Abstract

Visualization of gas signalings in vivo

Mayumi Kajimura, Satoshi Kashiwagi, Masaru Shimoyama and Makoto Suematsu

Nitric oxide (NO) and carbon monoxide (CO) are known to induce diverse cellular effects by controlling the function of their receptor protein, soluble guanylate cyclase (sGC). While the studies in vitro have shown that both NO and CO activate purified sGC (Stone & Marletta, 1994), confounded results exist in the literature as to roles of CO to control sGC functions in vivo. Indeed very little is known about how the gases interact with each other and transduce their effects at the complex in vivo level. This could have arisen from the lack of experimental systems to evaluate amounts and distribution of these molecules and to assess their receptor functions in their native environment. Thus we have taken this challenge and sought to visualize the sGC function to revisit the question if effects of these monoxides on this cGMP-producing enzyme are synergistic or antagonistic. The rat retina was chosen as an experimental system because its well-defined anatomical layers consisting of specific cell types enabled us to examine spatial relationships between NO- or COgenerating enzyme and sGC. Development of a new probing method using two unique monoclonal antibodies (mAbs) against sGC, mAb28131 and mAb3221 enabled us to evaluate activation states of this enzyme. We found that mAb3221 increased its affinity to purified sGC by 100- and 10-folds upon application of NO and CO, respectively, while mAb28131 did not alter the affinity. Direct detection of the gas-mediated alterations in sGC function with mAb3221 led us to postulate that the effect of CO on modulating sGC activity is not static but dynamic in that low tissue availability of NO makes CO a stimulatory modulator of sGC, while high tissue availability of NO makes CO an inhibitory one (Kajimura et al., 2003). Another area of investigation involves the visualization of NO in microcirculation. Here we have taken advantage of 4, 5-diamonofluorescein diacetate (DAF- 2DA) which enabled us to assess NO generation at the microvasculature. DAF-2DA is a membrane permeable fluorescence precursor sensing intracellular NO generation (Kojima et al, 1998). It has been believed that most NO at microvascular walls is derived solely from endothelial cells which express NOS3. On the contrary, we found evidence that two different sources, NO synthase-1 and -3, play a major role in maintaining NO at arteriolar and venuler walls, respectively (Kashiwagi et al, 2002). We believe that the idea that different gases possessing similar structure (e.g. O2, NO, CO) interact with one another to alter protein functions and thus cellular and organ functions in vivo is worth pursuing and to do so visualizing generation and reception of gaseous mediators is needed.

Development of novel bioimaging fluorescence probes based on rational design strategies

Yasuteru Urano

Fluorescence imaging is the most powerful technique currently available for continuous observation of dynamic intracellular processes in living cells. Suitable fluorescence probes are naturally of critical importance for fluorescence imaging, but only a very limited range of biomolecules can currently be visualized because of the lack of flexible design strategies for fluorescence probes. At present, design is largely empirical. Very recently, we demonstrated that the fluorescein molecule, which has been widely employed as a core of fluorescence probes, could be understood as a directly linked electron donorflacceptor system, and that their fluorescence properties could be controlled and anticipated by using the concept of intramolecular photoinduced electron transfer (PeT).1-3 Based on these photo-physical findings, we could construct the first and totally rational design strategies for novel fluorescence probes, and could develop so far the following novel fluorescence probes; (a) DPAXs and DMAXs for singlet oxygen,3 (b) DAFs and DAMBOs4 for nitric oxide, (c) HPF and APF for highly reactive oxygen species including hydroxyl radical and peroxynitrite,5 and so on. We next focused on the role of the carboxylic group of fluorescein. In the light of the above findings, the carboxylic group could be replaced with another functional group, and indeed we could develop novel fluorescein derivatives, called TokyoGreen, by breaking out of the traditional structure of fluorescein which has methyl or methoxy group instead. Further, by precisely controlling the oxidation potential of the benzene moiety, we succeeded in developing novel and useful scaffolds which are applicable for a wide range of fluorescence probes. The value of this approach is exemplified by its application to develop a novel, highly sensitive and membrane-permeable fluorescence probe for bgalactosidase, which is the most widely used reporter enzyme.

Spatio-temporal dynamics of intracellular signaling

Atsushi Miyawaki

"Why bio-imaging, i.e. real time fluorescence imaging?" Currently, this is a topic of great interest in the bioscience community. Many molecules involved in signal transduction have been identified, and the hierarchy among those molecules has also been elucidated. It is not uncommon to see a signal transduction diagram in which arrows are used to link molecules to show enzyme reactions and intermolecular interactions. To obtain a further understanding of a signal transduction system, however, the diagram must contain the three axes in space as well as a fourth dimension, time, because all events are controlled ingeniously in space and time. Since the isolation of green fluorescent protein (GFP) from the bioluminescent jellyfish, Aequorea victoria, in 1992 and later with its relatives (GFP-like proteins from Anthozoa), researchers have been awaiting the development of a tool which enables the direct visualization of biological functions. This has been increasingly enhanced by the marriage of GFP with fluorescence resonance energy transfer (FRET), and is further expanded upon by the need for "post-genomic analyses." It is not my intent to discourage the trend seeking the visualization of biological function. I would like to propose that it is time to evaluate the true asset of "bio-imaging" for its potential and limitations in order to utilize and truly benefit from this novel technique. My talk will cover GFP-based FRET technology to develop fluorescent indicators that report concentrations of second messenger molecules and activation of signaling components for visualization of signaling cascades. These indicators can be called "molecular spies". There will be discussion on how to design molecular spies, how to smuggle them into cells, and how to interpret their signals. Thus, we may move towards a more comprehensive understanding of signal transduction systems.

In vivo observations of gene expression and protein structures by magnetic resonance

Masahiro Shirakawa, Tetsuro Kokubo, Fuminori Sugihara, Se-won Ki, Tomomi Sakai, Yutaka Ito and Yosuke Yoshinari

Owing to its non-invasiveness and ability for depth observation, magnetic resonance (MR) is suitable for in vivo observations of functional molecules in living organisms.1 We are trying to develop technologies for elucidation of functions, localizations and tertiary structures of proteins in living organisms by using MR imaging, multi-dimensional NMR spectroscopy, and magnetic resonance force microscopy (MRFM). One of the techniques that attract the largest attention in the field of molecular imaging is visualization of gene expression inside intact organ and organisms.2 We have developed a reporter system for MR-detection of gene expression in yeast, in which polyphosphate is used as a molecular probe and its related genes as reporter genes. Because 31P-NMR signal of polyphosphate is isolated from those of other phosphoruscontaining compounds that give observable signals in the spectrum of living cells, 31P-MRI can distinguish colonies of polyphosphate-containing cells from others in a quantitative manner. Now attempt is being made to apply this method to other organisms. "In cell NMR" is a two- or higher dimensional NMR observation of target proteins in living cells. This methodology has been successfully applied for conformational characterization of proteins in E. coli and frog cells.3 We measured NMR and ESR spectra of a protein injected to Xenopus laevis egg cells. Future application of in cell NMR and ESR will also be discussed.

Molecular imaging of drug targets in living brain

Tetsuya Suhara

Recent progress in molecular medicine discovered new targets of drugs. These targets often play important role in the pathophysiology of disease. In vivo validation of specific ligand binding to these targets in human is needed to assess their function. Positron emission tomography (PET) techniques has made it possible to validate these targets. PET is a tracer technique that makes it possible to detect accurately and noninvasively in vivo concentration of radio-labeled compounds. Since various biological and pharmacological compounds can be labeled with positron-emitting nuclides, PET can be used to investigate the biochemical regional characteristics of various molecular targets. One of the key components of the PET technique is radio-labeled compounds often called as a molecular probe. Various biochemical substrates and drugs which can specifically bind to certain protein such as receptors and transporters or a substrate of specific enzyme such as acetylcholine esterase have been labeled with positron emitter. The sensitivity of PET technique is extremely high and combined with high specific radioactivities (10-100 Ci/umol), the total mass of the compounds can be sub nano mol. Thus in PET study, the underlying biological systems would not be perturbed by mass effects. The distribution and density of drug targets such as serotonin transporter can be measured using specific ligand like [11C]McN5652 or [11C]DASB in the living human brain. The function of certain drug target like p-gycoprotein can be measure using its labeled substrate like [11C]verapamil. As an indirect index, occupany has been used as a reliable index of therapeutic drug at the specific binding site. Several antipsychotic drugs have been evaluated using PET at fixed time point. However, based on the pharmacokinetic studies, the kinetic profile of antipsychotics at dopamine D2 receptor sites is also an important index for antipsychotic action and dosing schedule. We have simulated the time course of dopamine D2 receptor occupancy from plasma pharmacokinetics and the apparent in vivo affinity parameter (ED50; concentration to induce 50% occupancy).

A new molecular probe for hypoxic tumors: PET diagnosis to radiotherapy

Yasuhisa Fujibayashi, Takako Furukawa, Takeshi Tanaka, Shingo Kasamatsu and Yoshiharu Yonekura

Tumor is a tissue of uncontrolled growth, and sometimes become hypoxic caused by insufficient angiogenesis. However, it is also well known that some tumor cells are hypoxia tolerant and have glycolysis-dominant energy production system. To make matter worse, hypoxic tumor is registant to radiation therapy as well as some kind of anti-tumor drugs. Thus, detection of hypoxic tumor region is of great importance in tumor therapy. We have developed a new molecular probe for hypoxic tumors, copper(II)-diacetyl-bis (N4-methylthiosemicarbazone) (Cu-ATSM), labeled with shortlived radio-copper. Cu-ATSM is a small molecularweight and lipophilic compound, so that easily accessible into intracellular enzymes through cell membrane including blood-brain barrier. Only in hypoxic tissues, Cu-ATSM can be reduced by electron transport enzymes. Reduced Cu(I) is released from the ligand and retained in the cells. As a result, hypoxic tumor cells can be visualized using positron emission tomography (PET). Fortunately, there are several available radioactive Cu isotopes, such as Cu-60, 61 and 62 for PET imaging, and Cu-64 for internal radiation therapy. The hypoxia selectivity of Cu-ATSM is useful not only for the detection of hypoxic tumors using Cu-60, 61 and 62, but also for tumor selective delivery of therapeutic Cu-64. When Cu-64-ATSM was intravenously injected into tumor-bearing hamsters, mean survival time was extended from 3 weeks to 30 weeks. In in-virto cell studies, the mechanism of Cu-64 toxicity was considered to be post-mitotic apoptosis. Multi-centers study on PET diagnosis using Cu- ATSM will start from next April. Clinical usefulness and limitation of Cu-ATSM will be clarified.

Breeding and building molecules to spy on cells and tumors

Roger Y. Tsien

Genetically encoded tags and indicators are molecular spies that reveal specific gene products and biochemical processes in living cells and organisms. Fluorescent proteins from jellyfish and corals have been bred to eliminate multimerization and cover the entire visible spectrum. Somatic hypermutation in B lymphoma cells can offer a powerful new way to evolve protein properties. Indicators constructed from fluorescent proteins can report local dynamic signals such as redox potential, kinase vs. phosphatase activities, protein-protein interactions, and ion and neurotransmitter concentrations. Short tetracysteine motifs are complementary tags, which can be labeled in live cells with membranepermeant biarsenical dyes. Unique applications include green vs. red pulse-chase labeling of old vs. new copies of the same protein, electron-microscopic localization, chromophore-assisted light inactivation of a chosen protein without the problems of antibody penetration, and measurement of local Ca2+ within nanometers of proteins such as Ca2+ channels. For clinical applications one would prefer not to have to introduce genes or be limited to optical detection. Arginine-rich sequences are known to mediate uptake of a wide variety of contrast agents into cells and tissues in vivo. We find that such uptake can be prevented by appending certain polyanionic sequences and selectively re-activated by cleavage of the linker. This new mechanism offers the exciting possibility that radioactive, magnetic, and infrared contrast agents and therapeutic drugs may be concentrated in diseased tissues expressing particular extracellular proteases.