Gil-Ja Jhon

Phosphorescent sensor for biological mobile zinc

A new phosphorescent zinc sensor (ZIrF) was constructed, based on an Ir(III) complex bearing two 2-(2,4-difluorophenyl)pyridine (dfppy) cyclometalating ligands and a neutral 1,10-phenanthroline (phen) ligand. A zinc-specific di(2-picolyl)amine (DPA) receptor was introduced at the 4-position of the phen ligand via a methylene linker. The cationic Ir(III) complex exhibited dual phosphorescence bands in CH(3)CN solutions originating from blue and yellow emission of the dfppy and phen ligands, respectively. Zinc coordination selectively enhanced the latter, affording a phosphorescence ratiometric response. Electrochemical techniques, quantum chemical calculations, and steady-state and femtosecond spectroscopy were employed to establish a photophysical mechanism for this phosphorescence response. The studies revealed that zinc coordination perturbs nonemissive processes of photoinduced electron transfer and intraligand charge-transfer transition occurring between DPA and phen. ZIrF can detect zinc ions in a reversible and selective manner in buffered solution (pH 7.0, 25 mM PIPES) with K(d) = 11 nM and pK(a) = 4.16. Enhanced signal-to-noise ratios were achieved by time-gated acquisition of long-lived phosphorescence signals. The sensor was applied to image biological free zinc ions in live A549 cells by confocal laser scanning microscopy. A fluorescence lifetime imaging microscope detected an increase in photoluminescence lifetime for zinc-treated A549 cells as compared to controls. ZIrF is the first successful phosphorescent sensor that detects zinc ions in biological samples.

HSP25 inhibits radiation-induced apoptosis through reduction of PKCδ-mediated ROS production

Since radiation-induced caspase-dependent apoptosis and ROS generation were partially prevented by HSP25 overexpression, similar to the treatment of control cells with antioxidant agents such as DPI and tiron, questions arise whether radiation-mediated ROS generation contributes to the apoptotic cell death, and also whether HSP25 overexpression can reduce ROS mediated apoptotic cell death. In the present study, radiation-induced cytochrome c release from mitochondria and activation of caspases accompanied by a decrease of mitochondrial membrane potential in Jurkat T cells were shown to be inhibited by mitochondrial complex I inhibitor rotenone, suggesting that mitochondrial ROS might be important in radiation-induced caspase-dependent apoptosis. When HSP25 was overexpressed, effects similar to the treatment of cells with the antioxidants were obtained, indicating that HSP25 suppressed radiation-induced mitochondrial alteration that resulted in apoptosis. Furthermore, activation of p38 MAP kinase by radiation was associated with radiation-induced cell death and ROS production and PKCδ was an upstream molecule for p38 MAP kinase activation, ROS generation and subsequent caspase-dependent apoptotic events. However, in the HSP25 overexpressed cells, the above-described effects were blocked. In fact, radiation-induced membrane translocation of PKCδ and tyrosine phosphorylation were inhibited by HSP25. Based on the above data, we suggest that HSP25 downregulates PKCδ, which is a key molecule for radiation-induced ROS generation and mitochondrial-mediated caspase-dependent apoptotic events.

Bonghan System as Mesenchymal Stem Cell Niches and Pathways of Macrophages in Adipose Tissues

A new technique for visualizing Bonghan ducts (BHDs) and Bonghan corpuscles (BHCs) was developed by using a vivi-staining dye, Trypan blue. The dye stains BHDs and BHCs preferentially to adipocytes so that tracking a BHD and a BHC, even inside adipose tissues, is possible. Concerning the functions of the BHD and the BHC in adipose tissues, we propose conjectures: the Bonghan system may be niches for mesenchymal stem cells, which can differentiate into adipocytes, and pathways for macrophages involved in adipogenesis.

Flavonoids and aromatic compounds from the rhizomes ofZingiber zerumbet

Repeated column chromatography of the CHCI3-soluble fraction ofZingiber zerumbet led to the isolation and identification of two aromatic compounds,p-hydroxybenzaldehyde (1) and vanillin (2), and six kaempferol derivatives, kaempferol-3,4′,7-O-trimethylether (3), kaempferol-3-O-methylether (4), kaempferol-3,4′-O-dimethylether (5), 4″-O-acetylafzelin (6), kaempferol-3-O-(4-O-acetyl-α-L-rhamnopyranoside)], 2″,4″-O-diacetylafzelin (7), kaempferol-3-O-(2,4-O-diacetyl-α-L-rhamnopyranoside)], and 3″,4″-O-diacetylafzelin (8), kaempferol-3-O-(3,4-O-diacetyl-α-L-rhamnopyranoside)]. The structures of1–8 were identified by analysis of spectro-scopic data as well as by comparison with published values. This is the first report on the isolation of compounds1–3 from this plant.

Separation of gangliosides using cyclodextrin in capillary zone electrophoresis.

Gangliosides are glycosphingolipids containing sialic acid. These glycolipids have been suggested to play important roles in biological processes such as cell growth, differentiation and malignant transformation. Based on these proposed biological functions, gangliosides can be used as diagnostic tools and therapeutics for various human diseases. In this study, capillary zone electrophoresis (CZE) was used to determine the major gangliosides GM1, GD1a, GD1b and GT1b in mammalian brains, in addition to GM3 and Lac. Enhancement of selectivity and efficiency of separation was obtained by using 50 mM borate-phosphate buffer containing 16.5 mM alpha-cyclodextrin (alpha-CD). Under this condition, several forms of gangliosides were successfully separated from extracts of deer antler, apricot seed and rat brain. The results demonstrate that the CD-modified CZE is a useful method for detecting glycolipids from various biological matrices.

Monoacetyldiglycerides as new Ca2+ mobilizing agents in rat pancreatic acinar cells

Several monoacetyldiglycerides were synthesized from glycerol in search for new Ca2+ mobilizing agent in vitro. All monoacetyldiglycerides except linolenoyl and phenlycyclopropylcarbonyl derivatives showed activity toward Ca2+ release in pancreatic acinar cells. Linoleoyl and docosahexaenoyl derivatives were chosen for further test and exhibited unique activity.

Fluorescence ratiometric zinc sensors based on controlled energy transfer

The high-fidelity detection of labile zinc is of central importance for understanding the molecular mechanisms that link zinc homeostasis and human pathophysiology. Fluorescence ratiometric sensors are most suitable for the detection and trafficking of intracellular zinc ions. Here, we report the development of fluorescence ratiometric zinc sensors (HN1 and HN2) based on two-fluorophore platforms. The sensor constructs include blue fluorescent umbelliferone and an energy-accepting chromophore that absorbs the blue fluorescence. Zinc binding was found to promote fluorescence turn-on of the umbelliferone emission by suppression of intramolecular photoinduced electron transfer, thereby facilitating resonance energy transfer to the energy acceptors. The net observables were the fluorescence ratiometric changes, the extent of which depended strongly on the chemical structures of the acceptors. Photophysical investigations, including steady-state and transient photoluminescence spectroscopy, suggested a mechanism for the fluorescent zinc response that involved a combination of the intramolecular electron transfer and the interchromophoric energy transfer. The zinc probes displayed sensing capability that is suitable for the detection of biological zinc ions, with good selectivity, pH tolerance, and appropriate Kd values. Finally, zinc detection was demonstrated by fluorescence ratiometric visualization of exogenously supplied zinc ions in live HeLa cells. The probes enabled the reliable monitoring of zinc equilibration across the cell membrane.

Real-Time in Vivo Simultaneous Measurements of Nitric Oxide and Oxygen Using an Amperometric Dual Microsensor

This paper reports a real-time study of the codynamical changes in the release of endogenous nitric oxide (NO) and oxygen (O(2)) consumption in a rat neocortex in vivo upon electrical stimulation using an amperometric NO/O(2) dual microsensor. Electrical stimulation induced transient cerebral hypoxia due to the increased metabolic demands that were not met by the blood volume inside the stimulated cortical region. A NO/O(2) dual microsensor was successfully used to monitor the pair of real-time dynamic changes in the tissue NO and O(2) contents. At the onset of electrical stimulation, there was an immediate decrease in the cortical tissue O(2) followed by a subsequent increase in the cortical tissue NO content. The averages of the maximum normalized concentration changes induced by the stimulation were a 0.41 (±0.04)-fold decrease in the O(2) and a 3.6 (±0.9)-fold increase in the NO concentrations when compared with the corresponding normalized basal levels. The peak increase in NO was always preceded by the peak decrease in O(2) in all animals (n = 11). The delay between the maximum decrease in O(2) and the maximum increase in NO varied from 3.1 to 54.8 s. This rather wide variation in the temporal associations was presumably attributed to the sparse distribution of NOS-containing neurons and the individual animals differences in brain vasculatures, which suggests that a sensor with fine spatial resolution is needed to measure the location-specific real-time NO and O(2) contents. In summary, the developed NO/O(2) dual microsensor is effective for measuring the NO and O(2) contents in vivo. This study provides direct support for the dynamic role of NO in regulating the cerebral hemodynamics, particularly related to the tissue oxygenation.

Reactive oxygen species regulate M-CSF-induced monocyte/macrophage proliferation through SHP1 oxidation

Macrophage colony-stimulating factor (M-CSF) stimulation results in the production of reactive oxygen species (ROS) that participate in the proliferation of monocyte/macrophage. However, the molecular mechanisms whereby ROS modulate the signaling processes of M-CSF remain poorly defined. We report here that the redox-sensitive Src homology region 2 domain-containing phosphatase 1 (SHP1) is a critical regulator of M-CSF-mediated signaling in bone marrow monocyte/macrophage lineage cells (BMMs). Application of diphenylene iodonium (DPI) inhibited the responses of BMMs to M-CSF, including ROS production, cell proliferation, and phosphorylation of c-Fms as well as Akt kinase, but not of MAP kinases such as ERK, p38, and JNK. Dysregulation of SHP1 by overexpression or RNA interference in BMMs showed that SHP1 specifically regulates PI3 kinase (PI3K)/Akt signaling, but not MAP kinases in a redox-dependent manner, thereby regulating proliferation of BMMs through cyclins D1 and D2. These findings demonstrate that M-CSF-mediated ROS generation leads to SHP1 oxidation, which promotes cell proliferation through the PI3K/Akt-dependent signaling pathway.

Identification of triacylglycerols containing two short-chain fatty acids at sn-2 and sn-3 positions from bovine udder by fast atom bombardment tandem mass spectrometry

Several triacylglycerol (TAG) molecular species, that contain two short-chain fatty acids (C4 to C8) at the sn-2 and sn-3 positions of the glycerol backbone, were isolated from bovine udder by using solvent extraction and silica gel column chromatography. Their structures were identified by fast atom bombardment (FAB) tandem mass spectrometry (MS/MS), based on the information obtained from collision-induced dissociation (CID) spectra of sodium-adducted molecules ([M + Na](+)) of model TAG compounds which had been synthesized from glycerol and appropriate fatty acids. For each species, the relative positions of the three fatty acids on the glycerol backbone, as well as fatty acid composition and double-bond position in the fatty acyl group, were determined. A majority of sodium-adducted molecules observed in the FAB mass spectrum were mixtures of at least two components that have different fatty acid composition but the same molecular mass. In addition, all the components present in mixtures of all the species contain a long-chain fatty acid (C12 to C18) at the sn-1 position, a short-chain fatty acid (C4 to C8) at the sn-2 position, and a butyric acid uniquely at the sn-3 position.