Design of Biochemical Probes
Department of Applied Chemistry, Faculty of Science and Technology, Keio University, Yokohama 223-8522, Japan
The creation of original chemical sensors is one of the main concerns in our research group. In biological research fields, chemical sensors such as synthetic biochemical probes play an important role in the investigation of biological functions, and in cellular or in vivo imaging.
One of our recent research highlights was the creation of a magnesium-imaging probe (KMG series fluorescent molecules) for living cells.The KMG-series molecules for magnesium (Mg) imaging were designed and synthesized based on our prior knowledge of Mg ionophore design. While the first example (KMG-20) was based on a coumarin fluorophore, we later developed the fluorescein derivative KMG-104, which is the best magnesium fluorescent probe for imaging applications in the cytoplasm of living cells and also developed KCM-1 for simultaneous imaging of Mg and Ca ions in living cells. More recently, a flash-type Mg fluorescent probe was developed for specific protein labeling.
To obtain bright fluorescent probes, we have developed a set of fluorescent dyes (BODIPY-based KFL series dyes), which have excellent optical properties like sharp fluorescence spectra with high quantum yields, and moreover, the wavelength is finely tunable over a wide spectral range including the NIR region by introducing proper electron-donating groups into the furan moieties of the chromophore.
By linking an ion recognizing ionophore with a fluorescent dye as a transducer, a chemical probe (fluorescent probe) is obtained, transforming a simple molecular recognition ligand into a sensor ligand. For instance, a KFL fluorescent dye combined with a BAPTA chelating group resulted in a bright fluorescent probe for calcium ions. We also have developed a set of chemiluminescent (CL) dyes (BODIPY-based KCL and KBI series dyes) with excellent CL properties. The luciferin-based CL probe KBI is a useful probe for highly sensitive detection of ROS such as O2-?.
Several bioluminescent (BL) systems have been investigating based on synthetic coelenterazine (CTZ) derivatives as substrates in combination with Renilla luciferase (Rluc) variants or artificial luciferases (Aluc) as the enzyme.It was found that extending the conjugated system at the 6-carbon position of CTZ is more effective compared to extensions at the 2-, 5-, or 8-carbon positions. The 6-position carbon variants of CTZ combined with the known Rluc mutant Rluc8.6 resulted in the most intense bioluminescence in the blue spectral region. In addition, with the system consisting of a CTZ derivative and the artificial luciferase Aluc, we have succeeded in the development of the most high-intensity artificial bioluminescence system.
Flow Field-flow Fractionation for Proteomic & Lipidomic Analysis
Myeong Hee Moon
Dept. of Chemistry, Yonsei University, 50 Yonsei-ro, Seodaemun-gu,
Seoul, 120-749, Korea
Flow field-flow fractionation (FlFFF) is an elution-based separation method that is capable of size-sorting particles and biological macromolecules like proteins, DNA, cells, and etc, without relying on a partition or an interaction of sample components with the packing materials. Since separation in FlFFF takes place in an unobstructed channel with a biological buffer solution, a gentle but high speed separation of biological macromolecules can be achieved without shear induced deformation or degradation. Especially for the biological macromolecules, high performance separation/isolation methods are crucial to reduce the sample complexity even though sophisticated use of mass spectrometry (MS) is available.
This presentation will show recent advances of FlFFF for the size separation of subcellular species and lipoproteins as well as the subsequent characterization of collected fractions using nanoflow liquid chromatography (nLC) and MS. A high speed, semi-preparative scale, biocompatible size sorting of subcellular organelle species from homogenate mixtures of HEK 293T cells using FlFFF system will be introduced with confirmation of collected fractions containing nuclei, lysosomes, mitochondria, and peroxisomes using SEM, Western blot, and proteomic analysis with nLC-MS/MS. FlFFF can be powerfully utilized for lipidomic analysis of plasma lipoproteins by both bottom-up and top-down approaches. Lipoproteins in blood are globular complexes containing phospholipids (PLs), cholesterols, triacylglycerols (TGs), and etc. Among lipoproteins, low density lipoprotein (LDL) is known to be strongly related to coronary artery disease (CAD). In bottom-up approach, different lipoproteins can be size-sorted by FlFFF and lipids including oxidized PLs extracted from each collected lipoprotein were analyzed by nLC-MS/MS with structural determination. Ox-PLs are known to cause an accumulation of plaque at the interior wall of blood vessels, leading to a blockage of blood circulation. It was applied to plasma samples from CAD patients for the development of candidate marker lipids. The top-down approach was developed for high speed screening of targeted lipids by direct hyphenation of a miniaturized FlFFF channel with MS (mAF4-ESI-MS/MS) so that lipids in different lipoproteins can be directly quantified using selected reaction monitoring (SRM) without extraction. This presentation demonstrates both a comprehensive analysis of lipids in lipoproteins by off-line combination of FlFFF and nLC-MS/MS, and a high speed on-line analytical method that can be applied for targeted lipid content in aqueous solution without the need for organic solvent extraction of lipids.
In cells and in vitro studies of ubiquitin, polyubiquitin chains and proteins attached to them.
Kyoto University, Kyoto, Japan
In-cell NMR is an isotope-aided multi-dimensional NMR technique that enables observations of conformations, interactions and dynamics of proteins in living cells at the atomic level. We have established a method to measure high-resolution two-dimensional heteronuclear NMR spectra of proteins inside living human cells1. The in-cell NMR spectra of proteins demonstrate the possibility for broad application of this technique to studying interactions and protein processing. The in-cell NMR spectra of FKBP12 show the formation of specific complexes between the protein and extracellularly administered immunosuppressants, showing the potent utility of this technique in drug screening programs. Moreover, an in-cell NMR-related method revealed that ubiquitin has much higher hydrogen exchange rates in the intracellular environment1. After this observation, we analyzed the folding stability of ubiquitin chains and found that they decrease with increasing chain length, resulting in the formation of amyloid-like fibrils2. We also found that polyubiquitin chains covalently linked to EGFP also form aggregates depending on chain length, when they are expressed in cells2. The folding stabilities of (poly-)ubiquitin-attached proteins in vitro will be discussed also3.
Monitroing Neural Events with Carbon Fiber Microelectrodes
R. Mark Wightman
Department of Chemistry and Neuroscience Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-3290 USA.
Neurons within the intact brain secrete chemical substances to communicate with neighboring cells.
These substances, termed neurotransmitters, comprise an important way in which information is relayed and processed during behavior. However, until recently, this chemical communication had not been characterized because chemical sensors suitable to monitor subsecond chemical events in micron dimensions were unavailable. We have employed cyclic voltammetry at carbon-fiber microelectrodes to examine the dynamics of neurotransmitter concentrations within the brain of rats.1 Measurements with subsecond time resolution within the intact brain give a real time view of neurotransmitters during goal-directed behaviors. These findings reveal an unanticipated spatial and temporal heterogeneity of dopamine as well as norepinephrine transmission within the brain that encodes specific responses.
To examine these events in further detail, we have used a device that allows simultaneous iontophoresis, cyclic voltammetry and electrophysiology.2 Fast-scan cyclic voltammetry records the local dopamine concentration in 10 ms, and, in the time between cyclic voltammograms, we record the local electrical activity arising from neurons firing in close proximity to the electrode tip. The coupled iontophoretic barrels allow introduction of drugs that can block the different types of dopamine receptors. Measurements with this technique in rats undergoing a reward based task have confirmed the segregation of D1 and D2 dopamine receptors.
Immune Sensing of Cytosolic Bacteria and Bacterial Virulence
National Institute of Biological Sciences, Beijing, 102206, China
Canonical inflammasomes are large cytoplasmic complexes that mediate caspase-1 activation, cytokine maturation and macrophage inflammatory death. We identify the NAIP family of NLR (Nucleotide-binding domain (NBD) and Leucine-rich Repeat) proteins that are inflammasome receptors for various bacterial products including flagellin and also the needle and rod subunit of bacterial toxin-injecting type III secretion system (T3SS). Ligation of the NAIPs by the corresponding ligands promotes their physical association with another NLR protein NLRC4, resulting in caspase-1 activation and anti-bacteria defense. We also discover that Pyrin, encoded by the familial Mediterranean fever disease gene MEFV, forms an inflammasome complex in response to bacterial modifications and inactivation of host Rho proteins. These include glucosylation by Clostridium difficile cytotoxin TcdB, adenylylation by FIC-domain bacterial effectors, ADP-ribosylation by C. botulinum C3 toxin and deamidation by B. cenocepacia, which all occur in the switch I region in Rho-subfamily GTPases. Loss of the Pyrin inflammasome causes elevated intra-macrophage growth of B. cenocepacia and diminished lung inflammation in mice. The NAIP and Pyrin inflammasomes serve as a mechanism for distinguishing pathogenic bacteria from non-pathogenic ones such as commensals. Lastly but most importantly, we demonstrate that inflammatory caspases including caspase-4/5 in human and caspase-11 in mice are cytosolic innate immune receptors for bacterial LPS (endotoxin). LPS directly binds to these caspases, leading to their oligomerization and catalytic activation and eventually necrotic cell death. Caspase-4/5/11-mediated non-canonical inflammasome activation not only serves as a general anti-bacterial defense mechanism but also plays a critical role in septic shock, providing an attractive new target for anti-sepsis drug development.
Emerging Lodinated Disinfection By-products in Drinking Water
Susan D. RICHARDSON
Department of Chemistry and Biochemistry, University of South Carolina, Columbia, SC 29205
Iodinated disinfection by-products (DBPs) are highly cytotoxic and genotoxic and increase in formation in drinking water treated with chloramine, which has become a popular disinfectant in the United States. Of these iodo-DBPs, iodoacetic acid is the most genotoxic of all DBPs studied to-date, more than the regulated DBPs. It was also recently shown to be tumorigenic in mice. Other iodo-DBPs identified include iodo-trihalomethanes, iodo-amides, and iodoacetaldehyde, as well as chloroiodo-, and bromoiodo-acids. In addition, we have new results indicating the formation of iodo-nitriles, iodo-tetrahalomethanes, and iodo-trihaloacetic acids.
The primary source of iodine in the iodo-DBP structures is believed to be naturally occurring iodide in source waters. However, our recent research has shown that iodinated compounds used for medical imaging (X-ray contrast media) can also form them. Iodinated X-ray contrast media (ICM) are resistant to removal by wastewater treatment and are found at the highest levels of any pharmaceutical in the environment, up to 100 ppb in river waters. We are also currently exploring other sources of iodine and are using advanced mass spectrometry tools to uncover the structures of these important new compounds. High resolution mass spectrometry (MS) using time-of-flight (TOF)-MS and Orbitrap-MS have been beneficial for revealing molecular formulas, and MS/MS has been useful for determining the chemical structures. In addition, a new, highly sensitive gas chromatography (GC)-MS/MS method has enabled the detection of iodo-trihaloacetic acids for the first time. These methods, along with a new total organic halogen-inductively coupled plasma (ICP)-MS method for sensitive detection of total organic iodine species, will be presented. And, new toxicity data showing the importance of these iodo-DBPs in chloraminated drinking water will be presented.
Innovations in Analytical Strategies and Methods for the Establishment of Si-traceable Bioassays
Division of Metrology for Quality of Life, Korea Research institute of Standards and Science, Daejeon 34113, Republic of Korea
Modern Biotechnologies promise our long and healthy lives with a variety of new products and services in near future. At the same time, however, there are growing concerns on the safety and efficacies of such unprecedented products and services, which should be ousted only through very scientific quality control and quality assurance. Numerous bioassays are being developed and applied for this purpose, but their reliability and credibility are not readily agreed and often lead to exhausting controversies. Scientists at national metrology institutes, in particular, have worked to provide solutions to this problem. The best desirable way is to provide measurement traceability to SI to as many bioassays as possible. The complexity of biological materials or quantities impedes the applicability of conventional chemical methods that rather new approaches should be sought out. Here are some examples of success in this regard: enumeration of single molecules; reductive analysis of individual chemical entities; and best practices of measurement protocols. Some technical details of such examples are discussed in this lecture. It is highly desired that more pronounced scientists get involved in this effort to facilitate rapid advancement of scientific quality control and quality assurance of biotechnology based products and services that are supposed to greatly enhance the quality of human life.