Comparative proteomic analysis of advanced serous epithelial ovarian carcinoma: possible predictors of chemoresistant disease

To identify specific proteins associated with chemotherapeutic responses, we analyzed protein expression patterns in stage IIIc primary serous epithelial ovarian cancer tissues displaying differential responses to first-line postoperative adjuvant chemotherapy. The expression profiles of five chemoresistant tissues [progression-free survival (PFS) ≤12 months] and five chemosensitive tissues (PFS ≥48 months) were analyzed with 2D electrophoresis, and the spot intensities of differentially expressed proteins were quantified. To validate these proteins as markers for chemoresistant disease, we analyzed tissues from an additional 17 patients. All the patients were allocated to the over- or underexpressing group according to protein spot intensity, and survival analysis was performed. In chemoresistant tissues, four proteins (thioredoxin domain containing four, similar to RIKEN cDNA 1700016G05, tubulin α 1A chain, and the pyruvate dehydrogenase E1-β subunit precursor) were overexpressed, and seven proteins [keratin 1, vitamin D-binding protein, creatine kinase B, annexin V, SH3-containing guanine nucleotide exchange factor (SGEF), tryptophan-aspartate repeat protein-1 (WDR 1), and WDR 1 isoform 1] were underexpressed. The underexpression of keratin 1, creatine kinase B, annexin V, SGEF, WDR1, and WDR1 isoform 1 were significantly correlated with poor overall survival. A combination of keratin 1 and SGEF showed the highest sensitivity of 0.800, specificity of 0.917, PPV of 0.800, and NPV of 0.917 in predicting chemoresistant disease. These proteins may be useful as predictive markers of chemoresistant disease. However, further analyses in large-scale should be performed before they can be considered reliable predictive markers of chemoresistant disease.     

Targeted mutagenesis of zebrafish: use of zinc finger nucleases

The modeling of human disease in the zebrafish (Danio rerio) is moving away from chemical mutagensis and transient downregulation using morpholino oligomers to more targeted and stable transgenic methods. In this respect, zinc finger nucleases offer a means of introducing mutations at targeted sites at high efficiency. We describe here the development of zinc finger nucleases and their general use in model systems with a focus on the zebrafish.     

Characterization of TioQ, a type II thioesterase from the thiocoraline biosynthetic cluster

An antitumor agent thiocoraline is a thiodepsipeptide marine product derived from two Micromonospora sp. strains that inhibits protein synthesis by binding of its key 3-hydroxyquinaldic acid (3HQA) chromophores to duplex DNA. There are at least two potential pathways via which the 3HQA moiety could be biosynthesized from L-Trp. By biochemical characterization and by preparation of knockouts of an adenylation-thiolation enzyme, TioK, and of two type II thioesterases, TioP and TioQ, found in the thiocoraline biosynthetic gene cluster, we gained valuable insight into the pathway followed for the production of 3HQA.     

Deficient spindle assembly checkpoint in multiple myeloma

Multiple myeloma (MM) is a hematological disease characterized by an abnormal accumulation of plasma cells in the bone marrow. These cells have frequent cytogenetic abnormalities including translocations of the immunoglobulin heavy chain gene and chromosomal gains and losses. In fact, a singular characteristic differentiating MM from other hematological malignancies is the presence of a high degree of aneuploidies. As chromosomal abnormalities can be generated by alterations in the spindle assembly checkpoint (SAC), the functionality of such checkpoint was tested in MM. When SAC components were analyzed in MM cell lines, the RNA levels of most of them were conserved. Nevertheless, the protein content of some key constituents was very low in several cell lines, as was the case of MAD2 or CDC20 in RPMI-8226 or RPMI-LR5 cells. The recovery of their cellular content did not substantially affect cell growth, but improved their ability to segregate chromosomes. Finally, SAC functionality was tested by challenging cells with agents disrupting microtubule dynamics. Most of the cell lines analyzed exhibited functional defects in this checkpoint. Based on the data obtained, alterations both in SAC components and their functionality have been detected in MM, pointing to this pathway as a potential target in MM treatment.     

Oxazolo[3,2-a]pyridine. A new structural scaffold for the reversal of multi-drug resistance in Leishmania

Compounds belonging to three different classes of fused heterocyclic systems, structurally related to Calcium-channel blockers of the 1,4-dihydropyridine family, were evaluated in their ability to overcome leishmanial resistance to common drugs in a MDR Leishmania tropica strain. Compounds with the skeletal basis of oxazolo[3,2-a]pyridine displayed significant reversion of resistance to daunomycin and miltefosine, with reversion indexes up to 6.7-fold and 8.7-fold, respectively. Most interestingly, the enantiopure compound 20S attained to revert the resistance to both drugs and fairly more significantly than its enantiomer 20R.     

Succinate production in Escherichia coli

Succinate has been recognized as an important platform chemical that can be produced from biomass. While a number of organisms are capable of succinate production naturally, this review focuses on the engineering of Escherichia coli for the production of four-carbon dicarboxylic acid. Important features of a succinate production system are to achieve an optimal balance of reducing equivalents generated by consumption of the feedstock, while maximizing the amount of carbon channeled into the product. Aerobic and anaerobic production strains have been developed and applied to production from glucose and other abundant carbon sources. Metabolic engineering methods and strain evolution have been used and supplemented by the recent application of systems biology and in silico modeling tools to construct optimal production strains. The metabolic capacity of the production strain, the requirement for efficient recovery of succinate, and the reliability of the performance under scaleup are important in the overall process. The costs of the overall biorefinery-compatible process will determine the economic commercialization of succinate and its impact in larger chemical markets.     

RNA aptamers that functionally interact with green fluorescent protein and its derivatives

Green Fluorescent Protein (GFP) and related fluorescent proteins (FPs) have been widely used to tag proteins, allowing their expression and subcellular localization to be examined in real time in living cells and animals. Similar fluorescent methods are highly desirable to detect and track RNA and other biological molecules in living cells. For this purpose, we have developed a group of RNA aptamers that bind GFP and related proteins, which we term Fluorescent Protein-Binding Aptamers (FPBA). These aptamers bind GFP, YFP and CFP with low nanomolar affinity and binding decreases GFP fluorescence, whereas slightly augmenting YFP and CFP brightness. Aptamer binding results in an increase in the pKa of EGFP, decreasing the 475 nm excited green fluorescence at a given pH. We report the secondary structure of FPBA and the ability to synthesize functional multivalent dendrimers. FPBA expressed in live cells decreased GFP fluorescence in a valency-dependent manner, indicating that the RNA aptamers function within cells. The development of aptamers that bind fluorescent proteins with high affinity and alter their function, markedly expands their use in the study of biological pathways.     

Transport and cytotoxicity of the anticancer drug 3-bromopyruvate in the yeast Saccharomyces cerevisiae

We have investigated the cytotoxicity in Saccharomyces cerevisiae of the novel antitumor agent 3-bromopyruvate (3-BP). 3-BP enters the yeast cells through the lactate/pyruvate H⁺ symporter Jen1p and inhibits cell growth at minimal inhibitory concentration of 1.8 mM when grown on non-glucose conditions. It is not submitted to the efflux pumps conferring Pleiotropic Drug Resistance in yeast. Yeast growth is more sensitive to 3-BP than Gleevec (Imatinib methanesulfonate) which in contrast to 3-BP is submitted to the PDR network of efflux pumps. The sensitivity of yeast to 3-BP is increased considerably by mutations or chemical treatment by buthionine sulfoximine that decrease the intracellular concentration of glutathione.