Ligand binding inhibitors of A1 adenosine receptor from Rana rugosa are phospholipase A2s.

Inhibitors of the A1 adenosine receptor were isolated from the skin extract of Korean frog, Rana rugosa. The frog-skin extract was prepared by an electrical shock and fractionated with C4 followed by C18 reverse-phase HPLC. Two A1 receptor inhibitors were isolated using a filter binding assay and the molecular masses of the proteins were estimated by matrix-assisted laser desorption ionization time-of-flight mass spectrometry to be 15 347 and 15 404 Da, respectively. The inhibitory activity was also measured against other membrane receptors, such as the A2 adenosine receptor, muscarinic acetylcholine receptor and capsaicin receptor. Ligand binding to the A2 and muscarinic receptors was also severely inhibited by these proteins. However, they did not inhibit the functional activation of the capsaicin receptor by its ligand, capsaicin, suggesting that inhibition of ligand-receptor binding occurs specifically. Their N-terminal sequences were determined by Edman degradation. Surprisingly, they showed sequence similarity to the secretory protein, phospholipase A2 from various organisms. The phospholipase A2 activity of both proteins was tested using Dole's assay technique. Both proteins showed phospholipase A2 activity, and therefore, they were designated as PLA2-R1 and PLA2-R2, respectively. In addition, their ligand-binding inhibitory activity depended on their phospholipase A2 activity. This is the first finding that the frog secretes a phospholipase A2 similar to that of snake venoms, which posess inhibitory activity against the adenosine A1, adenosine A2 and muscarinic receptors.     

Functional compensation for adipose differentiation-related protein (ADFP) by Tip47 in an ADFP null embryonic cell line

Ectopic accumulation of lipid droplets in non-adipose tissues correlates with the degree of insulin resistance in these tissues. Emerging evidence indicates that lipid droplets are specialized organelles that participate in lipid metabolism and intracellular trafficking. These properties are thought to derive from the lipid droplet-associated PAT protein family (perilipin, ADFP, and Tip47). The functions of the ubiquitously distributed adipose differentiation-related protein (ADFP) and Tip47 remain unknown. To evaluate the roles of ADFP and Tip47 in lipid biogenesis and metabolism, ADFP null and wild type (wt) clonal cell lines were established from ADFP null and wt mice, respectively. In ADFP null cells, Tip47 was identified as the sole lipid droplet-associated protein from the PAT family by mass spectroscopy, which was further confirmed by immunoblotting and immunocytochemistry. Following incubation with oleic acid, ADFP null cells were able to form lipid droplets to the same extent as wt cells. No statistical differences between the two cell types were observed in NEFA uptake or lipolysis. Small interference RNAs (siRNAs) against Tip47 were found to down-regulate protein levels for Tip47 by 85%. ADFP null cells treated with Tip47 siRNA retained the ability to form lipid droplets but to a lesser extent and shunted the utilization of exogenously added NEFA from triglycerides to phospholipids. These data support the hypothesis that Tip47 plays an important role in lipid metabolism. Tip47 and ADFP in peripheral tissues may play a critical role in regulating the formation and turnover, and hence metabolic consequences, of ectopic fat.     

Anisotropic nucleation growth of actin bundle: a model for determining the well-defined thickness of bundles

Biopolymers such as DNA, F-actins, and microtubules, which are highly charged, rodlike polyelectrolytes, are assembled into architectures with defined morphology and size by electrostatic interaction with multivalent cations (or polycations) in vivo and in vitro. The physical origin to determine their morphology and size is not clearly understood yet. Our results show that the actin bundle formation consists of two stages: the thickness of actin bundles is determined nearly at the initial stage, while the length of actin bundles is determined later on. It is also found that the thickness of actin bundles decreases with the increase of polycation-mediated attraction between F-actins. From these results, we propose the anisotropic nucleation-growth mechanism, in which the thickness of actin bundles is determined by critical nucleus size, whereas the length of actin bundles is determined by the concentration of free actins relative to nucleus concentration. Observing that polycations are concentrated in some sites of actin bundles, which are thought to be nucleation sites to initiate the formation of actin bundles, supports this model. This anisotropic nucleation-growth mechanism of actin bundles can be broadly applied to the self-assembly of rodlike polyelectrolytes.     

Memory-enhancing effect of a supercritical carbon dioxide fluid extract of the needles of Abies koreana on scopolamine-induced amnesia in mice

Abies koreana Wilson (A. koreana) is a shrub or broadly pyramidal evergreen tree endemic in the mountainous regions of South Korea. We obtained the essential oil (EO) from alpine needle leaves of A. koreana by the supercritical fluid extraction (SFE) method. EO was analyzed by gas chromatography-mass spectrometry (GC-MS), and 68 compounds were identified constituting 95.66% of the oil. The major components were elemol (11.17%), terpinen-4-ol (9.77%), sabinene (8.86%), 10(15)-cadien-4-ol (7.16%), alpha-terpineol (6.13%), alpha-pinene (6.07%) and gamma-terpinene (4.71%). To investigate the memory-enhancing effects, we conducted a passive avoidance test using a scopolamine (1 mg/kg, ip)-induced amnesia mouse model. A peritoneal injection of EO from A. koreana (100 mg/kg) showed a memory enhancing effect of 72.7% compared with the control. These results suggest that EO of A. koreana may be a useful therapeutic agent against such amnesia-inducing diseases as Alzheimer and vascular dementia.     

The SL1 stem-loop structure at the 5'-end of potato virus X RNA is required for efficient binding to host proteins and for viral infectivity

The 5'-region of Potato virus X (PVX) RNA, which contains an AC-rich, single-stranded region and stem-loop structure 1 (SL1), affects RNA replication and assembly. Using Systemic Evolution of Ligands by EXponential enrichment (SELEX) and the electrophoretic mobility shift assay, we demonstrate that SL1 interacts specifically with tobacco protoplast protein extracts (S100). The 36 nucleotides that correspond to the top region of SL1, which comprises stem C, loop C, stem D, and the tetra loop (TL), were randomized and bound to the S100. Remarkably, the wild-type (wt) sequence was selected in the second round, and the number of wt sequences increased as selection proceeded. All of the selected clones from the fifth round contained the wt sequence. Secondary structure predictions (mFOLD) of the recovered sequences revealed relatively stable stem-loop structures that resembled SL1, although the nucleotide sequences therein were different. Moreover, many of the clones selected in the fourth round conserved the TL and C-C mismatch, which suggests the importance of these elements in host protein binding. The SELEX clone that closely resembled the wt SL1 structure with the TL and C-C mismatch was able to replicate and cause systemic symptoms in plants, while most of the other winners replicated poorly only on inoculated leaves. The RNA replication level on protoplasts was also similarly affected. Taken together, these results indicate that the SL1 of PVX interacts with host protein(s) that play important roles related to virus replication.     

Enhanced nuclear factor-kappa B-associated Wnt-1 expression in hepatitis B- and C-related hepatocarcinogenesis: identification by functional proteomics

Summary Chronic infections with hepatitis B and C viruses (HBV and HCV) are etiologically linked to hepatitis, liver cirrhosis, and hepatocellular carcinoma (HCC). Both viruses may induce activation of nuclear factor-kappa B (NF-κB) in hepatocytes that plays a crucial role in the regulation of cell growth and apoptosis. Functional proteomics analysis of proteins associated with NF-κB signaling complexes in both viruses-related HCC tumor and non-tumor tissues may disclose possible common mechanisms in hepatocarcinogenesis. By functional proteomics, we analyzed proteins associated with NF-κB-signaling complexes in four-paired human HCC tumor and non-tumor tissues from HBV- and HCV-infected patients, respectively, and in one-paired tissue with dual viral infection. The quantity of NF-κB-associated proteins was semi-quantitatively measured by protein spot intensity on the gels of two-dimensional polyacrylamide gel electrophoresis. The results showed that overexpression of NF-κB-associated Wnt-1 protein in tumor part was detected in the␣majority of HBV- and HCV-infected HCC samples. These data suggest that enhanced expression of NF-κB-associated Wnt-1 protein might be a mechanism of hepatocarcinogenesis common to HBV- and HCV-infected patients. NF-κB signaling pathway and Wnt-1 protein could be potential targets for designing highly effective therapeutic agents in treating HCC and for chemoprevention of hepatocarcinogenesis.     

Detergent-free caveolae proteome suggests an interaction with ER and mitochondria

Recent proteomic studies of detergent resistant membrane fractions have begun to characterize the protein composition of caveolae and lipid rafts. The methods used in most of these studies, however, are not able to distinguish between plasma membrane and internal membrane lipid domains. Here we used a non-detergent method for obtaining fractions enriched in caveolae derived from the plasma membrane of multiple cell types. Unexpectedly, the proteins in the caveolae proteome suggest these lipid domains may interact with elements of ER and mitochondria. A comparison of the partial proteome we obtained with other published reports identifies 26 proteins that are candidate marker proteins for identifying caveolae in multiple cell types.     

Dendrimer-like DNA-based fluorescence nanobarcodes

A major challenge in clinical diagnostics and environmental analysis is the difficulty in rapid and sensitive detection of multiple target molecules simultaneously (i.e., multiplexed detections). Our group has designed and synthesized a dendrimer-like DNA (DL-DNA) that is multivalent and anisotropic; using this unique DNA structure, we have developed a fluorescence-tagged nanobarcode system for multiplex detection. This nanobarcode system allows the rapid and sensitive detection of multiple pathogens simultaneously using the ratios of two different fluorescent dyes, green and red, with which different DL-DNAs are labeled. The key step of our nanobarcode model lies in the monodisperse preparation of DL-DNA. Two methods, solution phase and solid phase, are presented here. With slight modifications, this platform technology can also be extended to the multiplexed detection of RNA and proteins. This protocol can be completed in 2-5 d.     

Conversion of alloantigen-specific CD8+ T cell anergy to CD8+ T cell priming through in vivo ligation of glucocorticoid-induced TNF receptor

In this study, we investigated the effect of an agonistic mAb (DTA-1) against glucocorticoid-induced TNF receptor (GITR) in a murine model of systemic lupus erythematosus-like chronic graft-vs-host disease (cGVHD). A single dose of DTA-1 inhibited the production of anti-DNA IgG1 autoantibody and the development of glomerulonephritis, typical symptoms of cGVHD. DTA-1-treated mice showed clinical and pathological signs of acute GVHD (aGVHD), such as lymphopenia, loss of body weight, increase of donor cell engraftment, and intestinal damage, indicating that DTA-1 shifted cGVHD toward aGVHD. The conversion of cGVHD to aGVHD occurred because DTA-1 prevented donor CD8+ T cell anergy. Functionally active donor CD8+ T cells produced high levels of IFN-gamma and had an elevated CTL activity against host Ags. In in vitro MLR, anergic responder CD8+ T cells were generated, and DTA-1 stimulated the activation of these anergic CD8+ T cells. We further confirmed in vivo that donor CD8+ T cells, but not donor CD4+ T cells, were responsible for the DTA-1-mediated conversion of cGVHD to aGVHD. These results indicate that donor CD8+ T cell anergy is a restriction factor in the development of aGVHD and that in vivo ligation of GITR prevents CD8+ T cell anergy by activating donor CD8+ T cells that otherwise become anergic. In sum, our data suggest GITR as an important costimulatory molecule regulating cGVHD vs aGVHD and as a target for therapeutic intervention in a variety of related diseases.