Dramatic down-regulation of oxidoreductases in human hepatocellular carcinoma hepG2 cells: proteomics and gene ontology unveiling new frontiers in cancer enzymology

Background

Oxidoreductases are enzymes that catalyze many redox reactions in normal and neoplastic cells. Their actions include catalysis of the transformation of free, neutral oxygen gas into oxygen free radicals, superoxide, hydroperoxide, singlet oxygen and hydrogen peroxide. These activated forms of oxygen contribute to oxidative stress that modifies lipids, proteins, DNA and carbohydrates. On the other hand, oxidoreductases constitute one of the most important free radical scavenger systems typified by catalase, superoxide dismutase and glutathione peroxidase. In this work, proteomics, Gene Ontology mapping and Directed Acyclic Graphs (DAG) are employed to detect and quantify differential oxidoreductase enzyme expressions between HepG2 cells and normal human liver tissues.

Results

For the set of bioinformatics calculations whose BLAST searches are performed using the BLAST program BLASTP 2.2.13 [Nov-27-2005], DAG of the Gene Ontology's Molecular Function annotations show that oxidoreductase activity parent node of the liver proteome contains 331 annotated protein sequences, 7 child nodes and an annotation score of 188.9, whereas that of HepG2 cells has 188 annotated protein sequences, 3 child nodes and an annotation score of only 91.9. Overwhelming preponderance of oxidoreductases in the liver is additionally supported by the isomerase DAGs: nearly all the reactions described in the normal liver isomerase DAG are oxidoreductase isomerization reactions, whereas only one of the three child nodes in the HepG2 isomerase DAG is oxidoreductase. Upon normalization of the annotation scores to the parent Molecular Function nodes, oxidoreductases are down-regulated in HepG2 cells by 58%. Similarly, for the set of bioinformatics calculations whose BLAST searches are carried out using BLASTP 2.2.15 [Oct-15-2006], oxidoreductases are down-regulated in HepG2 cells by 56%.

Conclusion

Proteomics and Gene Ontology reveal, for the first time, differential enzyme activities between HepG2 cells and normal human liver tissues, which may be a promising new prognostic marker of Hepatocellular carcinoma. Two independent sets of bioinformatics calculations that employ two BLAST program versions, and searched different databases, arrived at essentially the same conclusion: oxidoreductases are down-regulated in HepG2 cells by approximately 57%, when compared to normal human liver tissues. Down-regulation of oxidoreductases in hepatoma is additionally supported by Gene Ontology analysis of isomerises.     

Sample prep for proteomics of breast cancer: proteomics and gene ontology reveal dramatic differences in protein solubilization preferences of radioimmunoprecipitation assay and urea lysis buffers

Background

An important step in the proteomics of solid tumors, including breast cancer, consists of efficiently extracting most of proteins in the tumor specimen. For this purpose, Radio-Immunoprecipitation Assay (RIPA) buffer is widely employed. RIPA buffer's rapid and highly efficient cell lysis and good solubilization of a wide range of proteins is further augmented by its compatibility with protease and phosphatase inhibitors, ability to minimize non-specific protein binding leading to a lower background in immunoprecipitation, and its suitability for protein quantitation.

Results

In this work, the insoluble matter left after RIPA buffer extraction of proteins from breast tumors are subjected to another extraction step, using a urea-based buffer. It is shown that RIPA and urea lysis buffers fractionate breast tissue proteins primarily on the basis of molecular weights. The average molecular weight of proteins that dissolve exclusively in urea buffer is up to 60% higher than in RIPA. Gene Ontology (GO) and Directed Acyclic Graphs (DAG) are used to map the collective biological and biophysical attributes of the RIPA and urea proteomes. The Cellular Component and Molecular Function annotations reveal protein solubilization preferences of the buffers, especially the compartmentalization and functional distributions. It is shown that nearly all extracellular matrix proteins (ECM) in the breast tumors and matched normal tissues are found, nearly exclusively, in the urea fraction, while they are mostly insoluble in RIPA buffer. Additionally, it is demonstrated that cytoskeletal and extracellular region proteins are more soluble in urea than in RIPA, whereas for nuclear, cytoplasmic and mitochondrial proteins, RIPA buffer is preferred. Extracellular matrix proteins are highly implicated in cancer, including their proteinase-mediated degradation and remodelling, tumor development, progression, adhesion and metastasis. Thus, if they are not efficiently extracted by RIPA buffer, important information may be missed in cancer research.

Conclusion

For proteomics of solid tumors, a two-step extraction process is recommended. First, proteins in the tumor specimen should be extracted with RIPA buffer. Second, the RIPA-insoluble material should be extracted with the urea-based buffer employed in this work.     

2-Hydroxyphenacyl azoles and related azolium derivatives as antifungal agents

2-Hydroxyphenacyl azole and 2-hydroxyphenacyl azolium compounds have been described as a new class of azole antifungals. Most target compounds showed significant in vitro antifungal activities against tested fungi (Candida albicans, Saccharomyces cerevisiae, Aspergillus niger, and Microsporum gypseum) with low MICs values included in the range of 0.25-32 microg/mL comparable to reference drug fluconazole. The most active compounds were also assessed for their cytotoxicity using MTT colorimetric assay on normal mouse fibroblast (NIH/3T3) cells. The results of antifungal activity and toxicity tests indicated that these compounds display antifungal activity at non-cytotoxic concentrations.     

Cell division requires a direct link between microtubule-bound RacGAP and Anillin in the contractile ring

The mitotic microtubule array plays two primary roles in cell division. It acts as a scaffold for the congression and separation of chromosomes, and it specifies and maintains the contractile-ring position. The current model for initiation of Drosophila and mammalian cytokinesis [1-5] postulates that equatorial localization of a RhoGEF (Pbl/Ect2) by a microtubule-associated motor protein complex creates a band of activated RhoA [6], which subsequently recruits contractile-ring components such as actin, myosin, and Anillin [1-3]. Equatorial microtubules are essential for continued constriction, but how they interact with the contractile apparatus is unknown. Here, we report the first direct molecular link between the microtubule spindle and the actomyosin contractile ring. We find that the spindle-associated component, RacGAP50C, which specifies the site of cleavage [1-5], interacts directly with Anillin, an actin and myosin binding protein found in the contractile ring [7-10]. Both proteins depend on this interaction for their localization. In the absence of Anillin, the spindle-associated RacGAP loses its association with the equatorial cortex, and cytokinesis fails. These results account for the long-observed dependence of cytokinesis on the continual presence of microtubules at the cortex.     

A comparison of alternative methods to compute conditional genotype probabilities for genetic evaluation with finite locus models

An increased availability of genotypes at marker loci has prompted the development of models that include the effect of individual genes. Selection based on these models is known as marker-assisted selection (MAS). MAS is known to be efficient especially for traits that have low heritability and non-additive gene action. BLUP methodology under non-additive gene action is not feasible for large inbred or crossbred pedigrees. It is easy to incorporate non-additive gene action in a finite locus model. Under such a model, the unobservable genotypic values can be predicted using the conditional mean of the genotypic values given the data. To compute this conditional mean, conditional genotype probabilities must be computed. In this study these probabilities were computed using iterative peeling, and three Markov chain Monte Carlo (MCMC) methods – scalar Gibbs, blocking Gibbs, and a sampler that combines the Elston Stewart algorithm with iterative peeling (ESIP). The performance of these four methods was assessed using simulated data. For pedigrees with loops, iterative peeling fails to provide accurate genotype probability estimates for some pedigree members. Also, computing time is exponentially related to the number of loci in the model. For MCMC methods, a linear relationship can be maintained by sampling genotypes one locus at a time. Out of the three MCMC methods considered, ESIP, performed the best while scalar Gibbs performed the worst.     

Penetration of the Blood-Brain Barrier by Bacillus anthracis Requires the pXO1-Encoded BslA Protein

Anthrax is a zoonotic disease caused by the gram-positive spore-forming bacterium Bacillus anthracis. Human infection occurs after the ingestion, inhalation, or cutaneous inoculation of B. anthracis spores. The subsequent progression of the disease is largely mediated by two native virulence plasmids, pXO1 and pXO2, and is characterized by septicemia, toxemia, and meningitis. In order to produce meningitis, blood-borne bacteria must interact with and breach the blood-brain barrier (BBB) that is composed of a specialized layer of brain microvascular endothelial cells (BMEC). We have recently shown that B. anthracis Sterne is capable of penetrating the BBB in vitro and in vivo, establishing the classic signs of meningitis; however, the molecular mechanisms underlying the central nervous system (CNS) tropism are not known. Here, we show that attachment to and invasion of human BMEC by B. anthracis Sterne is mediated by the pXO1 plasmid and an encoded envelope factor, BslA. The results of studies using complementation analysis, recombinant BslA protein, and heterologous expression demonstrate that BslA is both necessary and sufficient to promote adherence to brain endothelium. Furthermore, mice injected with the BslA-deficient strain exhibited a significant decrease in the frequency of brain infection compared to mice injected with the parental strain. In addition, BslA contributed to BBB breakdown by disrupting tight junction protein ZO-1. Our results identify the pXO1-encoded BslA adhesin as a critical mediator of CNS entry and offer new insights into the pathogenesis of anthrax meningitis.Bacillus anthracis, the etiologic agent of anthrax, is a gram-positive spore-forming bacterium that is commonly found in soil (29). The bacterium can infect animals and humans by ingestion, inhalation, or cutaneous inoculation of B. anthracis spores (8). Spores are taken up by resident macrophages that migrate to the lymph nodes (15). Here, the spores germinate into vegetative bacteria, multiply, and then disseminate throughout the host, causing septicemia and toxemia (8). Systemic disease can be complicated by the onset of a fulminant and rapidly fatal hemorrhagic meningitis and meningoencephalitis (27). Anthrax meningitis is associated with a high mortality rate despite intensive antibiotic therapy (24). Biopsy studies after an outbreak of inhalational anthrax and experimental studies of inhalational infection in rhesus monkeys demonstrated the presence of bacilli in the central nervous system (CNS) and pathologies consistent with suppurative and hemorrhagic meningitis in the majority of cases (1, 12). The intentional release of B. anthracis spores (19) during the 2001 bioterrorism event resulted in a case of meningitis (19), necessitating a need for a better understanding of the pathogenesis of anthrax meningitis and CNS infection.To cause meningitis, blood-borne bacteria must interact with and breach the blood-brain barrier (BBB). The majority of the BBB is anatomically represented by the cerebral microvascular endothelium; brain microvascular endothelial cells (BMEC) are joined by tight junctions and display a paucity of pinocytosis, thereby effectively limiting the passage of substances and maintaining the CNS microenvironment (4, 5). Despite its highly restrictive nature, certain bacterial pathogens are still able to penetrate the BBB and gain entry into the CNS. The presence of bacilli in the brains of patients (1, 24) and in experimental models of anthrax infection (42, 44) suggests that vegetative B. anthracis cells are able to cross the BBB to initiate meningeal inflammation and the classic pathology associated with meningitis.B. anthracis harbors two large virulence plasmids, pXO1 and pXO2 (8), which are required for full virulence, as strains lacking these plasmids are attenuated in animal models of infection (29). B. anthracis Sterne (pXO1⁺ pXO2⁻) has been utilized as a vaccine strain (41) but is still widely used in both in vitro and in vivo studies of anthrax infection since it causes lethal disease in mouse models of infection (46). Despite the crucial roles of pXO1 and pXO2 in anthrax disease pathogenesis, very few plasmid-encoded factors have been characterized. The best described are the antiphagocytic polyglutamyl capsule, encoded by biosynthetic enzymes on pXO2, and the anthrax toxin complex comprised of protective antigen, lethal factor (LF), and edema factor (EF), encoded by pXO1 (8, 29). Sequence analysis of the pXO1 plasmid revealed that the majority of plasmid-encoded factors, ∼70%, were of unknown function (31). More recently, in silico analysis identified novel pXO1-encoded proteins with immunogenic potential and relevance for pathogenesis. These included factors with putative adherent and invasive properties (2). Interestingly, two of the immunoreactive proteins were predicted surface layer (S-layer) proteins (2), one of which, B. anthracis S-layer protein A (BslA, pXO1-90), has recently been described and shown to mediate adherence of the vegetative form to host cells (20).Using in vitro and in vivo model systems, we have recently shown that B. anthracis Sterne adheres to and invades brain endothelium (44). This interaction was partially dependent on the pXO1-encoded anthrax toxins; however, the molecular mechanisms that contribute to B. anthracis penetration of the BBB are currently unknown. In this study, we investigate the role of pXO1 in B. anthracis Sterne''s interaction with brain endothelium and identify the encoded BslA adhesin as a critical mediator for BBB attachment and penetration during the pathogenesis of anthrax meningitis.     

You can't always get what you want: size assortative mating by mutual mate choice as a resolution of sexual conflict

Background  

Assortative mating patterns for mate quality traits like body size are often observed in nature. However, the underlying mechanisms that cause assortative mating patterns are less well known. Sexual selection is one important explanation for assortment, suggesting that i) one (usually the female) or both sexes could show preferences for mates of similar size or ii) mutual mate choice could resolve sexual conflict over quality traits into assortment. We tested these hypotheses experimentally in the socially monogamous cichlid fish Pelvicachromis taeniatus, in which mate choice is mutual.     

Neutrophil Gelatinase-Associated Lipocalin induces the expression of heme oxygenase-1 and superoxide dismutase <Subscript>1, 2</Subscript>

Lipocalin-2 (Lcn2, NGAL) is a member of the lipocalin super family with diverse function such as the induction of apoptosis, the suppression of bacterial growth, and modulation of inflammatory response. Much interest has recently been focused on the physiological/pathological role of the lipocalin-2 that is considered to be a novel protective factor against oxidative stress. However, its precise biological roles in this protection are not fully understood. In this report we intended to test the effect of lipocalin-2 on the expression of heme oxygenase _{(1, 2)} and superoxide dismutase _{(1, 2)} which are two strong antioxidants. NGAL was cloned to pcDNA3.1 plasmid by using genetic engineering method. The recombinant vector was transfected to CHO and HEK293T to establish stable cell expressing NGAL and the expression of HO-1, 2 and SOD_{1, 2} were compared with appropriate controls by RT-PCR and western blot. On the other hand, expression of NGAL was suppressed by siRNA transfection in order to study the effect of lipocalin-2 on mentioned genes/proteins. The results showed that the expression of HO-1 and SOD_{1, 2} enzymes were higher in cells expressing recombinant lipocalin-2 compared with the control cells. Although the expression of HO-1 was lower in NGAL silencing cells, the expression of SOD₁ and SOD₂ were higher. Our data suggest that NGAL is a potent inducer of HO-1 and somewhat SOD₁ and SOD₂ and it appears that part of antioxidant property of NGAL could be attributed to the induction of HO-1and SOD_{1, 2}.     

Identification of GABA A receptor modulators in Kadsura longipedunculata and assignment of absolute configurations by quantum-chemical ECD calculations

A petroleum ether extract of Kadsura longipedunculata enhanced the GABA-induced chloride current (I_GABA) by 122.5 ± 0.3% (n = 2) when tested at 100 μg/ml in Xenopuslaevis oocytes expressing GABA A receptors (α₁β₂γ_2S subtype) in two-microelectrode voltage clamp measurements. Thirteen compounds were subsequently identified by HPLC-based activity profiling as responsible for GABA A receptor activity and purified in preparative scale. 6-Cinnamoyl-6,7-dihydro-7-myrceneol and 5,6-dihydrocuparenic acid were thereby isolated for the first time. The determination of the absolute stereochemistry of these compounds was achieved by comparison of experimental and calculated ECD spectra. All but one of the 13 isolated compounds from K. longipedunculata potentiated I_GABA through GABA A receptors composed of α₁β₂γ_2S subunits in a concentration-dependent manner. Potencies ranged from 12.8 ± 3.1 to 135.6 ± 85.7 μM, and efficiencies ranged from 129.7 ± 36.8% to 885.8 ± 291.2%. The phytochemical profiles of petroleum ether extracts of Kadsura japonica fruits (114.1 ± 2.6% potentiation of I_GABA at 100 μg/ml, n = 2), and Schisandra chinensis fruits (inactive at 100 μg/ml) were compared by HPLC-PDA-ESIMS with that of K. longipedunculata.     

Mass and relative elution time profiling: two-dimensional analysis of sphingolipids in Alzheimer's disease brains

Current lipidomic profiling methods rely mainly on MS to identify unknown lipids within a complex sample. We describe a new approach, involving LC×MS/MS (liquid chromatography×tandem MS) analysis of sphingolipids based on both mass and hydrophobicity, and use this method to characterize the SM (sphingomyelin), ceramide and GalCer (galactosylceramide) content of hippocampus from AD (Alzheimer's disease) and control subjects. Using a mathematical relationship we exclude the influence of sphingolipid mass on retention time, and generate two-dimensional plots that facilitate accurate visualization and characterization of the different ceramide moieties within a given sphingolipid class, because related molecules align horizontally or vertically on the plots. Major brain GalCer species that differ in mass by only 0.04 Da were easily differentiated on the basis of their hydrophobicity. The importance of our method's capacity to define all of the major GalCer species in the brain samples is illustrated by the novel observation that the proportion of GalCer with hydroxylated fatty acids increased approximately 2-fold in the hippocampus of AD patients, compared with age- and gender-matched controls. This suggests activation of fatty acid hydroxylase in AD. Our method greatly improves the clarity of data obtained in a lipid profiling experiment and can be expanded to other lipid classes.