Injae Shin

A near-infrared fluorescent sensor for detection of cyanide in aqueous solution and its application for bioimaging

A new NIR fluorescent sensor based on an amine-substituted heptamethine cyanine dye displayed a highly selective fluorescence enhancement with cyanide in aqueous solutions, and was applied for the imaging of anthropogenic and biogenic cyanide.

A Rhodamine−Hydroxamic Acid-Based Fluorescent Probe for Hypochlorous Acid and Its Applications to Biological Imagings

A new rhodamine-hydroxamic acid-based fluorescent chemosensor for the rapid detection of HOCl in aqueous media was developed. The system, which utilizes an irreversible HOCl-promoted oxidation reaction, responds instantaneously at room temperature with linear proportionality to the amount of HOCl. This system is highly selective for HOCl over other reactive oxygen species (ROS) and highly sensitive in aqueous solutions. Biological imaging studies using living cells and organisms (A549 cells and zebrafish) to detect HOCl are successfully demonstrated.

Synthesis of a highly metal-selective rhodamine-based probe and its use for the in vivo monitoring of mercury.

This protocol describes detailed procedures for the preparation of a rhodamine-based mercury probe and for its applications to the detection of mercury in cells and vertebrate organisms. The mercury probe 1, which is prepared in two steps from rhodamine 6G, responds rapidly to Hg2+ in aqueous solutions with a 1:1 stoichiometry. Owing to the fact that the probe reacts with Hg2+ in an irreversible manner, it has advantages over other reversible mercury probes in in vivo assays with respect to both sensitivity and selectivity. In addition, fluorescent imaging assays of Hg2+ in live cells and zebrafish by using this mercury probe are detailed in this protocol. The approximate time frame for the preparation of the probe is 24 h and for its use in imaging assays is 1.5 h.

Construction of Carbohydrate Microarrays by Using One-Step, Direct Immobilizations of Diverse Unmodified Glycans on Solid Surfaces

Carbohydrate microarrays have received great attention as high-throughput analytic tools in studies of carbohydrate-mediated biological processes. Most of the methods employed to fabricate glycan microarrays rely on the immobilization of modified glycans on the properly derivatized surfaces. This immobilization strategy requires the availability of modified glycans whose syntheses in many cases are time-consuming and difficult. We have developed a simple and direct immobilization technique that involves a one-step, site-specific attachment of diverse unmodified glycans to the hydrazide-derivatized glass surface. To demonstrate the generality of this direct immobilization method, we examined its use for the construction of carbohydrate microarrays containing a variety of glycans. The results of protein and cell-binding experiments indicate that the glycan microarrays, prepared by using this methodology, are applicable to the rapid evaluation of glycan-mediated biomolecular interactions and the determination of quantitative binding affinities between carbohydrates and proteins.

Tom40 protein import channel binds to non-native proteins and prevents their aggregation.

Mitochondria contain the translocator of the outer mitochondrial membrane (TOM) for protein entry into the organelle, and its subunit Tom40 forms a protein-conducting channel. Here we report the role of Tom40 in protein translocation across the membrane. The site-specific photocrosslinking experiment revealed that translocating unfolded or loosely folded precursor segments of up to 90 residues can be associated with Tom40. Purified Tom40 bound to non-native proteins and suppressed their aggregation when they are prone to aggregate. A denatured protein bound to the Tom40 channel blocked the protein import into mitochondria. These results indicate that, in contrast to the nonstick tunnel of the ribosome for polypeptide exit, the Tom40 channel offers an optimized environment to translocating non-native precursor proteins by preventing their aggregation.

An apoptosis-inducing small molecule that binds to heat shock protein 70.

Apoptosis (or programmed cell death) is a fundamental biological process that regulates a variety of normal physiological processes, ranging from development to aging. Damaged or unwanted cells in organisms are removed by the intrinsic and/or extrinsic apoptotic pathways. The intrinsic apoptotic pathway occurs by the release of cytochrome c from mitochondria. The extrinsic apoptotic pathway is caused by the binding of death ligands, such as TNF (tumor necrosis factor), Fas, and TRAIL (TNF-related-apoptosis-inducing ligand), to their corresponding receptors. Although programmed cell death is involved in a number of key biological phenomena, aberrant apoptosis results in diverse human diseases. For example, the dysregulation of apoptosis disrupts tissue homeostasis by prolonging cell survival and contributes to the progression of diverse human tumors. In addition, retarded apoptosis causes the elimination of autoreactive lymphocytes to fail, leading to autoimmunity. Moreover, excessive apoptosis results in cell-loss disorders such as neurodegenerative (Alzheimer,s and Parkinson,s diseases) and cardiovascular diseases. Since apoptosis is involved in both normal physiology and various human diseases, research on apoptosis has become a central area in basic biological studies and in the development of therapeutic agents. Small molecules that either induce or prevent apoptotic cell death have significant potential as therapeutic agents to treat apoptosis-related diseases. In addition, these agents could also be employed to understand the roles that apoptotic regulatory proteins play in biological processes. Herein we describe a novel apoptosis-inducing small molecule which interacts with Hsc70 and Hsp70. Cell-based screening with a small molecule library is an attractive approach to identify bioactive compounds that regulate protein functions in cells or affect processes such as cell differentiation or morphology. We applied this approach to select molecules with apoptosis-inducing activity, using a recently prepared imidazole library on a solid support to identify bioactive compounds that induce interesting cellular events (Scheme 1a). The amine-conjugated diethylene glycol linker was introduced into the library for facile solid-phase synthesis and the identification of target protein(s) by affinity chromatography. To search for molecules that induce apoptosis in cells, 216 imidazole derivatives (1 mm) were incubated with the highly proliferative P19 embryonic carcinoma cell line for 3 h and subsequently treated with a mixture of annexin V-fluorescein (0.5 mgmL ) and propidium iodide (PI, 2 mgmL ) to rapidly screen for apoptosis inducers. The exposure of phosphatidylserine on the outer leaflet of the cell plasma membrane is a key feature of the early stages (2–4 h) of apoptosis. Phosphatidylserine can be detected fluorescently by using annexin Vfluorescein. Propidium iodide (PI) can be used to monitor membrane-perturbed cells which result from the plasma membrane becoming permeable (a feature of necrosis) or late-stage apoptosis. Therefore, the combined use of annexin V-fluorescein and PI allows for the rapid evaluation of apoptosis in cells treated with the compound library. In our screen, compounds that exhibited positive annexin V and negative PI staining in P19 cells after 3 h incubation were selected as inducers of apoptosis. However, compounds that showed positive annexin Vand positive PI staining in the cells were not selected as “hits” because it is possible that the treated cells underwent necrosis rather than apoptosis. One compound apoptozole-linker (Az-linker) showed a high level of positive annexin V and negative PI staining in P19 cells (Scheme 1b). For further studies, Az (without linker) was resynthesized and purified (see the Supporting Information). Scheme 1. Structures of a) an imidazole library (see the Supporting Information for substituents R–R) and b) apoptozole (Az).

Chemoselective ligation of maleimidosugars to peptides/protein for the preparation of neoglycopeptides/neoglycoprotein

Two types of maleimidosugars as thiol-selective carbohydrates, 1-maleimidosugars and acetyl-linked maleimidosugars, were efficiently synthesized. They were coupled to glutathione, Fas peptide and bovine serium albumin (BSA) to prepare the corresponding glycosylated peptides and a protein via stable thioether linkages in a chemoselective manner.

Carbohydrate Arrays for Functional Studies of Carbohydrates

Carbohydrates, as components of glycoproteins, glycolipids and proteoglycans, play an important biological role as recognition markers through carbohydrate-protein interactions. For the most part, biophysical and biochemical methods have been used to analyze these biomolecular interactions. In contrast, less attention has been given to the development of high-throughput procedures to elucidate carbohydrate-protein recognition events. Recently, carbohydrate arrays were developed and employed as a novel high-throughput analytic tool for monitoring carbohydrate-protein interactions. This technique has been used to profile protein binding and enzymatic activity. The results have shown that carbohydrate binding to the corresponding lectins is highly selective and that the relative binding affinities are well correlated with those obtained from solution-based assays. In addition, this effort demonstrated that carbohydrate arrays could be also utilized to identify and characterize novel carbohydrate-binding proteins or carbohydrate-processing enzymes. Finally, the results of this investigation showed that lectin-carbohydrate binding affinities could be quantitatively assessed by determining IC50 values for soluble carbohydrates with the carbohydrate arrays. The results of these studies suggest that carbohydrate arrays have the potential of playing an important role in basic researches, the diagnoses of diseases and drug discovery.

Fluorescent high-throughput screening of chemical inducers of neuronal differentiation in skeletal muscle cells

This protocol describes detailed procedures for the fluorescent high-throughput screening of small molecules that induce neurogenesis in cultures of skeletal muscle cells. The detection of neurogenesis relies on a fluorescent dye, FM 1-43, which is used to study the neuronal property of depolarization-induced synaptic vesicle recycling. Thus, small molecules with neurogenesis-inducing activity in skeletal muscle cells can be rapidly identified by measuring the fluorescence intensity of the treated cells using a fluorescent microplate reader. This protocol uses murine myoblast C2C12 cells for screening, which are readily available and relatively easy to culture. Neurogenesis of PC12 cells induced by nerve growth factor is employed as a positive control for this screening. The screening time for this protocol is 8 d, which also includes the procedure to detect depolarization-induced synaptic vesicle recycling using FM 1-43.

One-step, acid-mediated method for modification of glass surfaces with N-hydroxysuccinimide esters and its application to the construction of microarrays for studies of biomolecular interactions

Microarray technologies have received considerable attention owing to the fact that they serve as powerful tools for the high-throughput analysis of biomolecular interactions and the identification of bioactive substances that bind to biomolecules. Most of the current methods used to construct microarrays rely on the immobilization of substances on properly derivatized surfaces. Among various functional groups used for this purpose, the N-hydroxysuccinimide (NHS) ester group has been largely employed because it can be readily reacted with amine or hydrazide functionalities in substances of interest. However, the NHS ester group is usually introduced onto the surface of a glass slide by employing inconvenient and time-consuming multistep processes. In recent studies, we have developed an efficient, single step method for derivatization of glass surfaces with NHS ester groups that takes advantage of an acid-mediated reaction of NHS ester functionalized dimethallylsilanes with silanols on the glass surface. Conditions for the surface modification procedure that utilize TfOH rather than Sc(OTf)(3) were found to be superior. Protein and RNA-binding experiments show that glass surfaces modified by employing this method are suitable for efficient immobilization of various substances that are appended by amine, hydrazide, and alcohol functionalities. The microarrays, generated in this way, are applicable to procedures for rapid analysis of protein-protein, protein-glycan, protein-small molecule, and peptide-RNA interactions, as well as for profiling enzyme activities. The newly developed acid-mediated, glass surface modification method should be generally applicable to the preparation of various functional group-modified surfaces.