropyrimidinases are important biotechnological enzymes involved in the production of α- and β-amino acids. Their isolation from new sources with different substrate specificities, improved activity, enantioselectivity, or higher stability continues to be of great industrial interest. Here, we provide a detailed description of how to produce high quantities of the recombinant hydantoinase/dihydropyrimidinase enzyme from Sinorhizobium meliloti CECT4114 (SmeDhp). Several techniques are combined to obtain this goal, from cloning to activity measurement by HPLC. (Source: Springer protocols feed by Biochemistry) Springer protocols feed by Biochemistry news http://www.medworm.com/rss/comments.php?id=5281186 Tue, 04 Oct 2011 06:15:15 +0100 5281186 http://www.medworm.com/index.php?rid=5281185&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-331-8_5 An enzymatic methodology for the preparation of β-hydroxy-α-amino acid derivatives is described. The method consists of the stereoselective aldol addition reaction of glycine to N-Cbz-amino aldehydes furnishing 3-hydroxy-2,4-diaminobutyric derivatives. (Source: Springer protocols feed by Biochemistry) Springer protocols feed by Biochemistry news http://www.medworm.com/rss/comments.php?id=5281185 Tue, 04 Oct 2011 06:15:15 +0100 5281185 http://www.medworm.com/index.php?rid=5281184&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-331-8_4 We describe here the application of aspartate aminotransferase and branched chain aminotransferase from E. coli for the synthesis of various glutamate analogues, molecules of particular interest regarding the neuroactive properties of glutamic acid. (Source: Springer protocols feed by Biochemistry) Springer protocols feed by Biochemistry news http://www.medworm.com/rss/comments.php?id=5281184 Tue, 04 Oct 2011 06:15:15 +0100 5281184 http://www.medworm.com/index.php?rid=5281183&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-331-8_3 Biocatalytic conversion of 5-substituted hydantoin derivatives is an efficient method for the production of unnatural enantiomerically pure amino acids. The enzymes required to carry out this hydrolysis occur in a wide variety of eubacterial species each of which exhibit variations in substrate selectivity, enantiospecificity, and catalytic efficiency. Screening of the natural environment for bacterial strains capable of utilizing hydantoin as a nutrient source (as opposed to rational protein design of known enzymes) is a cost-effective and valuable approach for isolating microbial species with novel hydantoin-hydrolysing enzyme systems. Once candidate microbial isolates have been identified, characterization and optimization of the activity of target enzyme systems can be achieved by subj... Springer protocols feed by Biochemistry news http://www.medworm.com/rss/comments.php?id=5281183 Tue, 04 Oct 2011 06:15:15 +0100 5281183 http://www.medworm.com/index.php?rid=5281182&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-331-8_2 The use of unnatural amino acids, particularly synthetic α-amino acids, for modern drug discovery research requires the availability of enantiomerically pure isomers. Starting from a racemate, one single enantiomer can be obtained using a deracemization process. The two more common strategies of deracemization are those obtained by stereoinversion and by dynamic kinetic resolution. Both techniques will be here described using as a substrate the d,l-3-(2-naphthyl)-alanine, a non-natural amino acid: the first one employing a multi-enzymatic redox system, the second one combining an hydrolytic enzyme together with a base-catalyzed substrate racemization. In both cases, the final product, l-3-(2-naphthyl)alanine, is recovered with good yield and excellent enantiomeric excess. (Source: Sp... Springer protocols feed by Biochemistry news http://www.medworm.com/rss/comments.php?id=5281182 Tue, 04 Oct 2011 06:15:15 +0100 5281182 http://www.medworm.com/index.php?rid=5281181&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-331-8_1 Ammonia-lyases catalyze a wide range of processes leading to α,β-unsaturated compounds by elimination of ammonia. In this chapter, ammonia-lyases are reviewed with major emphasis on their synthetic applications in stereoselective preparation of unnatural amino acids. Besides the synthesis of various unnatural α-amino acids with the aid of phenylalanine ammonia-lyases (PALs) utilizing the 3,5-dihydro-5-methylidene-4H-imidazol-4-one (MIO) prosthetic groups, the biotransformations leading to various unnatural β-amino acids with phenylalanine 2,3-aminomutases using the same catalytic MIO prosthetic group are discussed. Cloning, production, purification, and biotransformation protocols for PAL are described in detail. (Source: Springer protocols feed by Biochemistry) Springer protocols feed by Biochemistry news http://www.medworm.com/rss/comments.php?id=5281181 Tue, 04 Oct 2011 06:15:15 +0100 5281181 http://www.medworm.com/index.php?rid=5203859&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-295-3_9 Human HspB1 (also denoted Hsp27) is a well-known member, together with alphaB-crystallin, of the small heat-shock (or stress) proteins (sHsps) (20–40 kDa). In this chapter, I describe procedures for testing the oligomeric and phosphorylation patterns of HspB1 as well as its interaction with specific partner/client polypeptides using tissue culture cells genetically modified to express different levels of this protein. The procedures have been developed in my laboratory and could be used in any well-established cellular laboratory. In addition, the different procedures presented here could be extended to test the nine other human sHsp members as well as sHsps from other species. (Source: Springer protocols feed by Biochemistry) Springer protocols feed by Biochemistry news http://www.medworm.com/rss/comments.php?id=5203859 Sun, 11 Sep 2011 03:16:55 +0100 5203859 http://www.medworm.com/index.php?rid=5203858&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-295-3_8 CHIP, the carboxyl-terminus of Hsp70 interacting protein, is both an E3 ubiquitin ligase and an Hsp70 co-chaperone and is implicated in the degradation of cytosolic quality control and numerous disease substrates. CHIP has been shown to monitor the folding status of the CFTR protein, and we have successfully reconstituted this activity using a recombinant CFTR fragment consisting of the cytosolic NBD1 and R domains. We have found that efficient ubiquitination of substrates requires chaperone activity to either deliver the substrate to CHIP or to maintain the substrate in a ubiquitination-competent conformation. This chaperone activity can be provided by the Hsp70/Hsp40 molecular chaperone system as seen in the NBD1–R ubiquitination assay. Alternatively, heat treatment of CHIP can act... Springer protocols feed by Biochemistry news http://www.medworm.com/rss/comments.php?id=5203858 Sun, 11 Sep 2011 03:16:55 +0100 5203858 http://www.medworm.com/index.php?rid=5203857&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-295-3_7 The ATPase cycle of Hsp70 chaperones controls their transient association with substrate and, thus, governs their function in protein folding. Nucleotide exchange factors (NEFs) accelerate ADP release from Hsp70 which results in rebinding of ATP and release of the substrate. This chapter describes several methods suitable to study NEFs of Hsp70 chaperones. On the one hand, steady-state ATPase assays provide information on how the NEF influences progression of the Hsp70 through the entire ATPase cycle. On the other hand, nucleotide release can be measured directly using labeled nucleotides, which enables identification and further characterization of NEFs. (Source: Springer protocols feed by Biochemistry) Springer protocols feed by Biochemistry news http://www.medworm.com/rss/comments.php?id=5203857 Sun, 11 Sep 2011 03:16:55 +0100 5203857 http://www.medworm.com/index.php?rid=5203856&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-295-3_6 Molecular chaperones promote polypeptide folding in cells by protecting newly made and otherwise misfolded proteins against aggregation or degradation by the ubiquitin proteasome pathway. The roles of Saccharomyces cerevisiae Cdc37 and Ydj1 molecular chaperones are described in this chapter. We focus on biogenesis of protein kinases that require several different molecular chaperones for their proper folding. Specific among these is Cdc37, which binds directly to its kinase clients either during or shortly after translation and protects them against rapid proteasomal degradation. Ydj1 has a similar role, but is less specific for protein kinases in its role as a molecular chaperone. The method that we describe uses pulse chase and immunoprecipitation to analyze the fate of newly made protei... Springer protocols feed by Biochemistry news http://www.medworm.com/rss/comments.php?id=5203856 Sun, 11 Sep 2011 03:16:55 +0100 5203856 http://www.medworm.com/index.php?rid=5203855&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-295-3_5 Heat-shock protein 90 (HSP90) is an essential molecular chaperone in eukaryotes. It is important for chaperoning proteins that are important determinants of multistep carcinogenesis. HSP90’s ATPase activity is associated with its chaperone function. Co-chaperones as well as posttranslational modifications (phosphorylation, acetylation, and S-nitrosylation) are important for regulating its ATPase activity. Yeast can be used to express and purify HSP90 and also detect its phosphorylation by pan-phosphoserine or phosphothreonine antibodies. (Source: Springer protocols feed by Biochemistry) Springer protocols feed by Biochemistry news http://www.medworm.com/rss/comments.php?id=5203855 Sun, 11 Sep 2011 03:16:55 +0100 5203855 http://www.medworm.com/index.php?rid=5203854&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-295-3_4 Molecular chaperones are a diverse group of highly conserved proteins that transiently interact with partially folded polypeptide chains during normal cellular processes, such as protein translation, translocation, and disassembly of protein complexes (1). Prior to folding or after denaturation, hydrophobic residues that are normally sequestered within a folded protein are exposed to the aqueous environment and are prone to aggregation or misfolding. Multiple classes of molecular chaperones, such as Hsp70s and Hsp40s, recognize and transiently bind polypeptides with exposed hydrophobic stretches in order to prevent misfolding. Other types of chaperones, such as Hsp90, have more specialized functions in that they appear to interact with only a subset of cellular proteins. This chapter focus... Springer protocols feed by Biochemistry news http://www.medworm.com/rss/comments.php?id=5203854 Sun, 11 Sep 2011 03:16:55 +0100 5203854 http://www.medworm.com/index.php?rid=5203853&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-295-3_3 Heat-shock protein 90 (Hsp90) is a molecular chaperone that assists in the maturation of a limited set of substrate proteins that are collectively referred to as clients. The majority of identified Hsp90 clients are involved in signal transduction, including many steroid hormone receptors and kinases. A handful of Hsp90 clients can be classified as nonsignal transduction proteins, including telomerase, cystic fibrosis transmembrane conductance regulator, and antigenic peptides destined for major histocompatibility complex. Because Hsp90 clients are causative agents in cancer and cystic fibrosis, research on Hsp90 has intensified in recent years. We review the historical path of Hsp90 research within each class of client (kinase, hormone receptor, and nonsignal transduction clients) and hig... Springer protocols feed by Biochemistry news http://www.medworm.com/rss/comments.php?id=5203853 Sun, 11 Sep 2011 03:16:55 +0100 5203853 http://www.medworm.com/index.php?rid=5203852&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-295-3_2 Heat shock proteins (HSPs) are rapidly induced after stresses, such as heat shock, and accumulate at high concentrations in cells. HSP induction involves a family of heat shock transcription factors that bind the heat shock elements of the HSP genes and mediate transcription in trans. We discuss methods for the study of HSP binding to HSP promoters and the consequent increases in HSP gene expression in vitro and in vivo. (Source: Springer protocols feed by Biochemistry) Springer protocols feed by Biochemistry news http://www.medworm.com/rss/comments.php?id=5203852 Sun, 11 Sep 2011 03:16:55 +0100 5203852 http://www.medworm.com/index.php?rid=5203851&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-295-3_23 Directed cell migration is fundamental to both physiological and pathophysiological processes such as embryogenesis, wound healing, and cancer metastasis. A complex series of events are required for directional cell migration, which is initiated by a migration-promoting or chemotactic stimulus, resulting in cellular polarization and entry into a cyclical pattern of leading edge protrusion, adhesion, and retraction of the trailing edge allowing cell movement. Heat shock proteins such as Hsp27, Hsp90, alphaB-crystallin, as well as heat shock transcription factors, are important players in both physiological and pathophysiological cell migration. A variety of techniques are currently available to assess cell migration, and among the most commonly utilized are those that employ a two-chamber m... Springer protocols feed by Biochemistry news http://www.medworm.com/rss/comments.php?id=5203851 Sun, 11 Sep 2011 03:16:55 +0100 5203851 http://www.medworm.com/index.php?rid=5203850&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-295-3_22 Extracellular heat shock proteins (HSP) play important roles in cell signaling and immunity. Many of these effects are mediated by cell surface receptors expressed on a wide range of cell types. We have investigated the nature of such proteins by cloning candidate receptors into cells (CHO-K1) with the rare property of being null for HSP binding. Using this approach, we have discovered that Hsp70 binds to a least two classes of receptor: c-type lectin receptors (CLR) and scavenger receptors (SR). However, the nature of the receptor–ligand interactions is not yet clear. Hsp70 can bind to LOX-1 (a member of both the CLR and SR), with the c-type lectin binding domain (CTLD) as well as the SR family members SREC-I and FEEL-1/CLEVER-1/STABILIN-1, which by contrast have arrays of EGF-like ... Springer protocols feed by Biochemistry news http://www.medworm.com/rss/comments.php?id=5203850 Sun, 11 Sep 2011 03:16:55 +0100 5203850 http://www.medworm.com/index.php?rid=5203849&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-295-3_21 Large heat-shock proteins (HSPs), including hsp110 and grp170, are unique immunochaperones capable of carrying and introducing antigens into professional antigen-presenting cells for efficient cross-presentation. Therefore, reconstituted chaperone complexes of large HSPs and protein antigen may be exploited for augmentation of an antigen-specific immune response. The methods for the preparation of the recombinant protein antigen chaperone complex and characterization of its T-cell priming capability in both in vitro and in vivo settings are described. (Source: Springer protocols feed by Biochemistry) Springer protocols feed by Biochemistry news http://www.medworm.com/rss/comments.php?id=5203849 Sun, 11 Sep 2011 03:16:55 +0100 5203849 http://www.medworm.com/index.php?rid=5203848&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-295-3_20 Heat shock proteins (Hsp) are molecular chaperones with the capability to interact with a wide range of other proteins and are thus often found coupled with other heat shock and non-heat shock proteins. This can be an advantage to study specific interactions between a chaperone and other proteins and to generate an antitumoral immune response. In this chapter, we present two protocols to isolate Hsp. One involves column chromatography with hydroxyapatite and the other employs immunoprecipitation with antibodies coupled to magnetic beads. In both cases, we specifically want to isolate Hsp coupled with other proteins and use the Hsp complexes as intermediaries to present the coupled peptides/proteins to the immune system, or to explore the associations of a particular Hsp with other proteins... Springer protocols feed by Biochemistry news http://www.medworm.com/rss/comments.php?id=5203848 Sun, 11 Sep 2011 03:16:55 +0100 5203848 http://www.medworm.com/index.php?rid=5203847&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-295-3_1 Heat-shock transcription factors (Hsfs) regulate transcription of heat-shock proteins as well as other genes whose promoters contain heat-shock elements. There are at least five Hsfs in mammalian cells, Hsf1, Hsf2, Hsf3, Hsf4, and Hsfy. To understand the physiological roles of Hsf1, Hsf2, and Hsf4 in vivo, we generated knockout mouse lines for these factors. In this chapter, we describe the design of the targeting vectors, the plasmids used, and the successful generation of mice lacking the individual genes. We also briefly describe what we have learned about the physiological functions of these genes in vivo. (Source: Springer protocols feed by Biochemistry) Springer protocols feed by Biochemistry news http://www.medworm.com/rss/comments.php?id=5203847 Sun, 11 Sep 2011 03:16:55 +0100 5203847 http://www.medworm.com/index.php?rid=5203846&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-295-3_19 We have developed an enhanced molecular chaperone-based vaccine through rapid isolation of heat-shock protein 70 peptide complexes (Hsp70.PC) after the fusion of tumor and dendritic cells (DCs) (Hsp70.PC-F). In this approach, the tumor antigens are introduced into the antigen-processing machinery of dendritic cells through the cell fusion process and, thus, we can obtain antigenic tumor peptides or their intermediates that have been processed by dendritic cells. Our results show that Hsp70.PC-F has increased immunogenicity compared to preparations from tumor cells alone and, therefore, constitutes an improved formulation of chaperone protein-based tumor vaccine. (Source: Springer protocols feed by Biochemistry) Springer protocols feed by Biochemistry news http://www.medworm.com/rss/comments.php?id=5203846 Sun, 11 Sep 2011 03:16:55 +0100 5203846 http://www.medworm.com/index.php?rid=5203845&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-295-3_18 Hsp60 (also called Cpn60) is a chaperonin with essential functions for cell physiology and survival. Additionally, its involvement in the pathogenesis of a number of diseases (e.g., some autoimmune disorders and cancer) is becoming evident with new research. For example, the distribution and levels of Hsp60 in cells and tissues have been found altered in many pathologic conditions, and the significance of these alterations is being investigated in a number of laboratories. The aim of this ongoing research is to determine the meaning of these Hsp60 alterations with regard to pathogenetic mechanisms, diagnosis, classification of lesions, and assessing of prognosis and response to treatment. Hsp60 occurs in the mitochondria, i.e., its typical residence according to classic knowledge, and also... Springer protocols feed by Biochemistry news http://www.medworm.com/rss/comments.php?id=5203845 Sun, 11 Sep 2011 03:16:55 +0100 5203845 http://www.medworm.com/index.php?rid=5203844&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-295-3_17 Cell death (in particular, apoptosis and necrosis) is accompanied by appearance of certain hallmarks that are manifested as specific alterations in cellular membranes, cytoplasm, nucleus and mitochondria. Some of those hallmarks are easily detectable in situ and, therefore, they can be applied for the assessment of dying or dead cells. In turn, there are also signs of viable cells that include a set of features, such as normal functioning of their membranes and organelles, ability to proliferate, etc. The present chapter provides descriptions of several convenient methods for quantitative determination of dead (apoptotic and necrotic) cells and also methods for determination of survived and viable cells. Here, we describe in details the methods of annexin V/propidium iodide (PI) staining, ... Springer protocols feed by Biochemistry news http://www.medworm.com/rss/comments.php?id=5203844 Sun, 11 Sep 2011 03:16:54 +0100 5203844 http://www.medworm.com/index.php?rid=5203843&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-295-3_16 Heat shock protein 70 (Hsp70) is a powerful chaperone whose expression is induced in response to a wide variety of physiological and environmental insults, including anticancer chemotherapy, thus allowing the cell to survive to lethal conditions. Hsp70 cytoprotective properties may be explained by its anti-apoptotic function. Indeed, this protein can inhibit key effectors of the apoptotic machinery at the pre- and postmitochondrial level. In cancer cells, the expression of Hsp70 is abnormally high, and Hsp70 may participate in oncogenesis and in resistance to chemotherapy. In rodent models, Hsp70 overexpression increases tumor growth and metastatic potential. Depletion or inhibition of Hsp70 frequently reduces the size of the tumors and even can cause their complete involution. But Hsp70 c... Springer protocols feed by Biochemistry news http://www.medworm.com/rss/comments.php?id=5203843 Sun, 11 Sep 2011 03:16:54 +0100 5203843 http://www.medworm.com/index.php?rid=5203842&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-295-3_15 We describe a computational protocol to identify functional modules and pathway relationship of chaperones based on physical interaction data derived from high-throughput proteomic experiments. The protocol first identifies interacting proteins shared by the different chaperone systems to organize the chaperones into functional modules. The chaperone functional modules represent groups of chaperones that are involved in mediating the folding of the shared interacting proteins. Either the chaperones in a module can function along a single folding pathway of a given substrate protein or the substrate protein might have two or more different folding pathways that the chaperones act on independently. As described in our computational protocol, probabilities of these pathway relationships betwe... Springer protocols feed by Biochemistry news http://www.medworm.com/rss/comments.php?id=5203842 Sun, 11 Sep 2011 03:16:54 +0100 5203842 http://www.medworm.com/index.php?rid=5203841&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-295-3_14 Mass spectrometry (MS) is a powerful analytical tool for proteomics research and drug and biomarker discovery. MS enables identification and quantification of known and unknown compounds by revealing their structural and chemical properties. Proper sample preparation for MS-based analysis is a critical step in the proteomics workflow because the quality and reproducibility of sample extraction and preparation for downstream analysis significantly impact the separation and identification capabilities of mass spectrometers. The highly expressed proteins represent potential biomarkers that could aid in diagnosis, therapy, or drug development. Because the proteome is so complex, there is no one standard method for preparing protein samples for MS analysis. Protocols differ depending on the typ... Springer protocols feed by Biochemistry news http://www.medworm.com/rss/comments.php?id=5203841 Sun, 11 Sep 2011 03:16:54 +0100 5203841 http://www.medworm.com/index.php?rid=5203840&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-295-3_13 The use of flow cytometry in heat-shock protein (HSP) research is increasing rapidly due to the high sensitivity and versatility of the technique. The method allows the simultaneous analysis of multiple proteins within numerous cell types in a heterogeneous sample, providing advantages over alternative techniques, such as ELISA and Western blotting. As a result, flow cytometry is becoming the leading technique used in this area of research. The current chapter describes the methodology for preparing samples for this technique and outlines two protocols for the analysis of surface- and intracellular-localised HSPs. (Source: Springer protocols feed by Biochemistry) Springer protocols feed by Biochemistry news http://www.medworm.com/rss/comments.php?id=5203840 Sun, 11 Sep 2011 03:16:54 +0100 5203840 http://www.medworm.com/index.php?rid=5203839&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-295-3_12 The enzyme-linked immunosorbent assay (ELISA) is an immunological technique which is used to determine the presence or quantity of an antigen within a sample. ELISAs rely on the use of at least one antibody (Ab) specific for the antigen being measured. This antibody is covalently linked to an enzyme which is detected through the use of an enzymatic substrate, which can be colorimetric, fluorogenic, or chemiluminescent. The ELISA for Hsp72 described here is a typical indirect sandwich ELISA, which can be used for measuring Hsp72 from cellular/tissue extracts, tissue culture supernatant, and serum. Typically, a 96-well ELISA plate is coated with a specific antibody which captures Hsp72 from the sample, and another antibody specific for a different Hsp72 epitope is used to detect Hsp72. An en... Springer protocols feed by Biochemistry news http://www.medworm.com/rss/comments.php?id=5203839 Sun, 11 Sep 2011 03:16:54 +0100 5203839 http://www.medworm.com/index.php?rid=5203838&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-295-3_11 Stress-inducible heat-shock proteins (HSPs, like HSP70 and HSP27) are molecular chaperones that ­protect cells from stress damage by keeping cellular proteins in a folding competent state and preventing them from irreversible aggregation. HSP27 and HSP70 chaperone activities are useful indicators to test chemical products and physical stress impact on protein denaturation, to select HSP inhibitors, or to ­determine the implication of the chaperone function in other HSP activities, such as apoptosis. We have developed two simple and fast chaperone activity tests for HSP27 and HSP70 that we initially set up to test the effect of potential HSP inhibitors obtained after screening of chemical and small molecule libraries. These chaperone quantification tests are based on the capacity of... Springer protocols feed by Biochemistry news http://www.medworm.com/rss/comments.php?id=5203838 Sun, 11 Sep 2011 03:16:54 +0100 5203838 http://www.medworm.com/index.php?rid=5203837&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-295-3_10 Elevated heat shock protein 27 (Hsp27) expression has been found in a number of tumors, including breast, prostate, gastric, uterine, ovarian, head and neck, and tumor arising from the nervous system and urinary system, and determined to be a predictor of poor clinical outcome. Although the mechanism of action of Hsp27 has been well documented, there are currently no available inhibitors of Hsp27 in clinical trials. RNA interference (RNAi) has the potential to offer more specificity and flexibility than traditional drugs to silence gene expression. Not surprisingly, RNAi has become a major focus for biotechnology and pharmaceutical companies, which are now in the early stages of developing RNAi therapeutics, mostly based on short interfering RNA (siRNAs), to target viral infection, cancer,... Springer protocols feed by Biochemistry news http://www.medworm.com/rss/comments.php?id=5203837 Sun, 11 Sep 2011 03:16:54 +0100 5203837 http://www.medworm.com/index.php?rid=5179106&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-204-5_2 (Source: Springer protocols feed by Biochemistry) Springer protocols feed by Biochemistry news http://www.medworm.com/rss/comments.php?id=5179106 Tue, 09 Aug 2011 23:00:00 +0100 5179106 http://www.medworm.com/index.php?rid=5179105&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-204-5_1 (Source: Springer protocols feed by Biochemistry) Springer protocols feed by Biochemistry news http://www.medworm.com/rss/comments.php?id=5179105 Tue, 09 Aug 2011 23:00:00 +0100 5179105 http://www.medworm.com/index.php?rid=5168718&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-204-5_9 Vascular smooth muscle cell (VSMC) proliferation and migration represent key features in atherosclerosis and restenosis. The proprotein convertases (PCs) furin and PC5 are highly expressed in human atheroma and are putatively involved in vascular lesion formation via the activation of precursor proteins, essential for cell proliferation and migration. In vitro assays have identified these PCs to govern cell functions via endoproteolytic cleavage of key substrates, including pro-integrins and pro-matrix metalloproteinases. In vivo gene expression studies of furin/PC5 and their substrates demonstrate their coordinated regulation in animal models of vascular remodelling and in human atherosclerotic lesions. Here we describe in vitro and in vivo models to investigate the function of furin/PC5 ... Springer protocols feed by Biochemistry news http://www.medworm.com/rss/comments.php?id=5168718 Tue, 09 Aug 2011 23:00:00 +0100 5168718 http://www.medworm.com/index.php?rid=5168717&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-204-5_14 Proprotein convertases (PCs) are secretory proteolytic enzymes that activate precursor proteins into biologically active forms by limited proteolysis at one or multiple internal sites. PCs are implicated in the processing of multiple protein precursors, including hormones, proteases, growth factors, angiogenic factors, and receptors. PCs have been linked recently to various pathologies such as Alzheimer’s disease, tumorigenesis, and infections. The zebrafish has emerged as an attractive model for studying the role of PCs not only in substrate production but also in development. Herein we describe methods that are used to characterize DNA sequences of PCs in zebrafish, as well as to evaluate the ontogeny and tissue distribution of their transcripts. We also provide information on the ... Springer protocols feed by Biochemistry news http://www.medworm.com/rss/comments.php?id=5168717 Tue, 09 Aug 2011 23:00:00 +0100 5168717 http://www.medworm.com/index.php?rid=5168716&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-204-5_15 The Zebrafish (Danio rerio) is a powerful and well-established tool used extensively for the study of early vertebrate development and as a model of human diseases. Zebrafish genes orthologous to their mammalian counterparts generally share conserved biological function. Protein knockdown or overexpression can be effectively achieved by microinjection of morpholino antisense oligonucleotides (MOs) or mRNA, respectively, into developing embryos at the one- to two-cell stage. Correlating gene expression patterns with the characterizing of phenotypes resulting from over- or underexpression can reveal the function of a particular protein. The microinjection technique is simple and results are reproducible. We defined the expression pattern of the proprotein convertase PCSK5 within the lateral ... Springer protocols feed by Biochemistry news http://www.medworm.com/rss/comments.php?id=5168716 Tue, 09 Aug 2011 23:00:00 +0100 5168716 http://www.medworm.com/index.php?rid=5168715&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-204-5_16 With the development of mice in which individual proteolytic enzymes have been inactivated, it has been of great interest to see how loss of these enzymes alters the processing of neuropeptides. In the course of studying changes in the peptide cholecystokinin (CCK) and other neuropeptides in several of these knockout mice, it has become clear that neuropeptide processing is complex and regionally specific. The enzyme responsible for processing in one part of the brain may not be involved in other parts of the brain. It is essential to do a detailed dissection of the brain and analyze peptide levels in many brain regions to fully understand the role of the enzymes. Because loss of these proteases may trigger compensatory mechanisms which involve expression of the neuropeptides being studied... Springer protocols feed by Biochemistry news http://www.medworm.com/rss/comments.php?id=5168715 Tue, 09 Aug 2011 23:00:00 +0100 5168715 http://www.medworm.com/index.php?rid=5168714&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-204-5_18 Proprotein convertases (PCs) convert pro-proteins into their bioactive forms through limited proteolytic cleavage, thereby regulating the temporal and spatial activation of a large number of functionally important proteins. This “converting” process is involved in a wide range of essential physiological and pathological processes, making PCs valuable therapeutic targets. One of the challenges in the field of PC research has been to identify the physiological substrates of a particular PC in a specific tissue or cellular process. Proteomics provides an unprecedented opportunity to identify novel PC substrates in a physiological context. Here we provide a detailed practical procedure utilizing two-dimensional fluorescent differential gel electrophoresis (2D-DiGE) and tandem mass ... Springer protocols feed by Biochemistry news http://www.medworm.com/rss/comments.php?id=5168714 Tue, 09 Aug 2011 23:00:00 +0100 5168714 http://www.medworm.com/index.php?rid=5168713&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-204-5_17 We describe the method for quantitative peptidomics using isotopic labels based on trimethylammonium butyrate, which can be synthesized in five different isotopic forms; this permits multivariate analysis of five different groups of tissue extracts in a single liquid chromatography/mass spectrometry run. (Source: Springer protocols feed by Biochemistry) Springer protocols feed by Biochemistry news http://www.medworm.com/rss/comments.php?id=5168713 Tue, 09 Aug 2011 23:00:00 +0100 5168713 http://www.medworm.com/index.php?rid=5168712&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-204-5_19 Sociability in mice is a multidimensional adaptive and functional response. Due to its complexity, it is important that researchers use well-defined behavioral assays that are easily replicated with clearly defined ethograms. In the Mouse Behavioral and Neuroendocrine Analysis Core Facility at Duke University, we have developed a broad series of tests that examine different components of neonatal and adult social behaviors that include sociability, sexual behavior, aggressive and territorial responses, and maternal behaviors. While the purpose of this chapter is not to provide an exhaustive description of all mouse social tests available, we provide investigators with a description of basic procedures and considerations necessary to develop a successful social behavior testing program with... Springer protocols feed by Biochemistry news http://www.medworm.com/rss/comments.php?id=5168712 Tue, 09 Aug 2011 23:00:00 +0100 5168712 http://www.medworm.com/index.php?rid=5094097&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-261-8_9 Myosins are mechano-enzymes that convert the chemical energy of ATP hydrolysis into mechanical work. They are involved in diverse biological functions including muscle contraction, cell migration, cell division, hearing, and vision. All myosins have an N-terminal globular domain, or “head” that binds actin, hydrolyses ATP, and produces force and movement. The C-terminal “tail” region is highly divergent amongst myosin types, and this part of the molecule is responsible for determining the cellular role of each myosin. Many myosins bind to cell membranes. Their membrane-binding domains vary, specifying which lipid each myosin binds to. To directly observe the movement and localisation of individual myosins within the living cell, we have developed methods to visualis... Springer protocols feed by Biochemistry news http://www.medworm.com/rss/comments.php?id=5094097 Thu, 04 Aug 2011 16:20:16 +0100 5094097 http://www.medworm.com/index.php?rid=5094096&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-261-8_8 Recent advances in single-molecule labeling and detection techniques allow high-resolution imaging of the motion of single molecules. Molecular motors are biological machines that convert chemical energy into mechanical work. Myosin Va (MyoVa) is a well-characterized processive molecular motor, essential for cargo transport in living organisms. Quantum dots (Qdots) are fluorescent semiconductor nanocrystals that are extremely useful for single-molecule studies in biological sciences. High-resolution video microscopy and single-particle tracking of a Qdot-labeled MyoVa motor molecule allow the detection of individual steps in vitro and in live cells. (Source: Springer protocols feed by Biochemistry) Springer protocols feed by Biochemistry news http://www.medworm.com/rss/comments.php?id=5094096 Thu, 04 Aug 2011 16:20:11 +0100 5094096 http://www.medworm.com/index.php?rid=5094095&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-261-8_7 Optical tweezers offer the capability to directly observe nanometre displacements and apply piconewton forces to single proteins. This method has been applied to the study of many different biological systems. Optical tweezers have proven to be particularly useful in studying the fine details of the mechanisms of molecular motor proteins, and how their movement is coordinated with ATPase activity. This includes actin, microtubule, and also DNA- and RNA-based motor systems. Here, we provide the information necessary to reproduce the “three-bead geometry” widely applied to the study of actomyosin interactions, the “paradigm system” for motors that only interact intermittently with their filament substrate, and discuss how single-molecule interactions can be detected, ... Springer protocols feed by Biochemistry news http://www.medworm.com/rss/comments.php?id=5094095 Thu, 04 Aug 2011 16:20:03 +0100 5094095 http://www.medworm.com/index.php?rid=5094094&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-261-8_6 Atomic Force Microscopy (AFM) has gained increasing popularity over the years among biophysicists due to its ability to image and to measure pN to nN forces on biologically relevant scales (nm to μm). Continuous technical developments have made AFM capable of nondisruptive, subsecond imaging of fragile biological samples in a liquid environment, making this method a potent alternative to light microscopy. In this chapter, we discuss the basics of AFM, its theoretical limitations, and we describe how this technique can be used to get single protein resolution in liquids at room temperature. Provided imaging is done at low-enough forces to avoid sample disruption and conformational changes, AFM allows obtaining unique insights into enzyme dynamics. (Source: Springer protocols feed by Bioc... Springer protocols feed by Biochemistry news http://www.medworm.com/rss/comments.php?id=5094094 Thu, 04 Aug 2011 16:19:55 +0100 5094094 http://www.medworm.com/index.php?rid=5094093&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-261-8_5 Kinesin motors couple ATP hydrolysis to movement along microtubules, which act both as tracks and as activators of kinesin ATPase activity. Cryo-electron microscopy and image processing enables generation of three-dimensional snapshots of kinesin motors on their tracks at different stages of their ATPase cycle, and can reveal their motor mechanisms at secondary structure resolution. Here, we describe in detail the methods and conditions employed in our lab to prepare high-quality frozen-hydrated samples, which yield structural insights into kinesin motor mechanisms. (Source: Springer protocols feed by Biochemistry) Springer protocols feed by Biochemistry news http://www.medworm.com/rss/comments.php?id=5094093 Thu, 04 Aug 2011 16:19:51 +0100 5094093 http://www.medworm.com/index.php?rid=5094092&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-261-8_4 Traditional microscopy techniques are limited by the wave-like characteristics of light, which dictate that about 250 nm (or roughly half the wavelength of the light) is the smallest distance by which two identical objects can be separated while still being able to distinguish between them. Since most biological molecules are much smaller than this limit, traditional light microscopes are generally not sufficient for single-molecule biological studies. Fluorescence Imaging with One Nanometer Accuracy (FIONA) is a technique that makes possible localization of an object to approximately one nanometer. The FIONA technique is simple in concept; it is built upon the idea that, if enough photons are collected, one can find the exact center of a fluorophore’s emission to within a single nan... Springer protocols feed by Biochemistry news http://www.medworm.com/rss/comments.php?id=5094092 Thu, 04 Aug 2011 16:19:31 +0100 5094092 http://www.medworm.com/index.php?rid=5094091&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-261-8_3 Kinesin is an essential eukaryotic protein that drives intracellular transport of cargo, such as vesicles and organelles. It is the smallest motor protein known that converts free energy obtained from ATP hydrolysis into mechanical work, by stepping along microtubules. The enzymatic cycle of kinesin is tightly coupled to mechanical action. How kinesin’s two identical motor domains (that both bind and hydrolyze ATP and bind to a microtubule) bring about motility has been the subject of much research. Recently, we have developed and applied a single-motor motility assay based on confocal fluorescence microscopy to measure changes in distance and orientation of the two motor domains during processive walking using Förster resonance energy transfer. The key benefit of this approach ... Springer protocols feed by Biochemistry news http://www.medworm.com/rss/comments.php?id=5094091 Thu, 04 Aug 2011 16:19:16 +0100 5094091 http://www.medworm.com/index.php?rid=5094090&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-261-8_2 Molecular motors perform work in cells by moving in an ATP-dependent manner along filamentous tracks. In vitro, the mechanical action of such motor proteins can be investigated by attaching the molecules to surfaces in the so-called gliding or bead assays. Surface attachment protocols have to be used that do not interfere with the function of the molecule. Here, we describe a sandwich protocol that preserves functionality. The protocol can be used for a large variety of proteins, in particular kinesin motor proteins that are GFP-tagged. (Source: Springer protocols feed by Biochemistry) Springer protocols feed by Biochemistry news http://www.medworm.com/rss/comments.php?id=5094090 Thu, 04 Aug 2011 16:19:02 +0100 5094090 http://www.medworm.com/index.php?rid=5094089&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-261-8_1 Single-molecule enzymology has a longer history than is often supposed, with the first measurements being made as early as 1961. However, the development of new technologies has meant that most of the progress has been made in the last two decades. I review the development of single-molecule enzymology, focussing on five key papers which are milestones in the field. In particular, I discuss the 1961 paper by Boris Rotman, which made inventive use of what now seems like primitive technology, and continues to be influential to this day. (Source: Springer protocols feed by Biochemistry) Springer protocols feed by Biochemistry news http://www.medworm.com/rss/comments.php?id=5094089 Thu, 04 Aug 2011 16:18:45 +0100 5094089 http://www.medworm.com/index.php?rid=5094088&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-261-8_18 Single-molecule measurements of rotation catalyzed by the F1-ATPase or the FoF1 ATP synthase have provided new insights into the molecular mechanisms of the F1 and Fo molecular motors. We recently developed a method to record ATPase-driven rotation of F1 or FoF1 in a manner that solves several technical limitations of earlier approaches that were significantly hampered by time and angular resolution, and restricted the duration of data collection. With our approach it is possible to collect data for hours and obtain statistically significant quantities of data on each molecule examined with a time resolution of up to 5 μs at unprecedented signal-to-noise. (Source: Springer protocols feed by Biochemistry) Springer protocols feed by Biochemistry news http://www.medworm.com/rss/comments.php?id=5094088 Thu, 04 Aug 2011 16:18:30 +0100 5094088 http://www.medworm.com/index.php?rid=5094087&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-261-8_17 F1-ATPase is the smallest rotary molecular motor ever found. Unidirectional rotation of the γ-shaft is driven by precisely coordinated sequential ATP hydrolysis reactions in three catalytic sites arranged 120° apart in the cylinder. Single-molecule observation allows us to directly watch the rotation of the shaft using micron-sized plastic beads. Additionally, an advanced version of “total internal reflection fluorescence microscope (TIRFM)” enables us to detect binding and release of energy currency through fluorescently labeled ATP. In this chapter, we describe how to set up the system for simultaneous observation of these two critical events. This specialized optical setup is applicable to a variety of research, not only molecular motors but also other single-molec... Springer protocols feed by Biochemistry news http://www.medworm.com/rss/comments.php?id=5094087 Thu, 04 Aug 2011 16:17:59 +0100 5094087 http://www.medworm.com/index.php?rid=5094086&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-261-8_16 Eukaryotic chromosomes are highly packed into chromatin, the basic unit of which is the nucleosome. The presence of nucleosomes and the resulting organization of the genome into higher-order chromatin structures has profound consequences for virtually all aspects of DNA metabolism, including DNA transcription, repair, and chromosome segregation. We have developed novel approaches for nanofabricating “DNA curtains” for high-throughput single-molecule imaging, and we have begun adapting these new research tools in an effort to begin studying chromatin biology at the single-molecule level. In this protocol, we describe procedures for assembly and real-time single-molecule visualization of DNA curtains bound by reconstituted nucleosomes made from recombinant histones. (Source: Spri... Springer protocols feed by Biochemistry news http://www.medworm.com/rss/comments.php?id=5094086 Thu, 04 Aug 2011 16:17:33 +0100 5094086 http://www.medworm.com/index.php?rid=5094085&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-261-8_15 Magnetic tweezers provide a versatile tool enabling the precise application of force and torque on ­individual biomolecules. These properties make magnetic tweezers uniquely suited for the study of DNA topology and topoisomerases at the single-molecule level. Single-molecule approaches, which are complementary to ensemble biochemical and structural approaches, have provided remarkable insights into the mechanisms of topoisomerase activity and interactions with DNA. Here, we describe how to make single-molecule measurements of topoisomerase activity with a magnetic tweezers instrument. We provide detailed instructions for preparing and characterizing DNA substrates, flow cells, and supercoilable DNA tethers. We then describe magnetic tweezers measurements of supercoil relaxation by sing... Springer protocols feed by Biochemistry news http://www.medworm.com/rss/comments.php?id=5094085 Thu, 04 Aug 2011 16:16:57 +0100 5094085 http://www.medworm.com/index.php?rid=5094084&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-261-8_14 The dynamics of full protein synthesis and the co-translational folding processes are not fully understood. We have developed a novel method, using a combination of ribosome display and single-molecule techniques, for monitoring the synthesis, co-translational folding, and maturation of a complete polypeptide chain at the single-molecule level. This method enabled us to observe the appearance of green fluorescent protein fluorescence after de novo synthesis of the complete protein. Here, we provide the information necessary to reproduce this method, which will be valuable in revealing the dynamics of the co-translational folding and maturation of nascent polypeptides. (Source: Springer protocols feed by Biochemistry) Springer protocols feed by Biochemistry news http://www.medworm.com/rss/comments.php?id=5094084 Thu, 04 Aug 2011 16:16:33 +0100 5094084 http://www.medworm.com/index.php?rid=5094083&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-261-8_13 Almost all aspects of DNA metabolism involve separation of double-stranded DNA catalyzed by helicases. Observation and measurement of the dynamics of these events at the single-molecule level provide important mechanistic details of helicase activity and give the opportunity to probe aspects that are not revealed in bulk solution measurements. The assay, presented here, provides information about helicase unwinding rates and processivity. Visualization is achieved by using a fluorescent single-stranded DNA-binding protein (SSB), which allows the time course of individual DNA unwinding events to be observed using total internal reflection fluorescence microscopy. Observation of a prototypical helicase, Bacillus subtilis AddAB, shows that the unwinding process consists of bursts of unwinding... Springer protocols feed by Biochemistry news http://www.medworm.com/rss/comments.php?id=5094083 Thu, 04 Aug 2011 16:16:01 +0100 5094083 http://www.medworm.com/index.php?rid=5094082&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-261-8_12 RNA polymerase (RNAP) is a DNA-dependent motor protein that links ribonucleotide polymerization to force generation and DNA translocation through its active site, i.e., mechanical work. Single-molecule studies using optical tweezers have allowed researchers to probe the load-dependent ribonucleotide incorporation rate and processivity of both single-subunit viral and multisubunit prokaryotic and eukaryotic RNAPs engaged in transcription elongation. A single-molecule method is described here, which allows the complete transcription cycle (i.e., promoter binding, initiation, elongation and termination) to be followed in real-time using dual-trap optical tweezers and a unique “three-bead” geometry. This single-molecule transcription assay can be used to probe the mechanics of both... Springer protocols feed by Biochemistry news http://www.medworm.com/rss/comments.php?id=5094082 Thu, 04 Aug 2011 16:15:15 +0100 5094082 http://www.medworm.com/index.php?rid=5094081&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-261-8_11 The interconversion of nucleoside triphosphate (NTP) and diphosphate occurs in some of the most ­important cellular reactions. It is catalyzed by diverse classes of enzymes, such as nucleoside triphosphatases, kinases, and ATP synthases. Triphosphatases include helicases, myosins, and G-proteins, as well as many other energy-transducing enzymes. The transfer of phosphate by kinases is involved in many metabolic pathways and in control of enzyme activity through protein phosphorylation. To understand the processes catalyzed by these enzymes, it is important to measure the kinetics of individual elementary steps and conformation changes. Fluorescent nucleotides can directly report on the binding and release steps, and conformational changes associated with these processes. In single-mole... Springer protocols feed by Biochemistry news http://www.medworm.com/rss/comments.php?id=5094081 Thu, 04 Aug 2011 16:14:25 +0100 5094081 http://www.medworm.com/index.php?rid=5094080&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-261-8_10 Myosin is both an enzyme and a molecular motor that hydrolyzes ATP and interacts with actin filaments for force generation. Manipulation techniques with microneedles and laser traps have recently been developed to capture and manipulate the actomyosin interaction for the purpose of revealing the mechanics of this system. Combined with single-molecule imaging techniques, the coupling between chemical processes (ATP hydrolysis) and mechanical processes (myosin force generation) has been directly determined. In this chapter, we describe these two manipulation techniques, especially microneedle method, in detail. (Source: Springer protocols feed by Biochemistry) Springer protocols feed by Biochemistry news http://www.medworm.com/rss/comments.php?id=5094080 Thu, 04 Aug 2011 16:14:06 +0100 5094080 http://www.medworm.com/index.php?rid=5137654&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-194-9_7 Nitrogenase is one of the most complex enzymes known to date. The extensively studied molybdenum nitrogenase consists of two protein components and three metal centers that are critical for nitrogenase activity. The inherent complexity of this enzyme system, which is further compounded by the sensitivity of the metal clusters toward oxygen, makes the large-scale purification of fully active nitrogenase proteins a formidable task. This chapter highlights several methods that have been developed for the purification of nitrogenase proteins over the past few decades. Techniques used include weak anion exchange chromatography, size exclusion chromatography, and immobilized metal affinity chromatography. These methods can be selectively applied to nitrogenase variants and other related proteins... Springer protocols feed by Biochemistry news http://www.medworm.com/rss/comments.php?id=5137654 Tue, 02 Aug 2011 23:00:00 +0100 5137654 http://www.medworm.com/index.php?rid=5137653&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-194-9_6 Biological nitrogen fixation is a complex and tightly regulated process limited to a group of prokaryotic species known as diazotrophs. Among well-studied diazotrophs, Azotobacter vinelandii is the best studied for its convenience of aerobic growth, its high levels of nitrogenase expression, and its genetic tractability. This chapter includes protocols and strategies in the molecular biology and genetic engineering of A. vinelandii that have been used as valuable tools for advancing studies on the biosynthesis, mechanism, and regulation of nitrogen fixation. (Source: Springer protocols feed by Biochemistry) Springer protocols feed by Biochemistry news http://www.medworm.com/rss/comments.php?id=5137653 Tue, 02 Aug 2011 23:00:00 +0100 5137653 http://www.medworm.com/index.php?rid=5137652&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-194-9_5 Nitrogenase-like dark operative protochlorophyllide oxidoreductase (DPOR) is involved in the two-electron reduction of protochlorophyllide to form chlorophyllide during chlorophyll biosynthesis. Formation of bacteriochlorophyll additionally requires a structurally related enzyme system which is termed chlorophyllide oxidoreductase (COR). During DPOR catalysis, the homodimeric subunit ChlL2 transfers electrons to the corresponding heterotetrameric catalytic subunit (ChlN/ChlB)2. Analogously, subunit BchX2 of the COR enzymes delivers electrons to subunit (BchY/BchZ)2. The ChlL2 protein is a dynamic switch protein triggering the ATP-dependent transfer of electrons via a [4Fe–4S] cluster onto a second [4Fe–4S] cluster located on subunit (ChlN/ChlB)2. This initial electron transfer ... Springer protocols feed by Biochemistry news http://www.medworm.com/rss/comments.php?id=5137652 Tue, 02 Aug 2011 23:00:00 +0100 5137652 http://www.medworm.com/index.php?rid=5137651&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-194-9_4 Advances in sequencing technology in the past decade have enabled the sequencing of genomes of thousands of organisms including diazotrophs. Genomics have enabled thorough analysis of the gene organization of nitrogen-fixing species, the identification of new genes involved in nitrogen fixation, and the identification of new diazotrophic species. This chapter reviews key characteristics of nitrogen-fixing genomes and methods to identify and analyze genomes of new diazotrophs using genome scanning. This chapter refers to Azotobacter vinelandii, a well-studied nitrogen-fixing organism, as a model for studying nitrogen-fixing genomes. We discuss the main nitrogen fixation genes as well as accessory genes that contribute to diazotrophy. We also review approaches that can be used to modify geno... Springer protocols feed by Biochemistry news http://www.medworm.com/rss/comments.php?id=5137651 Tue, 02 Aug 2011 23:00:00 +0100 5137651 http://www.medworm.com/index.php?rid=5137650&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-194-9_3 Biosynthesis of MoFe protein and, particularly, that of its associated P-cluster and FeMoco has raised a significant amount of interest because of the biological importance and chemical exclusiveness of these unique clusters. Following a brief introduction to the properties of Azotobacter vinelandii MoFe protein, this chapter will focus on the recent progress toward understanding the assembly mechanism of MoFe protein, with an emphasis on studies that provide important structural or spectroscopic insights into this process. (Source: Springer protocols feed by Biochemistry) Springer protocols feed by Biochemistry news http://www.medworm.com/rss/comments.php?id=5137650 Tue, 02 Aug 2011 23:00:00 +0100 5137650 http://www.medworm.com/index.php?rid=5137649&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-194-9_2 Nitrogenase is the enzyme responsible for biological reduction of dinitrogen (N2) to ammonia, a form usable for life. Playing a central role in the global biogeochemical nitrogen cycle, this enzyme has been the focus of intensive research for over 60 years. This chapter provides an overview of the features of nitrogenase as a background to the subsequent chapters of this volume that detail the many methods that have been applied in an attempt to gain a deeper understanding of this complex enzyme. (Source: Springer protocols feed by Biochemistry) Springer protocols feed by Biochemistry news http://www.medworm.com/rss/comments.php?id=5137649 Tue, 02 Aug 2011 23:00:00 +0100 5137649 http://www.medworm.com/index.php?rid=5137648&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-194-9_1 The history of nitrogenase research dates all the way back to the 1800s. This chapter provides a brief account of the advances in this particular research area over the past few hundred years, which include such events as the initial discovery of biological nitrogen fixation, the preparation of active cell-free extracts, the purification of nitrogenase enzyme, the proposal of the Thorneley–Lowe model, and the report of x-ray crystallographic structures of the component proteins of nitrogenase. (Source: Springer protocols feed by Biochemistry) Springer protocols feed by Biochemistry news http://www.medworm.com/rss/comments.php?id=5137648 Tue, 02 Aug 2011 23:00:00 +0100 5137648 http://www.medworm.com/index.php?rid=5017606&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-188-8_9 Aptamers developed for applications in cancer therapy can improve the efficacy of drug treatment and enhance molecular imaging. Aptamers for these purposes are generated from SELEX (Systematic Evolution of Ligands by EXponential enrichment), more precisely cell-based SELEX, a process described in detail in this chapter. Experimental applications are also provided for aptamer-based drugs. (Source: Springer protocols feed by Biochemistry) Springer protocols feed by Biochemistry news http://www.medworm.com/rss/comments.php?id=5017606 Tue, 12 Jul 2011 19:59:32 +0100 5017606 http://www.medworm.com/index.php?rid=5017605&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-188-8_8 It is very clear that RNA interference (RNAi) is a potent and versatile tool for gene silencing. One of the hurdles to making siRNA/miRNA a human therapeutic includes effective in vivo delivery and being able to deliver drugs to target cells only. The commercial success of in vivo applications of RNAi hinges on the development of new delivery methods. Our strategy involves the use of antibody-based delivery agents to target and deliver siRNA into specific cell types. We have developed antibody-based agents for directed delivery into cultured cells and animal disease models. Using antibodies against various cell surface receptors, modified siRNAs are attached to antibody complexes using RNA carrier proteins. The complex can then be intravenously administered to in vivo models and taken up b... Springer protocols feed by Biochemistry news http://www.medworm.com/rss/comments.php?id=5017605 Tue, 12 Jul 2011 19:59:32 +0100 5017605 http://www.medworm.com/index.php?rid=5017604&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-188-8_7 A major barrier within the field of non-viral gene therapy toward therapeutic strategies, e.g., tumor therapy, has been lack of appropriate specific delivery strategies to the intended target tissues or cells. In this chapter, we describe a protocol for light-directed delivery of nucleic acids through the use of photochemical internalization (PCI) technology. PCI is based on a photosensitizing compound that localizes to endocytic membranes. Upon illumination, the photosensitizing compound induces damage to the endocytic membranes, resulting in release of endocytosed material, i.e., nucleic acids into cytosol. The main benefit of the strategy described is the possibility for site-specific delivery of nucleic acids to a place of interest. (Source: Springer protocols feed by Biochemistry) Springer protocols feed by Biochemistry news http://www.medworm.com/rss/comments.php?id=5017604 Tue, 12 Jul 2011 19:59:32 +0100 5017604 http://www.medworm.com/index.php?rid=5017603&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-188-8_6 The tissue-specific delivery nanoparticle consists of an antisense oligomer, a cell-penetrating peptide, and an antitumor antibody, each biotinylated and each linked via streptavidin. Within the nanoparticle, the antibody provides specific targeted delivery and binding to the target cells, the peptide improves cell membrane transport, and the antisense oligomer, through its mRNA-binding ability, provides specific retention of the radioactivity in the target cell nucleus. The use of streptavidin as linker eliminates the need for covalent conjugation without appearing to interfere with the in vitro and in vivo properties of each component. The delivery nanoparticle is under development to improve tumor targeting with unlabeled siRNAs as well as radiolabeled antisense oligomers in a variety o... Springer protocols feed by Biochemistry news http://www.medworm.com/rss/comments.php?id=5017603 Tue, 12 Jul 2011 19:59:32 +0100 5017603 http://www.medworm.com/index.php?rid=5017602&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-188-8_5 We describe here new CPPs allowing the delivery of splice redirecting steric-block ON using either chemical conjugation or non-covalent complexation. We also describe a convenient and robust splice redirecting assay which allows the quantitative assessment of ON nuclear delivery. (Source: Springer protocols feed by Biochemistry) Springer protocols feed by Biochemistry news http://www.medworm.com/rss/comments.php?id=5017602 Tue, 12 Jul 2011 19:59:32 +0100 5017602 http://www.medworm.com/index.php?rid=5017601&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-188-8_4 The dramatic acceleration in identification of new nucleic acid-based therapeutic molecules such as short interfering RNA (siRNA) and peptide–nucleic acid (PNA) analogues has provided new perspectives for therapeutic targeting of specific genes responsible for pathological disorders. However, the poor cellular uptake of nucleic acids together with the low permeability of the cell membrane to negatively charged molecules remain major obstacles to their clinical development. Several non-viral strategies have been proposed to improve the delivery of synthetic short oligonucleotides both in cultured cells and in vivo. Cell-penetrating peptides constitute very promising tools for non-invasive cellular import of oligonucleotides and analogs. We recently described a non-covalent strategy ba... Springer protocols feed by Biochemistry news http://www.medworm.com/rss/comments.php?id=5017601 Tue, 12 Jul 2011 19:59:31 +0100 5017601 http://www.medworm.com/index.php?rid=5017600&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-188-8_3 The synthesis of 2′-O,4′-C-methyleneoxymethylene bridged nucleoside (2′,4′-BNACOC) phosphoramidites and oligonucleotides containing 2′,4′-BNACOC are described. 2′,4′-BNACOC phosphoramidites bearing natural nucleobases, such as thymine, cytosine, 5-methylcytosine, adenine, and guanine were synthesized. Moreover, fully or partially 2′,4′-BNACOC-modified oligonucleotides can be prepared by using a standard protocol except for a prolonged coupling time on an automated DNA synthesizer. (Source: Springer protocols feed by Biochemistry) Springer protocols feed by Biochemistry news http://www.medworm.com/rss/comments.php?id=5017600 Tue, 12 Jul 2011 19:59:31 +0100 5017600 http://www.medworm.com/index.php?rid=5017599&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-188-8_2 The 3′-S-phosphorothiolate (3′-SP) linkage has proven to be a very useful analogue of the phosphodiester group in nucleic acid derivatives; it is achiral and also shows good resistance to nucleases. Whilst oligonucleotides containing a 3′-SP linkage are best prepared using phosphoramidite chemistry, the corresponding dinucleotides are most efficiently synthesised using a Michaelis–Arbuzov reaction between a nucleoside 5′-phosphite and a nucleoside 3′-S-disulphide. The method described here is for a thymidine dinucleotide and is based on the use of a silyl phosphite, which is more reactive than simple alkyl phosphites and also simplifies the deprotection strategy. Full experimental details and spectroscopic data for the synthetic intermediates and the tar... Springer protocols feed by Biochemistry news http://www.medworm.com/rss/comments.php?id=5017599 Tue, 12 Jul 2011 19:59:31 +0100 5017599 http://www.medworm.com/index.php?rid=5017598&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-188-8_21 Deoxyribozymes (DXZs) are catalytic oligodeoxynucleotides capable of performing diverse functions including the specific cleavage of a target RNA. These molecules represent a new type of therapeutic oligonucleotides combining the efficiency of ribozymes and the intracellular endurance and simplicity of modified antisense oligonucleotides. Commonly used DXZs include the 8–17 and 10–23 motifs, which have been engineered to destroy disease-associated genes with remarkable efficiency. Targeting DXZs to disease-associated transcripts requires extensive biochemical testing to establish target RNA accessibility, catalytic efficiency, and nuclease sensibility. The usage of modified nucleotides to render nuclease-resistance DXZs must be counterweighted against deleterious consequences o... Springer protocols feed by Biochemistry news http://www.medworm.com/rss/comments.php?id=5017598 Tue, 12 Jul 2011 19:59:31 +0100 5017598 http://www.medworm.com/index.php?rid=5017597&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-188-8_20 Target validation for small RNAs in cells can be a confusing task wrought with pitfalls and false leads. One technique for validating in vivo targets of small RNAs is immunoprecipitation of target RNAs using antibodies again the RNAi machinery. Antigene RNAs (agRNAs) regulate transcription in human cells using machinery from the RNAi regulatory pathway – namely argonaute proteins. Here we describe a technique for validating targets of agRNAs using RNA immunoprecipitation with antibodies against human argonaute proteins. This technique can be used to detect interactions of argonaute proteins in the cell nucleus with their targets, lowly expressed noncoding RNA transcripts. (Source: Springer protocols feed by Biochemistry) Springer protocols feed by Biochemistry news http://www.medworm.com/rss/comments.php?id=5017597 Tue, 12 Jul 2011 19:59:31 +0100 5017597 http://www.medworm.com/index.php?rid=5017596&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-188-8_1 A brief historical introduction describes early attempts to silence specific genes using the antisense oligonucleotides that flourished in the 1980s. Early aspirations for therapeutic applications were almost extinguished by the unexpected complexity of oligonucleotide pharmacology. Once the biochemistry and molecular biology behind some of the pharmacology was worked out, new approaches became apparent for using oligonucleotides to treat disease. The biochemistry of small nucleic acids is outlined in Section 2. Various approaches employing oligonucleotides to control cellular functions are reviewed in Section 3. These include antisense oligonucleotides and siRNA that bind to RNA, antigene oligonucleotides that bind to DNA, and aptamers, decoys, and CpG oligonucleotides that bind to protei... Springer protocols feed by Biochemistry news http://www.medworm.com/rss/comments.php?id=5017596 Tue, 12 Jul 2011 19:59:31 +0100 5017596 http://www.medworm.com/index.php?rid=5017595&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-188-8_19 Instruments based on the surface plasmon resonance (SPR) principle allow label-free detection of interactions between targets immobilized at a solid–liquid interface and partners in solution. This method is well suited to determine the kinetic parameters, the equilibrium constant and the stoichiometry of a reaction. Aptamers are ligands identified from random libraries of RNA, DNA or chemically modified oligonucleotides by in vitro selection (SELEX). Aptamers can be raised against a great variety of targets (small molecules, proteins, nucleic acids, cells, viruses, bacteria). SPR is routinely used in our laboratory for the analysis of RNA aptamer–RNA target complexes. To illustrate SPR investigation of such complexes, we describe here methods that were successfully used to moni... Springer protocols feed by Biochemistry news http://www.medworm.com/rss/comments.php?id=5017595 Tue, 12 Jul 2011 19:59:31 +0100 5017595 http://www.medworm.com/index.php?rid=5017594&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-188-8_18 Methods and protocols for automated synthesis and purification of immune modulatory oligonucleotides (IMOs), a novel class of Toll-like receptor 9 (TLR9) agonists, are described. IMOs containing two short identical sequences of 11-mers with phosphorothioate linkages can be synthesized in parallel synthetic strategy. A C3-linker that mimics the natural inter-nucleotide distance was commonly used for joining the two segments of IMOs. NittoPhase solid support bearing a symmetrical C3-linker (glycerol) and nucleoside-β-cyanoethyl-N,N-diisopropylphosphoramidites were used for IMO synthesis. The parallel synthesis was carried out in a 3′→ 5′ direction with removal of the final dimethoxytrityl (DMT) protecting group. After synthesis, the IMO was cleaved and deprotected by tr... Springer protocols feed by Biochemistry news http://www.medworm.com/rss/comments.php?id=5017594 Tue, 12 Jul 2011 19:59:31 +0100 5017594 http://www.medworm.com/index.php?rid=5017593&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-188-8_17 Viral single-stranded (ss) RNA is the natural ligand for TLR7 and TLR8. Synthetic ssRNA has been shown to act as a ligand for TLR7 and TLR8. We have previously reported a novel RNA structure, referred to as stabilized immune modulatory RNA (SIMRA), in which two short phosphorothioate oligoribonucleotides were linked through their 3′-ends via a linker. SIMRA compounds had greater stability in serum than unmodified ssRNA and induced immune responses via TLR7 and/or TLR8. SIMRA compounds were synthesized using phosphoramidite chemistry on controlled-pore glass solid support derivatized with a linker. After cleavage from the solid support and removal of protecting groups, SIMRA compounds were purified on an anion-exchange HPLC followed by desalting/dialysis, and lyophilization. SIMRA com... Springer protocols feed by Biochemistry news http://www.medworm.com/rss/comments.php?id=5017593 Tue, 12 Jul 2011 19:59:31 +0100 5017593 http://www.medworm.com/index.php?rid=5017592&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-188-8_16 We developed a simple, direct, and cost-effective approach to search for the most likely target genes of a known miRNA in vitro. We term this method “Labeled microRNA (miRNA) pull-down assay system,” or LAMP. Briefly, the pre-miRNA is labeled with digoxigenin (DIG), mixed with cell extracts, and immunoprecipitated by anti-DIG antiserum. We concluded that LAMP is an experimental approach for high-throughput identification of the target gene of known miRNAs from both Caenorhabditis elegans and zebrafish (Danio rerio), yielding fewer false-positive results than those produced by using only the bioinformatics approach. (Source: Springer protocols feed by Biochemistry) Springer protocols feed by Biochemistry news http://www.medworm.com/rss/comments.php?id=5017592 Tue, 12 Jul 2011 19:59:31 +0100 5017592 http://www.medworm.com/index.php?rid=5017591&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-188-8_15 Schistosomes are parasitic worms that infect over 200 million people and constitute an enormous public health problem worldwide. Molecular tools are being developed for use with these parasites in order to increase our understanding of their unique molecular and cell biology. Among the more promising methodologies is RNA interference (RNAi, or gene silencing), a mechanism by which gene-specific double-stranded RNA (dsRNA) triggers degradation of homologous mRNA transcripts. In this work we describe methods for applying RNAi to suppress gene expression in the intra-mammalian life stages of Schistosoma mansoni. These methods include isolating and culturing the parasites, preparing and delivering dsRNA targeting a specific gene and monitoring the outcome. Given the abundance of schistosome tr... Springer protocols feed by Biochemistry news http://www.medworm.com/rss/comments.php?id=5017591 Tue, 12 Jul 2011 19:59:31 +0100 5017591 http://www.medworm.com/index.php?rid=5017590&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-188-8_14 Cultured endothelial cells are renowned for being difficult to transfect, whether for the purpose of exogenous over-expression of plasmid DNA or for genetic knockdown via silencing RNA. Therefore, optimal conditions are absolutely necessary for achieving relatively high transfection efficiency coupled with low cellular toxicity in endothelial cells. This chapter will detail an optimized protocol that has been shown to knockdown gene expression using siRNA in primary cultures of human umbilical vein endothelial cells (HUVECs) – perhaps the most widely utilized endothelial cell line for vascular research. While developed for optimal siRNA transfection of HUVECs, aspects of this protocol can be empirically modified to yield efficient siRNA transfection in most other cell lines. (Source:... Springer protocols feed by Biochemistry news http://www.medworm.com/rss/comments.php?id=5017590 Tue, 12 Jul 2011 19:59:31 +0100 5017590 http://www.medworm.com/index.php?rid=5017589&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-188-8_13 RNA interference (RNAi) is a mechanism by which the introduction of small interfering RNAs (siRNAs) into cultured cells causes degradation of the complementary mRNA. Applications of RNAi include gene function analysis, pathway analysis, and target validation. While RNAi experiments have become common practice in research labs, multiple factors can influence the extent of siRNA-induced knockdown (and thus biological outcome). A properly designed and selected siRNA sequence, siRNA modification format, choice of transfection reagent/technique, optimized protocols of siRNA in vitro delivery, and an appropriate and optimized readout are all critical for ensuring a successful experiment. In this chapter, we describe a typical in vitro siRNA experiment with optimization of transfection conditions... Springer protocols feed by Biochemistry news http://www.medworm.com/rss/comments.php?id=5017589 Tue, 12 Jul 2011 19:59:31 +0100 5017589 http://www.medworm.com/index.php?rid=5017588&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-188-8_12 RNA interference (RNAi) is a regulatory mechanism of eukaryotic cells that uses small interfering RNAs (siRNA) to direct homology-dependent control of gene activity. Applications of RNAi include functional genomics, in vivo target validation, and gene-specific medicines. A key to in vivo application of siRNA is the advancement of efficient delivery to organs, tissues, or cell types of interest. There is a need to develop reliable and easy-to-use assays to evaluate siRNA delivery efficiency and distribution, study pathways, and stability of siRNAs in cells (post-transfection) and in animals (post- injection). We have adopted the Applied Biosystems TaqMan® based stem–loop RT-PCR technology, originally developed for quantification of endogenous microRNAs in cells, to fulfill these n... Springer protocols feed by Biochemistry news http://www.medworm.com/rss/comments.php?id=5017588 Tue, 12 Jul 2011 19:59:31 +0100 5017588 http://www.medworm.com/index.php?rid=5017587&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-188-8_11 Large numbers of diverse small non-coding RNAs have been discovered and characterized in eukaryotic RNA interference pathways. These small RNAs have distinctive characteristics and are associated with Argonaute family proteins to regulate gene expression and genomes at various levels. These small RNAs include the Dicer-dependent group such as microRNAs (miRNAs) and small interfering RNAs (siRNAs), and the Dicer-independent group such as Piwi-interacting RNAs (piRNAs). This review summarizes the various classes of eukaryotic small RNAs and the general knowledge of their characteristics, biogenesis, and functions, with emphasis on some of the recently identified small RNAs. (Source: Springer protocols feed by Biochemistry) Springer protocols feed by Biochemistry news http://www.medworm.com/rss/comments.php?id=5017587 Tue, 12 Jul 2011 19:59:30 +0100 5017587 http://www.medworm.com/index.php?rid=5017586&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-188-8_10 We describe a five-step process for selecting the best candidate antisense compound for altering IL-12Rb2 expression including (1) detecting mRNA splice products by RT-PCR, (2) measuring protein expression, (3) evaluating protein function, (4) checking cellular viability, and (5) validating efficacy of the final candidate compound. The significance of targeting exons composed of a number of base pairs divisible by 3 is also discussed. The five steps described here for selecting the best candidate P-PMO to alter IL-12Rb2 expression should be applied for designing and screening antisense compounds for other gene targets. (Source: Springer protocols feed by Biochemistry) Springer protocols feed by Biochemistry news http://www.medworm.com/rss/comments.php?id=5017586 Tue, 12 Jul 2011 19:59:30 +0100 5017586 http://www.medworm.com/index.php?rid=4931905&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-151-2_25 Site-specific immobilization of proteins and peptides on a sensor surface represents a significant challenge for bioanalytical applications such as surface plasmon resonance (SPR). The most common protocols for covalent protein immobilization usually result in heterogeneous presentation of the ligand at the surface, which can in some instances yield conflicting results with analogous data obtained in solution. Here, we discuss two complementary and generic bioconjugation methods that allow chemoselective immobilization of peptides and proteins via either their C-terminus (native chemical ligation) or their N-terminus (oxime ligation). While the protocols described in this chapter were designed for use in a Biacore instrument, the methods should also be applicable to other SPR instruments a... Springer protocols feed by Biochemistry news http://www.medworm.com/rss/comments.php?id=4931905 Thu, 16 Jun 2011 19:38:54 +0100 4931905 http://www.medworm.com/index.php?rid=4931904&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-151-2_24 Over the last decade, scanning probe microscopy (SPM) techniques, such as atomic force microscopy (AFM), have played an important role in a variety of biophysical research efforts. This straightforward technique has the capability to measure forces down to a few hundred piconewtons, which enables the observation of unique events within or between single molecules. However, in order to successfully carry out these types of biophysical measurements, the anchoring of the biomolecules of interest to the scanning probe cantilever tip needs to be of sufficient strength to avoid rupture prior to the analysis of the specific interaction to be probed. Hence, a covalent linkage of the biomolecule to the SPM probe tip is generally preferred. It is also advantageous to have a long-chain functional lin... Springer protocols feed by Biochemistry news http://www.medworm.com/rss/comments.php?id=4931904 Thu, 16 Jun 2011 19:38:53 +0100 4931904 http://www.medworm.com/index.php?rid=4931903&cid=s_37117_60_f&fid=37117&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-151-2_23 The modification of biologicals such as proteins/peptides, small molecules, and other polymers with lipids provides an efficient method for mediating their insertion into liposomes and lipid-core micellar nanocarriers. In this chapter, we describe several representative protocols developed in our laboratory for the bioconjugation of liposomes and lipid-core micelles for drug/gene delivery and diagnostic imaging applications. (Source: Springer protocols feed by Biochemistry) Springer protocols feed by Biochemistry news http://www.medworm.com/rss/comments.php?id=4931903 Thu, 16 Jun 2011 19:38:53 +0100 4931903