Apoptosis by aloe-emodin is mediated through down-regulation of calpain-2 and ubiquitin-protein ligase E3A in human hepatoma Huh-7 cells

Natural flavonoids are associated with anti-proliferation of cancer growth. However, the antioxidant and anti-proliferation effects of AE (aloe-emodin) have not been well studied. We have investigated how AE affects the proliferation of hepatic hepatocellular carcinoma cells and exerts an anti-cancer effect. The cytotoxic effect of AE was demonstrated using an MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide] assay and Huh-7 cells were inhibited by AE treatment in both dose- and time-dependent manners. The IC(50) level of AE was ～75 μM. AE also has anti-proliferative effects via induction of DNA damage and apoptosis. 2-DE (two-dimensional electrophoresis) revealed that several proteins were related to the anti-cancer effects of AE. CAPN2 (calpain-2) and UBE3A (ubiquitin-protein ligase E3A), which are associated with the apoptosis signalling pathway, were verified by Western blotting. AE exhibited potent anti-proliferative effects on Huh-7 cells via down-regulation of CAPN2 and UBE3A. The findings support the possibility of AE being a chemopreventative agent.     

Crystal structures of the two membrane-proximal Ig-like domains (D3D4) of LILRB1/B2: alternative models for their involvement in peptide-HLA binding

Leukocyte immunoglobulin-like receptors (LILRs), also called CD85s, ILTs, or LIRs, are important mediators of immune activation and tolerance that contain tandem immunoglobulin (Ig)-like folds. There are 11 (in addition to two pseudogenes) LILRs in total, two with two Ig-like domains (D1D2) and the remaining nine with four Ig-like domains (D1D2D3D4). Thus far, the structural features of the D1D2 domains of LILR proteins are well defi ned, but no structures for the D3D4 domains have been reported. This is a very important fi eld to be studied as it relates to the unknown functions of the D3D4 domains, as well as their relative orientation to the D1D2 domains on the cell surface. Here, we report the crystal structures of the D3D4 domains of both LILRB1 and LILRB2. The two Iglike domains of both LILRB1-D3D4 and LILRB2-D3D4 are arranged at an acute angle (~60°) to form a bent structure, resembling the structures of natural killer inhibitory receptors. Based on these two D3D4 domain structures and previously reported D1D2/HLA I complex structures, two alternative models of full-length (four Ig-like domains) LILR molecules bound to HLA I are proposed.     

Development and Evaluation of Genome-Probing Microarrays for Monitoring Lactic Acid Bacteria

The genome-probing microarray (GPM) was developed for quantitative, high-throughput monitoring of community dynamics in lactic acid bacteria (LAB) fermentation through the deposit of 149 microbial genomes as probes on a glass slide. Compared to oligonucleotide microarrays, the specificity of GPM was remarkably increased to a species-specific level. GPM possesses about 10- to 100-fold higher sensitivity (2.5 ng of genomic DNA) than the currently used 50-mer oligonucleotide microarrays. Since signal variation between the different genomes was very low compared to that of cDNA or oligonucleotide-based microarrays, the capacity of global quantification of microbial genomes could also be observed in GPM hybridization. In order to assess the applicability of GPMs, LAB community dynamics were monitored during the fermentation of kimchi, a traditional Korean food. In this work, approximately 100 diverse LAB species could be quantitatively analyzed as actively involved in kimchi fermentation.     

Kinetic isotope effect studies on the de novo rate of chromophore formation in fast- and slow-maturing GFP variants

The maturation process of green fluorescent protein (GFP) entails a protein oxidation reaction triggered by spontaneous backbone condensation. The chromophore is generated by full conjugation of the Tyr66 phenolic group with the heterocycle, a process that requires C-H bond scission at the benzylic carbon. We have prepared isotope-enriched protein bearing tyrosine residues deuterated at the beta carbon, and have determined kinetic isotope effects (KIEs) on the GFP self-processing reaction. Progress curves for the production of H 2O 2 and the mature chromophore were analyzed by global curve fitting to a three-step mechanism describing preoxidation, oxidation and postoxidation events. Although a KIE for protein oxidation could not be discerned ( k H/ k D = 1.1 +/- 0.2), a full primary KIE of 5.9 (+/-2.8) was extracted for the postoxidation step. Therefore, the exocyclic carbon is not involved in the reduction of molecular oxygen. Rather, C-H bond cleavage proceeds from the oxidized cyclic imine form, and is the rate-limiting event of the final step. Substantial pH-dependence of maturation was observed upon substitution of the catalytic glutamate (E222Q), indicating an apparent p K a of 9.4 (+/-0.1) for the base catalyst. For this variant, a KIE of 5.8 (+/-0.4) was determined for the intrinsic time constant that is thought to describe the final step, as supported by ultra-high resolution mass spectrometric results. The data are consistent with general base catalysis of the postoxidation events yielding green color. Structural arguments suggest a mechanism in which the highly conserved Arg96 serves as catalytic base in proton abstraction from the Tyr66-derived beta carbon.     

Irreversible oxidation of the active-site cysteine of peroxiredoxin to cysteine sulfonic acid for enhanced molecular chaperone activity

The thiol (-SH) of the active cysteine residue in peroxiredoxin (Prx) is known to be reversibly hyperoxidized to cysteine sulfinic acid (-SO(2)H), which can be reduced back to thiol by sulfiredoxin/sestrin. However, hyperoxidized Prx of an irreversible nature has not been reported yet. Using an antibody developed against the sulfonylated (-SO(3)H) yeast Prx (Tsa1p) active-site peptide (AFTFVCPTEI), we observed an increase in the immunoblot intensity in proportion to the H(2)O(2) concentrations administered to the yeast cells. We identified two species of hyperoxidized Tsa1p: one can be reduced back (reversible) with sulfiredoxin, and the other cannot (irreversible). Irreversibly hyperoxidized Tsa1p was identified as containing the active-site cysteine sulfonic acid (Tsa1p-SO(3)H) by mass spectrometry. Tsa1p-SO(3)H was not an autoxidation product of Tsa1p-SO(2)H and was maintained in yeast cells even after two doubling cycles. Tsa1p-SO(3)H self-assembled into a ring-shaped multimeric form was shown by electron microscopy. Although the Tsa1p-SO(3)H multimer lost its peroxidase activity, it gained approximately 4-fold higher chaperone activity compared with Tsa1p-SH. In this study, we identify an irreversibly hyperoxidized Prx, Tsa1p-SO(3)H, with enhanced molecular chaperone activity and suggest that Tsa1p-SO(3)H is a marker of cumulative oxidative stress in cells.     

Statistical superiority of genome-probing microarrays as genomic DNA-DNA hybridization in revealing the bacterial phylogenetic relationship compared to conventional methods

The genomic DNA-DNA hybridization (DDH) method has been widely used as a practical method for the determination of phylogenetic relationships between closely related biological strains. Traditional DDH methods have serious limitations including low reproducibility, a high background and a time-consuming procedure. The DDH method using a genome-probing microarray (GPM) has been recently developed to complement conventional methods and could be used to overcome the limitations that are typically encountered. It is necessary to compare the GPM-based DDH method to the conventional methods before using the GPM for the estimation of genomic similarities since all of the previous scientific data have been entirely dependent on conventional DDH methods. In order to address this issue we compared the DDH values obtained using the GPM, microplate and nylon membrane methods to multi-locus sequence typing (MLST) data for 9 Salmonella genomes and an Escherichia coli type strain. The results showed that the genome similarity values and the degrees of standard deviation obtained using the GPM method were lower than those obtained with the microplate and nylon membrane methods. The dendrogram from the cluster analysis of GPM DDH values was consistent with the phylogenetic tree obtained from the multi-locus sequence typing (MLST) data but was not similar to those obtained using the microplate and nylon membrane methods. Although the signal intensity had to be maximal when the targets were hybridized to their own probe, the methods using membranes and microplates frequently produced higher signals in the heterologous hybridizations than those obtained in the homologous hybridizations. Only the GPM method produced the highest signal intensity in homologous hybridizations. These results show that the GPM method can be used to obtain results that are more accurate than those generated by the other methods tested.     

Suppression of pepper SGT1 and SKP1 causes severe retardation of plant growth and compromises basal resistance

SGT1 associates with suppressor of kinetochore protein (Skp1)-Cullin-F-box (SCF)–ubiquitin-ligase complexes playing important roles in controlling developmental processes and defense responses in plants, yeast, and human. In this study, full-length cDNAs of Sgt1 and Skp1 orthologues were isolated from pepper ( Capsicum annuum L.) to characterize their functions. Protein sequences of CaSgt1 and CaSkp1 showed high degrees of similarities with their homologues in other plant species. Southern blot analyses revealed that CaSgt1 was a single copy gene in the pepper genome, whereas CaSkp1 corresponded to multi copy genes. Levels of CaSgt1 and CaSkp1 mRNAs increased in pepper leaves in response to incompatible pathogen challenge or salicylic acid treatment. Silencing of CaSgt1 or CaSkp1 using Tobacco rattle virus-based virus-induced gene-silencing (VIGS) system resulted in severe dwarfism and final damping-off symptom in the greenhouse. To identify factors determining damping-off symptom in CaSgt1 - or CaSkp1 -silenced plants, we employed VIGS under sterile conditions. Under such conditions, damping-off symptom was not observed suggesting that CaSgt1 and CaSkp1 play an essential role in plant growth and development as well as basal disease resistance in pepper plant.     

Hypochlorous acid induces apoptosis of cultured cortical neurons through activation of calpains and rupture of lysosomes

3-Chlorotyrosine, a bio-marker of hypochlorous acid (HOCl) in vivo, was reported to be substantially elevated in the Alzheimer's disease (AD) brains. Thus, HOCl might be implicated in the development of AD. However, its effect and mechanism on neuronal cell death have not been investigated. Here, we report for the first time that HOCl treatment induces an apoptotic-necrotic continuum of concentration-dependent cell death in cultured cortical neurons. Neurotoxicity caused by an intermediate concentration of HOCl (250 microm) exhibited several biochemical markers of apoptosis in the absence of caspase activation. However, the involvement of calpains was demonstrated by data showing that calpain inhibitors protect cortical neurons from apoptosis and the formation of 145/150 kDa alpha-fodrin fragments. Moreover, an increase in cytosolic Ca2+ concentration was associated with HOCl neurotoxicity and Ca2+ channel antagonists, and Ca2+ chelators prevented cleavage of alpha-fodrin and the induction of apoptosis. Finally, we found that calpain activation ruptured lysosomes. Stabilization of lysosomes by calpain inhibitors or imidazoline drugs, as well as inhibition of cathepsin protease activities, rescued cells from HOCl-induced neurotoxicity. Our results showed for the first time that HOCl induces apoptosis in cortical neurons, and that the cell death process involves calpain activation and rupture of lysosomes.     

Biotransformation of 1,2,3-tri- and 1,2,3,4,7,8-hexachlorodibenzo-p- dioxin by Sphingomonas wittichii strain RW1

Sphingomonas wittichii RW1 is able to catabolize 1,2,3,4-tetrachlorodibenzo-p-dioxin (H. B. Hong, Y. S. Chang, I. H. Nam, P. Fortnagel, and S. Schmidt, Appl. Environ. Microbiol. 68:2584-2588, 2002). Here we demonstrate the aerobic bacterial catabolism of the ubiquitous toxic diaryl ether pollutant 1,2,3,4,7,8-hexachlorodibenzo-p-dioxin by this strain. The products of this biotransformation were identified as tetrachlorocatechol and 2-methoxy-3,4,5,6-tetrachlorophenol by comparing mass spectra recorded before and after n-butylboronate and N,O-bis(trimethylsilyl)-trifluoroacetamide derivatization with those of authentic compounds. Additional experiments showed that the less-chlorinated 1,2,3,7,8-pentachlorodibenzo-p-dioxin was not transformed by the strain RW1. The importance of substitution patterns for the degradability of individual congeners was illustrated by the fact that the 1,2,3-trichlorodibenzo-p-dioxin was catabolized to yield 3,4,5-trichlorocatechol, whereas the 2,3,7-trichlorodibenzo-p-dioxin was not attacked.