Current Pharmaceutical Biotechnology - Volume 13, Issue 14, 2012

HIF-1α is regarded as a target for drug development in several diseases such as cancer. For high throughput screening of HIF-1α-targeted drug, we need to examine the activity quantitatively. In the present study, we proposed a method where stable expression system of HIF-1α was combined with image correlation analysis. When the stable transformants were labeled with DRAQ5, we could detect Co2+-induced nuclear translocation by the use of cross-correlation analysis of the dual labeling images. In the case of high throughput screening for HIF-1α-targeted drug, we should use Pearson's correlation coefficient to judge nuclear translocation.

Calcium-activated photoproteins are important and useful bioluminescent reagents for detecting the calcium ion (Ca2+) in biological systems. In conjunction with photon imaging technology, they can be used to observe Ca2+-related life processes in a living cell. To develop useful applications of calcium-activated photoproteins, we need to understand the molecular basis of the bioluminescence reaction. For this purpose, this review describes the oxygenation, chemiexcitation, and light emission processes of calcium-activated photoproteins in the bioluminescence reaction together with the fundamental chemistry of the luminous substrate, coelenterazine, based on recent results from mechanistic chemical studies of these primary processes. Finally, the whole reaction mechanism, including the active site structures of apoproteins, along with available information about the molecular mechanism and the crystallographic structures of calcium-activated photoproteins are summarized.

Fluorescence correlation spectroscopy is a powerful technique for studying the structures and dynamics of living cells. Dynamic light scattering (DLS) is also used to study dynamic characteristics and it has the potential to measure cell dynamics. However, it is difficult to apply DLS to highly scattering media. In this article, we review low-coherence dynamic light scattering (LC-DLS). It strongly suppresses the influence of multiple scattering and has a greater potential for measuring cell dynamics than conventional DLS. The properties of LC-DLS are described theoretically and experimentally. Measurement of the diffusion coefficients of macromolecules in turbid media and interparticle and molecular interactions by LC-DLS is demonstrated.

This paper describes a novel molecular design strategy for obtaining efficient two-photon absorption (TPA) materials. The most popular strategy for enhancing the TPA cross-section (σ(2)) of a molecule is to enhance its transition dipole moment. However, this strategy also red shifts the one-photon absorption (OPA) band. Consequently, molecules with large transition dipole moments typically exhibit strong OPA at visible wavelengths, making it difficult to use such molecules for TPA-related applications in the visible wavelength region. Therefore, an alternative molecular design principle for TPA materials to enhance the transition dipole moment is strongly required. The present paper describes a novel molecular design strategy for reducing the detuning energy by incorporating an azulenyl moiety in a large, planar π- electron system. This strategy enhances σ(2) without significantly red shifting the OPA band.

Experimental method of studying viscoelasticity, a common idea to understand properties of microscopic biological soft matter systems, especially single biopolymer chains, using atomic force microscopy (AFM) with magnetically- driven cantilever is surveyed. The experimental setup of applying well-characterized excitation to the cantilever and the analysis method to derive the viscoelasticity of the system under study are briefly introduced. Examples of measuring viscoelasticity of single peptide molecule and single titin molecule are shown. Considering the close relation of viscoelasticity and the time-scale for nonequilibrium dynamics in soft matter, extension of the method to a frequency-resolved analysis is attempted. A result of measuring viscoelasticity spectrum of a single dextran chain is shown. Challenges in further progress of the method are also described.

Atomic force microscopy (AFM) provides a novel way to understand the structure-function relationship of protein synthesis at a single-molecular level. High-resolution AFM imaging in air, liquid and vacuum allows for single DNA, RNA and proteins to be observed at the nano-scale in addition to their conformational transitions, bound states, temporal behavior, and assembly. The recent development of frequency modulation mode AFM has led to imaging hydration structures of DNA in water and molecular polarization of DNA complexes in vacuum. Real-time imaging in near-physiological environments captures transcriptional activation with characteristic conformation of DNA-protein complexes. We review current achievements and the future potential of methodological and biological AFM to reveal insights into DNA, RNA and their complexes.

Inotropic agents induce changes in the contraction amplitude and frequency of cardiomyocytes (CMs). However, it is unknown how local contractions of CMs treated by inotropic agents behave spatiotemporally. In this study, the effect of isoproterenol, a positive inotropic agent, on local contractions of isolated neonatal rat CMs was explored by atomic force microscopy (AFM). We observed that changes in local contraction amplitude of CM in the presence of isoproterenol were heterogeneous; they were unchanged or increased, at different positions, with respect to the amplitude of untreated CMs. Interestingly, spatial heterogeneities of local contraction amplitude of CM in the presence of isoproterenol did not obviously correlate with the local elasticity, indicating that the local contractions were facilitated by cooperative dynamics of the cytoskeletal structure in relatively large regions, rather than those just under AFM indentation. Moreover, local contraction amplitude of CM in the presence of isoproterenol was not proportional to that in the control condition, showing that the former change was no longer additive in local scales.

In many signaling pathways, various factors have been isolated by molecular genetic studies and wholegenome analysis. Understanding the interactions among these factors is crucial to understanding the signaling process as a whole. Recent studies on auxin signaling in the regulation of plant growth and development have discovered primary factors interacting with each other, and elucidated a very simple pathway modulating gene expression in response to auxin. However, these studies of auxin-signaling led to the question of how such a simple pathway generates multiple types of regulation in various processes of different cells throughout the life of a plant. Here, we provide an overview of recent progress in auxin biology focusing on protein-protein interactions in the signal transduction pathway and discuss various possibilities and approaches to resolve the issue.

Functional analysis of carbohydrates is needed to understand the initial interface between membranes and the outer world. For this analysis we need individual protocols such as a method to modify the surfaces of nanoparticles with a variety of carbohydrates effectively and exhaustively, to synthesize an oligosaccharide on each particle's surface by chemical or enzymatic sugar elongation reaction, and to analyze the binding properties of carbohydrates. In this article, we describe the basic strategies for scooping up proteins from crude sample mixtures via interaction with carbohydrates. This approach was used to identify proteins that interacted with GM2, a ganglioside that is abundant on the surfaces of human lung cancer cells.

Mitochondria are known to be one of major organelles within a cell and to play a crucial role in many cellular functions. These organelles show the dynamic behaviors such as fusion, fission and the movement along cytoskeletal tracks. Besides mitochondria, mitochondrial DNA is also highly motile. Molecular analysis revealed that several proteins are involved in mitochondria and mitochondrial DNA dynamics. In addition to the degeneration of specific nerves with high energy requirement, mutation of genes coding these proteins results in metabolic diseases. During the last few years, a significant amount of relevant data has been obtained on molecular basis of these diseases but mitochondrial dynamics in cells derived from the patients is poorly understood. So far time-lapse fluorescence microscopy, fluorescence recovery after photo bleaching and image correlation methods have been used to study organellar motion. Especially, image correlation method has possibility to evaluate diffusion coefficient of mitochondria and mitochondrial DNA simultaneously and directly. When we search candidates for compounds that modulate mitochondrial dynamics by high throughput screening, image correlation method may be useful although the careful interpretation is required for crowded and heterogeneous environment within a cell.

Rheological properties of living cells play important roles in regulating their various biological functions. Therefore, measuring cell rheology is crucial for not only elucidating the relationship between the cell mechanics and functions, but also mechanical diagnosis of single cells. Atomic force microscopy (AFM) is becoming a useful technique for single cell diagnosis because it allows us to measure the rheological properties of adherent cells at any region on the surface without any modifications. In this review, we summarize AFM techniques for examining single cell rheology in frequency and time domains. Recent applications of AFM for investigating the statistical analysis of single cell rheology in comparison to other micro-rheological techniques are reviewed, and we discuss what specificity and universality of cell rheology are extracted using AFM.

Tyrosine (Tyr) sulfation is a common posttranslational modification of secreted proteins or membrane-bound proteins that is implicated in numerous physiological and pathological processes. The Tyr sulfation modifies proteinprotein interactions involved in leukocyte adhesion, homeostasis, and receptor-mediated signaling. To data, 80 Tyrsulfated proteins have been identified. As new methodologies and bioinformatics for the detection of Tyr sulfation become available, the number of Tyr-sulfated acceptor proteins discovered is bound to increase. Further, recent advances in microscopy and fluorescence technology will provide information on the true spatial and temporal nature of Tyr-sulfated proteins within the intact cell. This review summarizes the methods for the detection of Tyr O-sulfation as well as the biological functions of sulfated Tyr. Further, illustrative examples of the impact of Tyr sulfation on the pharmacological properties are presented.

Methods for regulating peptide conformation by non-harmful light stimuli can be useful for remotely controlling cellular functions in vitro. Here, we synthesized a series of p-heteroatom-substituted azobenzenes and studied their photoisomerization properties. The trans-isomer of p-sulfur-substituted azobenzene was effectively isomerized by visible light irradiation and the cis-isomer was thermally stable at physiological temperature. We developed a novel visible light sensitive amino acid (AZO), via p-sulfur-substituted azobenzene, and utilized it as a photosensitive modulator of the SV40 nuclear localization signal (NLS). The cellular uptake of the AZO-NLS conjugate was controlled by visible light irradiation. Our technology can be utilized for regulating not only the cellular uptake, but also the function of peptides within cells by non-harmful visible light irradiation.

We have examined optical properties of a fluorene derivative with two positively charged substituents, 1,1'- diethyl-4,4'-(9,9-diethyl-2,7-fluorenediyl-2,1-ethanediyl)dipyridinium perchlorate (1), in water. The photoluminescence quantum yield of 1 was relatively high (35%) for use as a fluorescent probe in water. We also examined two-photon absorption (TPA) properties of 1 in methanol. The maximum value of the TPA cross-section (730 GM at 750 nm, 1 GM = 10-50 cm4 s photon-1 molecule-1) was larger than that for most two-photon-excited fluorescent dyes including a classical mitochondria-selective fluorescent dye rhodamine 123. Preliminary fluorescence imaging experiments of the mitochondria in living Paramecium caudatum and HeLa cells were carried out with 1. Bright green fluorescence was observed from the mitochondria in both living cells loaded 1 without toxicity effects. These our results indicate that water-soluble fluorene derivative 1 is a promising candidate as a two-photon-excited fluorescence probe for mitochondria in living cells.

Dielectric particles including living cells are trapped within focused laser beam spots, and as a result, they can be transferred by displacing the beam spots. Such the particle manipulating technique is called optical tweezers. Holographic optical tweezers (HOT) enables highly flexible and precise control of particles, introducing holography technique to them. HOT is one of the most expected techniques for investigations of cell-cell signaling which require precise arraying of living cells. We had developed a new highly controllable HOT system where two different intensity patterns, a carrier beam spot and a beam array, are generated quasi-simultaneously by time-division multiplexing. Particles are transferred to the beam array by the carrier beam spot displaced in real time by phase shifting of holograms. In this review, we introduce our work, the construction of the system, demonstration of manipulating particles and investigations of the spatio- temporal stability of trapped particles in our system.

Glycosphingolipids (GSLs) are present in all mammalian cell plasma membranes and intracellular membrane structures. They are especially concentrated in plasma membrane lipid domains that are specialized for cell signaling. Plasma membranes show typical structures called rafts and caveola domain structures, with large amounts of sphingolipids, cholesterol, and sphingomyelin in the cell membranes. Plasma membranes have two faces, many kinds of receptors for intercellular signal transducers such as GPI-anchored proteins on the exoplasmic faces of the rafts/caveolae and src family kinases on the cytosolic face. Thus they play a role in transmembrane signal transduction, following the phosphorylation of some substrates and gene expression. On the other hand, their functions have become clear through the study of gene-manipulated mice. For further advances, a visual method to display diversity of biological functions is necessary. For this purpose, the use of high-performance microscopes and live cell imaging technologies are useful for more detailed understanding.