Identification of secondary targets of N-containing bisphosphonates in mammalian cells via parallel competition analysis of the barcoded yeast deletion collection

Background

Nitrogen-containing bisphosphonates are the elected drugs for the treatment of diseases in which excessive bone resorption occurs, for example, osteoporosis and cancer-induced bone diseases. The only known target of nitrogen-containing bisphosphonates is farnesyl pyrophosphate synthase, which ensures prenylation of prosurvival proteins, such as Ras. However, it is likely that the action of nitrogen-containing bisphosphonates involves additional unknown mechanisms. To identify novel targets of nitrogen-containing bisphosphonates, we used a genome-wide high-throughput screening in which 5,936 Saccharomyces cerevisiae heterozygote barcoded mutants were grown competitively in the presence of sub-lethal doses of three nitrogen-containing bisphosphonates (risedronate, alendronate and ibandronate). Strains carrying deletions in genes encoding potential drug targets show a variation of the intensity of their corresponding barcodes on the hybridization array over the time.

Results

With this approach, we identified novel targets of nitrogen-containing bisphosphonates, such as tubulin cofactor B and ASK/DBF4 (Activator of S-phase kinase). The up-regulation of tubulin cofactor B may explain some previously unknown effects of nitrogen-containing bisphosphonates on microtubule dynamics and organization. As nitrogen-containing bisphosphonates induce extensive DNA damage, we also document the role of DBF4 as a key player in nitrogen-containing bisphosphonate-induced cytotoxicity, thus explaining the effects on the cell-cycle.

Conclusions

The dataset obtained from the yeast screen was validated in a mammalian system, allowing the discovery of new biological processes involved in the cellular response to nitrogen-containing bisphosphonates and opening up opportunities for development of new anticancer drugs.     

Identification of different isoforms of eEF1A in the nuclear fraction of human T-lymphoblastic cancer cell line specifically binding to aptameric cytotoxic GT oligomers.

GT oligomers, showing a dose-dependent cytotoxic effect on a variety of human cancer cell lines, but not on normal human lymphocytes, recognize and form complexes with nuclear proteins. By working with human T-lymphoblastic CCRF-CEM cells and by using MS and SouthWestern blotting, we identified eukaryotic elongation factor 1 alpha (eEF1A) as the main nuclear protein that specifically recognizes these oligonucleotides. Western blotting and supershift assays confirmed the nature of this protein and its involvement in forming a cytotoxicity-related complex (CRC). On the contrary, normal human lymphocytes did not show nuclear proteins able to produce CRC in a SouthWestern blot. Comparative bidimensional PAGE and Western-blotting analysis for eEF1A revealed the presence of a specific cluster of spots, focusing at more basic pH, in nuclear extracts of cancer cells but absent in those of normal lymphocytes. Moreover, a bidimensional PAGE SouthWestern blot demonstrated that cytotoxic GT oligomers selectively recognized the more basic eEF1A isoform expressed only in cancer cells. These results suggest the involvement of eEF1A, associated with the nuclear-enriched fraction, in the growth and maintenance of tumour cells, possibly modulated by post-translational processing of the polypeptide chain.     

Concentration Gradients of Free and Total γ-Aminobutyric Acid and Homocarnosine in Human CSF: Comparison of Suboccipital and Lumbar Sampling

Abstract: Concentrations of free and total γ-aminobutyric acid (GABA) and homocarnosine were determined in sequential aliquots of the first 30 ml of CSF obtained by lumbar puncture in five patients. Rostrocaudal gradients were calculated and compared to gradients estimated by determining concentrations of these substances in CSF obtained by simultaneous suboccipital and lumbar punctures in four more patients. In the lumbar fractions study, rostrocaudal mean gradients of 0.36, 36, and 21 pmol/ml for free GABA, total GABA, and homocarnosine, respectively, were calculated. In the suboccipital/lumbar study, gradients of 0.33, 30, and 24 pmol/ml for free GABA, total GABA, and homocarnosine, respectively, were estimated. These results indicate that valid comparison of CSF concentrations of these substances is restricted to similar fractions and suggest that in CSF the substances originate largely from brain rather than from peripheral sources.     

G-quadruplex DNA recognition by nucleophosmin: New insights from protein dissection

Background

Nucleophosmin (NPM1, B23) is a multifunctional protein that is involved in a variety of fundamental biological processes. NPM1/B23 deregulation is implicated in the pathogenesis of several human malignancies. This protein exerts its functions through the interaction with a multiplicity of biological partners. Very recently it is has been shown that NPM1/B23 specifically recognizes DNA G-quadruplexes through its C-terminal region.

Methods

Through a rational dissection approach of protein here we show that the intrinsically unfolded regions of NPM1/B23 significantly contribute to the binding of c-MYC G-quadruplex motif. Interestingly, the analysis of the ability of distinct NPM1/B23 fragments to bind this quadruplex led to the identifications of distinct NPM1/B23-based peptides that individually present a high affinity for this motif.

Results

These results suggest that the tight binding of NPM1/B23 to the G-quadruplex is achieved through the cooperation of both folded and unfolded regions that are individually able to bind it. The dissection of NPM1/B23 also unveils that its H1 helix is intrinsically endowed with an unusual thermal stability.

Conclusions

These findings have implications for the unfolding mechanism of NPM1/B23, for the G-quadruplex affinity of the different NPM1/B23 isoforms and for the design of peptide-based molecules able to interact with this DNA motif.

General observation

This study sheds new light in the molecular mechanism of the complex NPM1/G-quadruplex involved in acute myeloid leukemia (AML) disease.     

ELF magnetic fields in electric and gasoline‐powered vehicles

We conducted a pilot study to assess magnetic field levels in electric compared to gasoline‐powered vehicles, and established a methodology that would provide valid data for further assessments. The sample consisted of 14 vehicles, all manufactured between January 2000 and April 2009; 6 were gasoline‐powered vehicles and 8 were electric vehicles of various types. Of the eight models available, three were represented by a gasoline‐powered vehicle and at least one electric vehicle, enabling intra‐model comparisons. Vehicles were driven over a 16.3 km test route. Each vehicle was equipped with six EMDEX Lite broadband meters with a 40–1,000 Hz bandwidth programmed to sample every 4 s. Standard statistical testing was based on the fact that the autocorrelation statistic damped quickly with time. For seven electric cars, the geometric mean (GM) of all measurements (N = 18,318) was 0.095 µT with a geometric standard deviation (GSD) of 2.66, compared to 0.051 µT (N = 9,301; GSD = 2.11) for four gasoline‐powered cars (P < 0.0001). Using the data from a previous exposure assessment of residential exposure in eight geographic regions in the United States as a basis for comparison (N = 218), the broadband magnetic fields in electric vehicles covered the same range as personal exposure levels recorded in that study. All fields measured in all vehicles were much less than the exposure limits published by the International Commission on Non‐Ionizing Radiation Protection (ICNIRP) and the Institute of Electrical and Electronics Engineers (IEEE). Future studies should include larger sample sizes representative of a greater cross‐section of electric‐type vehicles. Bioelectromagnetics 34:156–161, 2013. © 2012 Wiley Periodicals, Inc.     

Monoclonal antibody detection of naphthalene dioxygenase from Pseudomonas aeruginosa 2NR

A monoclonal antibody, designated mAb alpha(CT), was generated against a peptide of the ISP(NAP) alpha-subunit of the naphthalene dioxygenase (NDO) enzyme of Pseudomonas aeruginosa. Since NDO expression is induced by aromatic hydrocarbons, its detection is important as a tool for environmental biomonitoring. This antibody is highly specific and works well both in an indirect ELISA assay and Western Blot analysis, allowing the detection of Pseudomonas spp. expressing the NDO inducible enzyme. The detection threshold for the ELISA assay developed in this work was 10(4) colony forming units (cfu) per ml. Thus, this mAb could represent a powerful tool to test for pollutants in soil, groundwater, and other natural environments.     

Detection of aromatic catabolic gene expression in heterogeneous organic matter used for reduction of volatile organic compounds (VOC) by biofiltration

A qualitative procedure of purified DNA/RNA co-extraction from complex organic matter, used as biofilter support for removing volatile organic compounds, was set up and applied to detect xylene monooxygenase gene expression by RT-PCR. A DNA/RNA extraction protocol based on a combination of sample lyophilization pre-treatment and CTAB––phenol/chloroform extraction procedure was optimized for the recovery of purified nucleic acids [100–500 ng DNA (10 kb) and 0.5–2 μg of rRNA 16S from 100 mg matrix]. PCR and RT-PCR protocols were established to detect xylene monooxygenase gene expression starting from differentially induced organic matrices obtained by biofiltration technology. This work allowed the microbial degradation activities in heterogeneous organic solid media to be studied and suggests a rapid method to follow specific biological activities during solid and/or semisolid organic substrates biotransformation.     

Increased gamma-aminobutyric acid (GABA), homocarnosine and beta-alanine in cerebrospinal fluid of patients treated with gamma-vinyl GABA (4-amino-hex-5-enoic acid)

γ-Vinyl GABA, an enzyme-activated irreversible inhibitor of GABA transaminase (GABA-T), was administered orally to 15 patients with various neurological conditions at daily doses of 0.5, 1, 2 or 6 g/day for 3 days. CSF samples were obtained by lumbar puncture before treatment and within 24 hours after the last dose and the CSF concentrations of free GABA, total GABA, homocarnosine, β-alanine and γ-vinyl GABA determined by ion-exchange chromatography with fluorometric detection. γ-Vinyl GABA treatment produced dose-dependent increases in free GABA, conjugated GABA (defined as total minus free GABA), homocarnosine and β-alanine. The concentrations of CSF γ-vinyl GABA also depended on the dose administered. These results indicate that γ-vinyl GABA enters the CNS after oral administration and alters GABA metabolism by inhibition of GABA-T and suggest that such treatment may achieve therapeutic benefit in conditions where such neurochemical alterations are desirable.