Curcumin induces apoptosis through mitochondrial hyperpolarization and mtDNA damage in human hepatoma G2 cells

Curcumin, a major pigment of turmeric, is a natural antioxidant possessing a variety of pharmacological activities and therapeutic properties. But its mechanisms are unknown. In our previous study, we found that a 2-h exposure to curcumin induced DNA damage to both the mitochondrial DNA (mtDNA) and the nuclear DNA (nDNA) in HepG2 cells and that mtDNA damage was more extensive than nDNA damage. Therefore, experiments were initiated to evaluate the role of mtDNA damage in curcumin-induced apoptosis. The results demonstrated that HepG2 cells challenged with curcumin for 1 h showed a transient elevation of the mitochondrial membrane potential (DeltaPsim), followed by cytochrome c release into the cytosol and disruption of DeltaPsim after 6 h exposure to curcumin. Apoptosis was detected by Hoechst 33342 and annexin V/PI assay after 10 h treatment. Interestingly, the expression of Bcl-2 remained unchanged. A resistance to apoptosis for the corresponding rho0 counterparts confirmed a critical dependency for mitochondria during the induction of apoptosis in HepG2 cells mediated by curcumin. The effects of PEG-SOD in protecting against curcumin-induced cytotoxicity suggest that curcumin-induced cytotoxicity is directly dependent on superoxide anion O2- production. These data suggest that mitochondrial hyperpolarization is a prerequisite for curcumin-induced apoptosis and that mtDNA damage is the initial event triggering a chain of events leading to apoptosis in HepG2 cells.     

Comparative and functional genomics reveals genetic diversity and determinants of host specificity among reference strains and a large collection of Chinese isolates of the phytopathogen Xanthomonas campestris pv. campestris

Background

Xanthomonas campestris pathovar campestris (Xcc) is the causal agent of black rot disease of crucifers worldwide. The molecular genetic diversity and host specificity of Xcc are poorly understood.

Results

We constructed a microarray based on the complete genome sequence of Xcc strain 8004 and investigated the genetic diversity and host specificity of Xcc by array-based comparative genome hybridization analyses of 18 virulent strains. The results demonstrate that a genetic core comprising 3,405 of the 4,186 coding sequences (CDSs) spotted on the array are conserved and a flexible gene pool with 730 CDSs is absent/highly divergent (AHD). The results also revealed that 258 of the 304 proved/presumed pathogenicity genes are conserved and 46 are AHD. The conserved pathogenicity genes include mainly the genes involved in type I, II and III secretion systems, the quorum sensing system, extracellular enzymes and polysaccharide production, as well as many other proved pathogenicity genes, while the AHD CDSs contain the genes encoding type IV secretion system (T4SS) and type III-effectors. A Xcc T4SS-deletion mutant displayed the same virulence as wild type. Furthermore, three avirulence genes (avrXccC, avrXccE1 and avrBs1) were identified. avrXccC and avrXccE1 conferred avirulence on the hosts mustard cultivar Guangtou and Chinese cabbage cultivar Zhongbai-83, respectively, and avrBs1 conferred hypersensitive response on the nonhost pepper ECW10R.

Conclusion

About 80% of the Xcc CDSs, including 258 proved/presumed pathogenicity genes, is conserved in different strains. Xcc T4SS is not involved in pathogenicity. An efficient strategy to identify avr genes determining host specificity from the AHD genes was developed.     

Assessment of algorithms for high throughput detection of genomic copy number variation in oligonucleotide microarray data

Background

Genomic deletions and duplications are important in the pathogenesis of diseases, such as cancer and mental retardation, and have recently been shown to occur frequently in unaffected individuals as polymorphisms. Affymetrix GeneChip whole genome sampling analysis (WGSA) combined with 100 K single nucleotide polymorphism (SNP) genotyping arrays is one of several microarray-based approaches that are now being used to detect such structural genomic changes. The popularity of this technology and its associated open source data format have resulted in the development of an increasing number of software packages for the analysis of copy number changes using these SNP arrays.

Results

We evaluated four publicly available software packages for high throughput copy number analysis using synthetic and empirical 100 K SNP array data sets, the latter obtained from 107 mental retardation (MR) patients and their unaffected parents and siblings. We evaluated the software with regards to overall suitability for high-throughput 100 K SNP array data analysis, as well as effectiveness of normalization, scaling with various reference sets and feature extraction, as well as true and false positive rates of genomic copy number variant (CNV) detection.

Conclusion

We observed considerable variation among the numbers and types of candidate CNVs detected by different analysis approaches, and found that multiple programs were needed to find all real aberrations in our test set. The frequency of false positive deletions was substantial, but could be greatly reduced by using the SNP genotype information to confirm loss of heterozygosity.     

The evaluation of FHB resistance QTLs introgressed into elite Canadian spring wheat germplasm

FHB resistance QTL alleles from Nyuubai, Sumai-3, and Wuhan-1 were evaluated for their effect on Fusarium head blight (FHB) index, Fusarium damaged kernels (FDK), deoxynivalenol (DON) accumulation, plant height, anthesis date, and numerous grain quality traits in three elite Canadian spring wheat backgrounds. The three FHB resistance parameters were negatively correlated with plant height in the three populations. The Wuhan-1 4B resistance allele was the most effective resistance allele but was associated with a 9.3 cm increase in plant height. The Wuhan-1 2D, Nyuubai 3BSc, Sumai-3 3BSc, Nyuubai 5AS, and Sumai-3 5AS alleles were also effective FHB resistance alleles in these populations. The Nyuubai and Sumai-3 3BS alleles were the least effective of the FHB resistance alleles in the FHB nursery tests. The Sumai-3 5AS resistance allele was significantly associated with reduced grain protein content, while the same trend was observed for the Nyuubai 5AS resistance allele but was not significant. FHB resistance tended to increase with more FHB resistance alleles introgressed into the elite genetic background, which suggested that marker-assisted selection (MAS) will prove useful for improving FHB resistance in Canadian germplasm.     

Phosphorylation site analysis of the anti-inflammatory and mRNA-destabilizing protein tristetraprolin

Tristetraprolin (TTP) is a member of the CCCH zinc finger proteins and is an anti-inflammatory protein. Mice deficient in TTP develop a profound inflammatory syndrome with erosive arthritis, autoimmunity and myeloid hyperplasia. TTP binds to mRNA AU-rich elements with high affinity for UUAUUUAUU nucleotides and causes destabilization of those mRNA molecules. TTP is phosphorylated extensively in vivo and is a substrate for multiple protein kinases in vitro. A number of approaches have been used to identify its phosphorylation sites. This article highlights the recent progress and different approaches utilized for the identification of phosphorylation sites in mammalian TTP. Important but limited results are obtained using traditional methods, including in vivo labeling, site-directed mutagenesis, phosphopeptide mapping and protein sequencing. Mass spectrometry (MS), including MALDI/MS, MALDI/MS/MS, liquid chromatography/MS/MS, immobilized metal ion affinity chromatography (IMAC)/MALDI/MS/MS and multidimensional protein identification technology has led the way in identifying TTP phosphorylation sites. The combination of these approaches has identified multiple phosphorylation sites in mammalian TTP, some of which are predicted by motif scanning to be phosphorylated by several protein kinases. This information should provide the molecular basis for future investigation of TTP's regulatory functions in controlling proinflammatory cytokines.     

Novel symmetric amphiphilic dendritic poly(L-lysine)-b-poly(L-lactide)-b-dendritic poly(L-lysine) with high plasmid DNA binding affinity as a biodegradable gene carrier

This study communicates the molecular design, preparation, and biological application of novel symmetric amphiphilic polycationic dendritic poly(L-lysine)-b-poly(L-lactide)-b-dendritic poly(L-lysine) D2-LLA15-D2 bearing two two-generation poly(L-lysine) PLL dendrons D2 and a central hydrophobic biodegradable poly(L-lactide) block LLA15. First, an amino-protected precursor of L1-OH was designed and synthesized and was further employed to prepare L1-LLA15 with an organic 4-(dimethylamino)-pyridine-mediated living-ring-opening polymerization of l-lactide. Subsequently, the hydroxy end-capped L1-LLA15 was coupled to synthesize a new triblock L1-LLA15-L1 with two one-generation amino-protected PLL dendrons L1. Furthermore, with a repeated trifluoroacetic-acid-mediated amino deprotection-protection cycle, new amphiphilic triblock D2-LLA15-D2 was successfully prepared. By means of NMR, mass spectrometry, and gel permeation chromatography, these synthetic precursors and final amphiphilic product were characterized to bear well-defined triblock structures. In addition, this synthesized amphiphilic triblock polycationic macromolecule was applied as a new polycationic plasmid DNA carrier, and its DNA binding affinity was examined via an agarose electrophoresis and a fluorescence titration assay along with two important references of hydrophilic dendritic D2-HEX-D2 and double-hydrophilic D2-PEG-4K-D2 bearing the same two D2 dendrons; much enhanced DNA binding affinity was interestingly revealed for the new amphiphilic structural D2-LLA15-D2. Moreover, the assembled polyplex microparticles of plasmid DNA/polycationic carrier were further analyzed by dynamic light scattering and transmission electron microscopy, indicating their averaged nanoparticle size around 150-200 nm. As for the cytotoxicity of the new D2-LLA15-D2, MTT assays were conducted with a human hepatocellular carcinoma cell line (SMMC-7721), indicating a very low cytotoxicity as compared with commercial linear PLL-23K and PEI-2K, and a DNase I degradation of the assembled polyplex particles was also done in the HBS buffer solution to evaluate their stabilities. Finally, employing the new amphiphilic D2-LLA15-D2 as gene carrier, in vitro gene transfection experiments were conducted with the SMMC-7721 cell line, indicating a transfection efficiency increase of at least 10 times higher than that of the naked plasmid DNA under a N/P charge ratio of 10. Therefore, these interesting results may provide a new possible way to construct efficient polycationic macromolecular gene carriers with low toxicity and less expensive low-generation PLL dendrons.     

Synthesis of alginic acid-poly[2-(diethylamino)ethyl methacrylate] monodispersed nanoparticles by a polymer-monomer pair reaction system

In this paper, alginic acid-poly(2-(diethylamino)ethyl methacrylate) (ALG-PDEA) nanoparticles were successfully prepared in aqueous medium using a polymer-monomer pair reaction system consisting of the anionic alginic acid (ALG) and the cationic 2-(diethylamino)ethyl methacrylate (DEA), without any aid of surfactants or organic solvents. The ALG-PDEA nanoparticles were monodispersed and stable in aqueous solution. Nanoparticles with desired size could be obtained by varying the amount of initiator or changing the concentration of reactants in solution, which renders this system highly controllable. After the ALG moiety was gelled by Ca(2+), the stability of the nanoparticles in basic or high salt concentration solutions could be notably enhanced. A pH-sensitive anticancer agent, hydroxycamptothecin (HCPT), was encapsulated in ALG-PDEA nanoparticles, and preliminary in vitro release as well as cytotoxicity experiments were carried out. It is found that this system seems to be a very promising carrier for the loading and delivery of labile drugs, taking into account that the preparation procedure is simple, mild, and organic solvent- and surfactant-free. Moreover, the abundant functional groups on the particle surface, such as carboxyls and hydroxyls, allow subsequent chemical modification, which may further unleash the potential of such a system in either biomedical applications or in the construction of other functional mesoscopic architectures.     

A new algorithm using cross-assignment for label-free quantitation with LC-LTQ-FT MS

A new algorithm is described for label-free quantitation of relative protein abundances across multiple complex proteomic samples. Q-MEND is based on the denoising and peak picking algorithm, MEND, previously developed in our laboratory. Q-MEND takes advantage of the high resolution and mass accuracy of the hybrid LTQ-FT MS mass spectrometer (or other high-resolution mass spectrometers, such as a Q-TOF MS). The strategy, termed "cross-assignment", is introduced to increase substantially the number of quantitated proteins. In this approach, all MS/MS identifications for the set of analyzed samples are combined into a master ID list, and then each LC-MS run is searched for the features that can be assigned to a specific identification from that master list. The reliability of quantitation is enhanced by quantitating separately all peptide charge states, along with a scoring procedure to filter out less reliable peptide abundance measurements. The effectiveness of Q-MEND is illustrated in the relative quantitative analysis of Escherichia coli samples spiked with known amounts of non-E. coli protein digests. A mean quantitation accuracy of 7% and mean precision of 15% is demonstrated. Q-MEND can perform relative quantitation of a set of LC-MS data sets without manual intervention and can generate files compatible with the Guidelines for Proteomic Data Publication.