Jae-Kyung Hyun

Inhibition of influenza virus internalization by (−)-epigallocatechin-3-gallate

(-)-Epigallocatechin-3-gallate (EGCG), one of the major flavonoid components of green tea, is known to have a broad antiviral activity against several enveloped viruses, including the influenza virus. However, its mode of action and the mechanism that allows it to target influenza virus molecules have not been fully elucidated. Thus, this study investigated the molecular mechanism by which EGCG suppresses influenza virus infections. EGCG was found to block an early step in the influenza viral life cycle, but it did not affect viral adsorption to target cells or viral RNA replication. However, EGCG inhibited hemifusion events between virus particles and the cellular membrane by reducing the viral membrane integrity, thereby resulting in the loss of the cell penetration capacity of the influenza virus. EGCG also marginally suppressed the viral and nonviral neuraminidase (NA) activity in an enzyme-based assay system. In conclusion, it is suggested that the anti-influenza viral efficacy of EGCG is attributable to damage to the physical properties of the viral envelope and partial inhibition of the NA surface glycoprotein. These results may facilitate future investigations of the antiviral activity of EGCG against other enveloped viruses as well as influenza virus.

De novo design and synthesis of ultra-short peptidomimetic antibiotics having dual antimicrobial and anti-inflammatory activities.

Background Much attention has been focused on the design and synthesis of potent, cationic antimicrobial peptides (AMPs) that possess both antimicrobial and anti-inflammatory activities. However, their development into therapeutic agents has been limited mainly due to their large size (12 to 50 residues in length) and poor protease stability. Methodology/Principal Findings In an attempt to overcome the issues described above, a set of ultra-short, His-derived antimicrobial peptides (HDAMPs) has been developed for the first time. Through systematic tuning of pendant hydrophobic alkyl tails at the N(π)- and N(τ)-positions on His, and the positive charge of Arg, much higher prokaryotic selectivity was achieved, compared to human AMP LL-37. Additionally, the most potent HDAMPs showed promising dual antimicrobial and anti-inflammatory activities, as well as anti–methicillin-resistant Staphylococcus aureus (MRSA) activity and proteolytic resistance. Our results from transmission electron microscopy, membrane depolarization, confocal laser-scanning microscopy, and calcein-dye leakage experiments propose that HDAMP-1 kills microbial cells via dissipation of the membrane potential by forming pore/ion channels on bacterial cell membranes. Conclusion/Significance The combination of the ultra-short size, high-prokaryotic selectivity, potent anti-MRSA activity, anti-inflammatory activity, and proteolytic resistance of the designed HDAMP-1, -3, -5, and -6 makes these molecules promising candidates for future antimicrobial therapeutics.

Non hemolytic short peptidomimetics as a new class of potent and broad-spectrum antimicrobial agents

Since the bacterial resistance to antibiotics is increasing rapidly, numerous studies have contributed to the design and synthesis of potent synthetic mimics of antimicrobial peptides (AMPs). In an attempt to find the pharmacophore of short antimicrobial peptidomimetics through systematic tuning of hydrophobic and hydrophilic patterns, we have identified a set of short histidine-derived antimicrobial peptides (SAMPs) with potent and broad-spectrum activity. A combination of high antimicrobial activity against methicillin-resistant Staphylococcus aureus (MRSA), without hemolytic activity and proteolytic stability makes these molecules promising candidates for novel antimicrobial therapeutics.

Pyrazole derived ultra-short antimicrobial peptidomimetics with potent anti-biofilm activity

In this study, we report on the first chemical synthesis of ultra-short pyrazole-arginine based antimicrobial peptidomimetics derived from the newly synthesized N-alkyl/aryl pyrazole amino acids. Through the systematic tuning of hydrophobicity, charge, and peptide length, we identified the shortest peptide Py11 with the most potent antimicrobial activity. Py11 displayed greater antimicrobial activity against antibiotic-resistant bacteria, including MRSA, MDRPA, and VREF, which was approximately 2-4 times higher than that of melittin. Besides its higher selectivity (therapeutic index) toward bacterial cells than LL-37, Py11 showed highly increased proteolytic stability against trypsin digestion and maintained its antimicrobial activity in the presence of physiological salts. Interestingly, Py11 exhibited higher anti-biofilm activity against MDRPA compared to LL-37. The results from fluorescence spectroscopy and transmission electron microscopy (TEM) suggested that Py11 kills bacterial cells possibly by integrity disruption damaging the cell membrane, leading to the cytosol leakage and eventual cell lysis. Furthermore, Py11 displayed significant anti-inflammatory (endotoxin-neutralizing) activity by inhibiting LPS-induced production of nitric oxide (NO) and TNF-α. Collectively, our results suggest that Py11 may serve as a model compound for the design of antimicrobial and antisepsis agents.

Poly-lysine peptidomimetics having potent antimicrobial activity without hemolytic activity

Diversity of sequence and structure in naturally occurring antimicrobial peptides (AMPs) limits their intensive structure–activity relationship (SAR) study. In contrast, peptidomimetics have several advantages compared to naturally occurring peptide in terms of simple structure, convenient to analog synthesis, rapid elucidation of optimal physiochemical properties and low-cost synthesis. In search of short antimicrobial peptides using peptidomimetics, which provide facile access to identify the key factors involving in the destruction of pathogens through SAR study, a series of simple and short peptidomimetics consisting of multi-Lys residues and lipophilic moiety have been prepared and found to be active against several Gram-negative and Gram-positive bacteria containing methicillin-resistant Staphylococcus aureus (MRSA) without hemolytic activity. Based on the SAR studies, we found that hydrophobicity, +5 charges of multiple Lys residues, hydrocarbon tail lengths and cyclohexyl group were crucial for antimicrobial activity. Furthermore, membrane depolarization, dye leakage, inner membrane permeability and time-killing kinetics revealed that bacterial-killing mechanism of our peptidomimetics is different from the membrane-targeting AMPs (e. g. melittin and SMAP-29) and implied our peptidomimetics might kill bacteria via the intracellular-targeting mechanism as done by buforin-2.

Analysis of nano-crystals: Evaluation of heavy metal-embedded biological specimen by high voltage electron microscopy

Heavy metal compounds are adsorbed onto biological specimen in order to enhance the contrast as well as to preserve the structural features of the specimen against electron beam-induced radiation damage. In particular, in combination with computational image processing, negative staining is widely used for structural analysis of protein complexes to moderate resolutions. Image analysis of negatively stained biological specimen is known to suffer from limited achievable resolution due to dehydration and large grain size of staining molecules although the extent of such effect remains somewhat dubious. Stain molecules exist as grains under electron beam. However, clear observation of the crystalline nature of the grains and their association with biological specimen has not been thoroughly demonstrated. In this study, we attempted high-resolution TEM (HRTEM) using high voltage electron microscopy and electron crystallography analysis for the detailed characterization of negatively stained biological specimen, focusing on physical state and chemical composition of the stain molecules. The electron crystallography analysis allowed for the identification of the crystal constituents of widely used stains, hence revealing the chemical nature and the morphology of the stain molecules at specimen level. This study re-evaluated generally accepted notions on negative staining, and may help correctly interpreting the structural analysis of stained biological specimen.

Cations Modulate Actin Bundle Mechanics, Assembly Dynamics, and Structure

Actin bundles are key factors in the mechanical support and dynamic reorganization of the cytoskeleton. High concentrations of multivalent counterions promote bundle formation through electrostatic attraction between actin filaments that are negatively charged polyelectrolytes. In this study, we evaluate how physiologically relevant divalent cations affect the mechanical, dynamic, and structural properties of actin bundles. Using a combination of total internal reflection fluorescence microscopy, transmission electron microscopy, and dynamic light scattering, we demonstrate that divalent cations modulate bundle stiffness, length distribution, and lateral growth. Molecular dynamics simulations of an all-atom model of the actin bundle reveal specific actin residues coordinate cation-binding sites that promote the bundle formation. Our work suggests that specific cation interactions may play a fundamental role in the assembly, structure, and mechanical properties of actin bundles.

Technical Advances in Intracellular Detection Using Immuno-Gold Particles: Simple Cryofixation with Metal Contact Quick Freezing.

The preparation of biological specimens using cryofixation techniques ensures excellent visibility of intracellular structures and preserves the antigenic sites of subcellular molecules. Hence, cryofixation is an effective method of preparing samples for analyses using antibodies conjugated to gold nanoparticles that are designed to detect the localization of specific target molecules within cells. However, cryofixation cannot be utilized easily because it requires expensive equipment and skilled technologists, resulting in a high level of expense for researchers. Here, we describe a simple technical approach to cryofixation that uses metal contact quick freezing followed by a modified freeze substitution technique and immuno-gold labeling electron microscopy. Micrograph images of cells prepared using this modified cryofixation method demonstrated its superiority over chemical fixation for high contrast visualization of the morphologies of cellular components and preservation of antigenicity for immuno-gold labeling. This report provides valuable technical information related to the advancement of metal contact quick freezing techniques, which can be used to visualize biomedical events of interest in an easy, simple, and rapid manner.

Enhanced Large-Area TEM Analysis on Mitochondrial Alteration in Brain Neuronal Cell of Parkinson's Disease Model Mouse Using Montage Function Installed in JEM 1400-Plus System

Despite the important role of transmission electron microscope (TEM) in providing tremendous ultrastructural information of cells, organelles, and molecules [1], one of the disadvantages of TEM is the difficulty associated with observing large-area with high resolution at low-magnification [2 and 3]. Although several analytical techniques and equipments including ultramicrotomy-combined serial block face scanning EM have been developed recently to overcome such problem, those are mostly concentrated in SEM applications [1, 2, 4, 5, and 6]. In this research, to acquire large-area TEM images with highresolution, we used the montage function in TEM Center, a software suite that controls JEM 1400-Plus TEM as described in Figure 1. Then, we compared the resolution of low mag. reference image (4,000X) acquired using normal imaging mode with that of 3X3 montage image (12,000X). The spatial resolution of montage image was improved without any image distortion during montage process (Figure 2). Next, using the montage function, we focused on the morphological alterations of the mitochondria in substantia nigra of hLRRK2 transgenic (G2019S) Parkinson’s disease model mice. Normal mitochondria in cell body and axon of wild-type mice mostly contained well-conserved ultrastructure. They maintained dense matrix, compacted with thin and uniform cristae, and surrounded by clear inner and outer membrane. However, majority of mitochondria in cell body and axon of the substantia nigra of hLRRK2 transgenic mice were swollen, different from the structure found in wildtype mice. Also, the mitochondria have deteriorated structure, with the cristae severely disrupted through the entire mitochondrial lumen, and the inner and outer mitochondrial membranes were partially disappeared. This result clearly shows the complex structural alteration of mitochondria in brain neuronal cells of Parkinson’s disease model mice, and demonstrates the contribution of montage-based large-area TEM image analysis system for the acquisition of high throughput statistical information for an accurate interpretation of various morpho-functional alterations of cellular organelles [7].

Critical importance of the correction of contrast transfer function fortransmission electron microscopy-mediated structural biology

AbstractsBackgroundTransmission electron microscopy (TEM) is an excellent tool for studying detailed biological structures. High-resolution structure determination is now routinely performed using advanced sample preparation techniques and image processing software. In particular, correction for contrast transfer function (CTF) is crucial for extracting high-resolution information from TEM image that is convoluted by imperfect imaging condition. Accurate determination of defocus, one of the major elements constituting the CTF, is mandatory for CTF correction.FindingsTo investigate the effect of correct estimation of image defocus and subsequent CTF correction, we tested arbitrary CTF imposition onto the images of two-dimensional crystals of Rous sarcoma virus capsid protein. The morphology of the crystal in calculated projection maps from incorrect CTF imposition was utterly distorted in comparison to an appropriately CTF-corrected image.ConclusionThis result demonstrates critical importance of CTF correction for producing true representation of the specimen at high resolution.