Hyaluronan- and RNA-binding deubiquitinating enzymes of USP17 family members associated with cell viability

BACKGROUND: Protein degradation by the ubiquitin system plays a crucial role in numerous cellular signaling pathways. Deubiquitination, a reversal of ubiquitination, has been recognized as an important regulatory step in the ubiquitin-dependent degradation pathway. RESULTS: While identifying putative ubiquitin specific protease (USP) enzymes that contain a conserved Asp (I) domain in humans, 4 USP17 subfamily members, highly homologous to DUB-3, have been found (USP17K, USP17L, USP17M, and USP17N), from human chorionic villi. Expression analysis showed that USP17 transcripts are highly expressed in the heart, liver, and pancreas and are expressed moderately in various human cancerous cell lines. Amino acid sequence analysis revealed that they contain the highly conserved Cys, His, and Asp domains which are responsible for the deubiquitinating activity. Biochemical enzyme assays indicated that they have deubiquitinating activity. Interestingly, the sequence analysis showed that these proteins, with exception of USP17N, contain the putative hyaluronan/RNA binding motifs, and cetylpyridinium chloride (CPC)-precipitation analysis confirmed the association between these proteins and intracellular hyaluronan and RNA. CONCLUSION: Here, we report that the overexpression of these proteins, with exception of USP17N, leads to apoptosis, suggesting that the hyaluronan and RNA binding motifs in these enzymes play an important role in regulating signal transduction involved in cell death

Superparamagnetic Iron Oxide Nanoparticles Coated with Galactose-Carrying Polymer for Hepatocyte Targeting

Our goal is to develop the functionalized superparamagnetic iron oxide nanoparticles (SPIONs) demonstrating the capacities to be delivered in liver specifically and to be dispersed in physiological environment stably. For this purpose, SPIONs were coated with polyvinylbenzyl-O-β-D-galactopyranosyl-D-gluconamide (PVLA) having galactose moieties to be recognized by asialoglycoprotein receptors (ASGP-R) on hepatocytes. For use as a control, we also prepared SPIONs coordinated with 2-pyrrolidone. The sizes, size distribution, structure, and coating of the nanoparticles were characterized by transmission electron microscopy (TEM), electrophoretic light scattering spectrophotometer (ELS), X-ray diffractometer (XRD), and Fourier transform infrared (FT-IR), respectively. Intracellular uptake of the PVLA-coated SPIONs was visualized by confocal laser scanning microscopy, and their hepatocyte-specific delivery was also investigated through magnetic resonance (MR) images of rat liver. MRI experimental results indicated that the PVLA-coated SPIONs possess the more specific accumulation property in liver compared with control, which suggests their potential utility as liver-targeting MRI contrast agent

Junctional membrane inositol 1,4,5-trisphosphate receptor complex coordinates sensitization of the silent EGF-induced Ca2+ signaling

Ca2+ is a highly versatile intracellular signal that regulates many different cellular processes, and cells have developed mechanisms to have exquisite control over Ca2+ signaling. Epidermal growth factor (EGF), which fails to mobilize intracellular Ca2+ when administrated alone, becomes capable of evoking [Ca2+]i increase and exocytosis after bradykinin (BK) stimulation in chromaffin cells. Here, we provide evidence that this sensitization process is coordinated by a macromolecular signaling complex comprised of inositol 1,4,5-trisphosphate receptor type I (IP3R1), cAMP-dependent protein kinase (PKA), EGF receptor (EGFR), and an A-kinase anchoring protein, yotiao. The IP3R complex functions as a focal point to promote Ca2+ release in two ways: (1) it facilitates PKA-dependent phosphorylation of IP3R1 in response to BK-induced elevation of cAMP, and (2) it couples the plasmalemmal EGFR with IP3R1 at the Ca2+ store located juxtaposed to the plasma membrane. Our study illustrates how the junctional membrane IP3R complex connects different signaling pathways to define the fidelity and specificity of Ca2+ signaling

Identification of compounds using HPLC-QTOF-MS online antioxidant activity mapping from aerial parts of Ligularia stenocephala

Abstract Inflammation, diabetes, and even malignancies are pharmacological effects connected by antioxidant capacity and free radicals. Many antioxidants scavenge free radicals originating from dietary sources such as fruits, vegetables, and teas. To identify the bioactive components of Ligularia stenocephala, an effective method combining HPLC-QTOF-MS and bioactivity evaluation was investigated for the first time. Antioxidant agents were isolated from L. stenocephala, a folk medicine used for edema and scrofula in Korea, Japan, and China. The phytochemical investigation of the aerial parts of L. stenocephala resulted in the separation and determination of six compounds (1–6). In particular, the chemical structures were identified as hyperoside (1), 3,5-dicaffeoylquinic acid (2), 3,5-dicaffeoylquinic acid methyl ester (3), trifolin (4), rutin (5), and 3,4-dicaffeoylquinic acid (6). Their structures were identified using 1D and 2D NMR spectroscopy and high-resolution electrospray ionization mass spectrometry (HR-ESI-MS) data analysis. The results showed that phenolic components were responsible for the antioxidant inhibitory activity of L. stenocephala. Additionally, to understand the mechanisms of the antioxidant inhibitory activity of L. stenocephala, a docking simulation study was performed to support the in vitro results. Taken together, this new method is rapid, inexpensive, and can be applied to identify the active components of medicinal herbs without separation

Hyaluronan- and RNA-binding deubiquitinating enzymes of USP17 family members associated with cell viability

Abstract Background Protein degradation by the ubiquitin system plays a crucial role in numerous cellular signaling pathways. Deubiquitination, a reversal of ubiquitination, has been recognized as an important regulatory step in the ubiquitin-dependent degradation pathway. Results While identifying putative ubiquitin specific protease (USP) enzymes that contain a conserved Asp (I) domain in humans, 4 USP17 subfamily members, highly homologous to DUB-3, have been found (USP17K, USP17L, USP17M, and USP17N), from human chorionic villi. Expression analysis showed that USP17 transcripts are highly expressed in the heart, liver, and pancreas and are expressed moderately in various human cancerous cell lines. Amino acid sequence analysis revealed that they contain the highly conserved Cys, His, and Asp domains which are responsible for the deubiquitinating activity. Biochemical enzyme assays indicated that they have deubiquitinating activity. Interestingly, the sequence analysis showed that these proteins, with exception of USP17N, contain the putative hyaluronan/RNA binding motifs, and cetylpyridinium chloride (CPC)-precipitation analysis confirmed the association between these proteins and intracellular hyaluronan and RNA. Conclusion Here, we report that the overexpression of these proteins, with exception of USP17N, leads to apoptosis, suggesting that the hyaluronan and RNA binding motifs in these enzymes play an important role in regulating signal transduction involved in cell death.</p

Characterization of Anti-Inflammatory and Antioxidant Constituents from Scutellaria baicalensis Using LC-MS Coupled with a Bioassay Method

An effective and previously demonstrated screening method for active constituents in natural products using LC-MS coupled with a bioassay was reported in our earlier studies. With this, the current investigation attempted to identify bioactive constituents of Scutellaria baicalensis through LC-MS coupled with a bioassay. Peaks at broadly 17&ndash;20 and 24&ndash;25 min on the MS chromatogram displayed an inhibitory effect on NO production in lipopolysaccharide-induced BV2 microglia cells. Similarly, peaks at roughly 17&ndash;19 and 22 min showed antioxidant activity with an 2,2&prime;-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS)/2,2-diphenyl-1- picrylhydrazyl (DPPH) assay. For confirmation of LC-MS coupled with a bioassay, nine compounds (1&ndash;9) were isolated from an MeOH extract of S. baicalensis. As we predicted, compounds 1, 8, and 9 significantly reduced lipopolysaccharide (LPS)-induced NO production in BV2 cells. Likewise, compounds 5, 6, and 8 exhibited free radical-scavenging activities with the ABTS/DPPH assay. In addition, the structural similarity of the main components was confirmed by analyzing the total extract and EtOAc fractions through molecular networking. Overall, the results suggest that the method comprised of LC-MS coupled with a bioassay can effectively predict active compounds without an isolation process, and the results of molecular networking predicted that other components around the active compound node may also be active

Mannosylated polyethylenimine coupled mesoporous silica nanoparticles for receptor-mediated gene delivery

Organic-inorganic nanohybrids have been studied for their use as non-viral transfection agents. The purpose of this study was to examine the ability of mesoporous silica nanoparticles (MSN) coupled with mannosylated polyethylenimine (MP) to transfect plasmid DNA in vitro. Although MSN is biocompatible and has low cytotoxicity, it is not easily transfected into a variety of cell types. To overcome this barrier, MP was coupled to MSN (abbreviated as MPS) to target macrophage cells with mannose receptors and enhance transfection efficiency. The DNA conveyance ability of MPS was examined by evaluating properties such as particle size, zeta potential, complex formation, protection of plasmid DNA against DNase-I, and the release of DNA upon cell entry. Particle sizes of the MPS/DNA complexes decreased with increasing weight ratio of MPS to DNA, while the zeta potential increased. Complete MPS/DNA complexes were formed at a weight ratio of five, and their resistance to DNase-I was evaluated. Cytotoxicity studies showed that MPS/DNA complexes resulted in a high percentage of cell viability, compared with PEI 25K as a vector. The transfection efficiency of MPS/DNA complexes was evaluated on Raw 264.7 and HeLa cell lines. It was found that MPS/DNA complexes showed enhanced transfection efficiency through receptor-mediated endocytosis via mannose receptors. These results indicate that MPS can be employed in the future as a potential gene carrier to antigen presenting cells. (C) 2008 Elsevier B.V. All rights reserved

Poly (amino ester) composed of poly (ethylene glycol) and aminosilane prepared by combinatorial chemistry as a gene carrier

Purpose. Application of combinatorial chemistry and high throughput screening for the synthesis and evaluation of mini-library of novel biodegradable poly (beta-amino ester)s (PAE)s composed of gamma-aminopropyl-triethoxysilane (APES) and poly (ethylene glycol) diacrylate (PEGDA) for gene delivery efficiency and safety in 293T and HeLa cells in the presence of and absence of serum. Materials and methods. PAEs were synthesized at different mole ratios of APES and PEGDA by Michael addition reaction and synthesis was confirmed by H-1 nuclear magnetic resonance (H-1-NMR). Ninety six ratios of polyplexes were evaluated for luciferase and MTS assay in 293T and HeLa cells in the presence of and absence of serum. Relationship between transfection efficiency and DNA binding ability of PAEs was studied by gel electrophoresis. Particle sizes and molecular weight of selected PAEs were measured by dynamic light scattering and gel permeation chromatography multi-angle light scattering, respectively. Results. H-1-NMR confirmed the synthesis of PAEs. In both cell lines, transfection efficiency and cell viability were increased for PAEs obtained from R106 (0.7:1, APES:PEGDA) to R121 (6:1, APES:PEGDA) with a marginal increase in APES concentration. Transfection pattern was uniform in the absence of and presence of serum. In both cell lines, PAE obtained from R121 demonstrated high transfection efficiency and low cytotoxicity as compared to polyethylenimine (25 KDa) and Lipofectamine. PAE obtained from R121 showed good DNA binding and condensation with average particle sizes of 133 nm. Conclusion. Addition of PEGDA over APES resulted in a novel PAE which has high safety and transfection efficiency. Transfection and cytotoxicity are very sensitive to monomer ratios and mainly governed by concentration of amine monomer