Seungah Lee

Caspase-independent death of human osteosarcoma cells by flavonoids is driven by p53-mediated mitochondrial stress and nuclear translocation of AIF and endonuclease G

Flavonoids have antioxidant and antitumor promoting effects. Rhus verniciflua Stokes (RVS) is a flavonoid-rich herbal medicine that has long been used in Korea as both a food additive and antitumor agent. It was previous reported that a purified flavonoid fraction prepared from RVS, herein named RCMF (the RVS chloroform-methanol fraction), inhibited the proliferation and induced apoptosis in human osteosarcoma (HOS) cells. This study examined the mechanisms involved in the RCMF-mediated apoptosis in HOS cells. RCMF was shown to be capable of inducing apoptosis in HOS cells by inducing p53 in the cells resulting in the decrease in Bcl-2 level, activation of Bax, and cytoplasmic release of cytochrome c, which led to the translocation of apoptosis-inducing factor (AIF) and endonuclease G (EndoG) into the nucleus. However, the RCMF-induced apoptosis was suppressed by transfecting the cells with antisense p53 oligonucleotides but not by treating them with a MAPK or caspase inhibitor. This suppression occurred through the regulation of Bcl-2 members as well as by preventing the nuclear translocation of the mitochondrial apoptogenic factors. Overall, it appears that p53-mediated mitochondrial stress and the nuclear translocation of AIF and EndoG are mainly required for the apoptosis induced by RCMF.

Quercetin inhibits α-MSH-stimulated melanogenesis in B16F10 melanoma cells.

Quercetin is known to inhibit tyrosinase activity and melanin production in melanocytes. However, several reports suggest that quercetin has different and opposite effects on melanogenesis. This study examined the precise effects of quercetin on melanogenesis using cell‐free assay systems and melanocytes. Quercetin inhibited the monophenolase and diphenolase activities of tyrosinase, and melanin synthesis in cell‐free assay systems. Quercetin induced mild stimulation of the tyrosinase activity and dihydroxyphenylalaminechrome tautomerase (TRP‐2) expression but only at low concentrations (<20 μm) in B16F10 melanoma cells. In contrast, the addition of 50 μm quercetin to the cells led to a significant decrease in the activity and synthesis of tyrosinase, as well as a decrease in the expression of tyrosinase‐related protein‐1 and TRP‐2 proteins, regardless of the presence or absence of α‐melanocyte stimulating hormone (α‐MSH). Quercetin also reduced the intracellular cAMP and the phosphorylated protein kinase A levels in α‐MSH‐stimulated B16F10 cells. Moreover, quercetin (20 μm) diminished the expression and activity of tyrosinase, and melanin content in cultured normal human epidermal melanocytes. These effects were not related to its cytotoxic action. Although the in vivo effects of quercetin are still unclear, these results suggest that quercetin could play important roles in controlling melanogenesis. Copyright

Super-resolution of fluorescence-free plasmonic nanoparticles using enhanced dark-field illumination based on wavelength-modulation

Super-resolution imaging of fluorescence-free plasmonic nanoparticles (NPs) was achieved using enhanced dark-field (EDF) illumination based on wavelength-modulation. Indistinguishable adjacent EDF images of 103-nm gold nanoparticles (GNPs), 40-nm gold nanorods (GNRs), and 80-nm silver nanoparticles (SNPs) were modulated at their wavelengths of specific localized surface plasmon scattering. The coordinates (x, y) of each NP were resolved by fitting their point spread functions with a two-dimensional Gaussian. The measured localization precisions of GNPs, GNRs, and SNPs were 2.5 nm, 5.0 nm, and 2.9 nm, respectively. From the resolved coordinates of NPs and the corresponding localization precisions, super-resolution images were reconstructed. Depending on the spontaneous polarization of GNR scattering, the orientation angle of GNRs in two-dimensions was resolved and provided more elaborate localization information. This novel fluorescence-free super-resolution method was applied to live HeLa cells to resolve NPs and provided remarkable sub-diffraction limit images.

Optical Super-Resolution Imaging of Surface Reactions

Optical super-resolution imaging has gained momentum in investigations of heterogeneous and homogeneous chemical reactions at the single-molecule level. Thanks to its exceptional spatial resolution and ability to monitor dynamic systems, much detailed information on single-molecule reaction/adsorption processes and single-particle catalytic processes has been revealed, including chemical kinetics and reaction dynamics; active-site distributions on single-particle surfaces; and size-, shape-, and facet-dependent catalytic activities of individual nanocatalysts. In this review, we provide an overview of recent advances in super-resolution chemical imaging of surface reactions.

Ultra-sensitive plasmonic nanometal scattering immunosensor based on optical control in the evanescent field layer.

Novel, fluorescence-free detection of biomolecules on nanobiochips was investigated based on plasmonic nanometal scattering in the evanescent field layer (EFL) using total internal reflection scattering (TIRS) microscopy. The plasmonic scattering of nanometals bonded to biomolecules was observed at different wavelengths by an electromagnetic field in the EFL. The changes in the scattering of nanometals on the gold-nanopatterned chip in response to the immunoreaction between silver nanoparticles and antibodies allowed fluorescence-free detection of biomolecules on the nanobiochips. Under optimized conditions, the TIRS immunoassay chip detected different amounts of immobilized antigen, i.e., human cardiac troponin I. The sandwich immuno-reaction was quantitatively analyzed in the dynamic range of 720 zM-167 fM. The limit of detection (S/N=4) was 600 zM, which was ~140 times lower than limits obtained by previous total internal reflection fluorescence and dark field methods. These results demonstrate the possibility for a fluorescence-free biochip nanoimmunoassay based on the scattering of nanometals in the EFL.

Quantitative analysis of human serum leptin using a nanoarray protein chip based on single-molecule sandwich immunoassay.

We report a method for the quantitative analysis of human serum leptin, which is a protein hormone associated with obesity, using a nanoarray protein chip based on a single-molecule sandwich immunoassay. The nanoarray patterning of a biotin-probe with a spot diameter of 150 nm on a self-assembled monolayer functionalized by MPTMS on a glass substrate was successfully accomplished using atomic force microscopy (AFM)-based dip-pen nanolithography (DPN). Unlabeled leptin protein molecules in human serum were detected based on the sandwich fluorescence immunoassay by total internal reflection fluorescence microscopy (TIRFM). The linear regression equation for leptin in the range of 100 zM-400 aM was determined to be y=456.35 x+80,382 (R=0.9901). The accuracy and sensitivity of the chip assay were clinically validated by comparing the leptin level in adult serum obtained by this method with those measured using the enzyme-linked immunosorbent assay (ELISA) performed with the same leptin standards and serum samples. In contrast to conventional ELISA techniques, the proposed chip methodology exhibited the advantages of ultra-sensitivity, a smaller sample volume and faster analysis time.

Chloroform Extract of Alfalfa (Medicago sativa) Inhibits Lipopolysaccharide-Induced Inflammation by Downregulating ERK/NF-κB Signaling and Cytokine Production

Alfalfa (Medicago sativa L.) is commonly used as a traditional medicine and functional food. This study investigated the anti-inflammatory potential of alfalfa and the mechanisms involved. The chloroform extract of alfalfa aerial parts inhibited lipopolysaccharide (LPS)-stimulated immune responses more than ether, butanol, or water soluble extracts. Treatment with 1 μg/mL LPS increased nitrite concentrations to 44.3 μM in RAW267.4 macrophages, but it was reduced to 10.6 μM by adding 100 μg/mL chloroform extract. LPS treatment also increased the concentrations of tumor necrosis factor-α, interleukin (IL)-6, and IL-1β to 41.3, 11.6, and 0.78 ng/mL in culture supernatants of the cells, but these cytokine levels decreased to 12.5, 3.1, and 0.19 ng/mL, respectively, by pretreating with 100 μg/mL of the extract. ICR mice injected with LPS (30 mg/kg body weight) alone showed a 0% survival rate after 48 h of the injection, but 48-h survival of the mice increased to 60% after oral administration of the extract. Subfractions of the chloroform extract markedly suppressed LPS-mediated activation of the extracellular signal-regulated kinase and nuclear factor kappa-B. Cinnamic acid derivatives and fatty acids were found to be active constituents of the extract. This research demonstrated that alfalfa aerial parts exert anti-inflammatory activity and may be useful as a functional food for the prevention of inflammatory disorders.

Real-Time Observations of Intracellular Mg2+ Signaling and Waves in a Single Living Ventricular Myocyte Cell

Despite the important regulatory role of Mg(2+) in metabolic pathways, its underlying mechanism is not completely understood at the single-cell level. This study examined the propagation and dynamics of Mg(2+) signaling across the cell membrane by employing the real-time visualization of intracellular Mg(2+) waves in living ventricular myocytes using a combination of total internal reflection fluorescence microscopy and Nomarski differential interference contrast. Real-time Mg(2+) waves and sparks in a living cell membrane were observed using a fluorescent Mg(2+) indicator (mag-fluo-4-AM) in the concentration range of 5 aM-5 muM. The intracellular locations of the fluorescent Mg(2+) indicator were confirmed by adding Na(+)ATP. The Mg(2+) sparks and waves showed random temporal propagation patterns in nonhomogeneous substructures. These results show that spatiotemporal intracellular Mg(2+) signaling information can be obtained for individual living cells.

Selective fluorescent-free detection of biomolecules on nanobiochips by wavelength dependent-enhanced dark field illumination

Individual silver nanoparticle-conjugated target protein (cTnI) molecules on gold-nanopatterned chip were selectively detected by wavelength dependent-enhanced dark field illumination. Using specific nanoparticles with unique sizes and materials, the immunotargeted nanoparticle on the chips was detected at the single-molecule level by monitoring changes in the plasmonic resonance based on wavelength dependence.

An ultra-sensitive nanoarray chip based on single-molecule sandwich immunoassay and TIRFM for protein detection in biologic fluids

This paper describes a single-molecule sandwich immunoassay method that utilizes total internal reflection fluorescence microscopy (TIRFM) at the single-molecule level for nanoarray protein chip applications. Nanoarray patterning of a biotin-probe with a spot diameter of 179 +/- 1 nm was performed successfully on a (3-mercaptopropyl)trimethoxysilane (MPTMS)-coated glass substrate by atomic force microscopy (AFM). The formation of biotin patterns was confirmed directly by observing the heights of bound streptavidin and biotin-antibody on glass substrates using an AFM in contact mode. Target protein molecules (or antigen) at the zepto-molar (zM) concentration level (x 10(-21) M) were detected on MPTMS-coated glass nanoarray protein chips by TIRFM. Finally, cytokine clinical samples (i.e. TNF-alpha and IL-1alpha) as cancer marker protein molecules were applied to nanoarray protein chips, and detection limits were at 600 zM.