Destabilizing Mutations Alter the Hydrogen Exchange Mechanism in Ribonuclease A

The effect of strongly destabilizing mutations, I106A and V108G of Ribonuclease A (RNase A), on its structure and stability has been determined by NMR. The solution structures of these variants are essentially equivalent to RNase A. The exchange rates of the most protected amide protons in RNase A (35ºC), the I106A variant (35ºC), and the V108G variant (10ºC) yield stability values of 9.9, 6.0, and 6.8 kcal/mol, respectively, when analyzed assuming an EX2 exchange mechanism. Thus, the destabilization induced by these mutations is propagated throughout the protein. Simulation of RNase A hydrogen exchange indicates that the most protected protons in RNase A and the V108G variant exchange via the EX2 regime, whereas those of I106A exchange through a mixed EX1 1 EX2 process. It is striking that a single point mutation can alter the overall exchange mechanism. Thus, destabilizing mutations joins high temperatures, high pH and the presence of denaturating agents as a factor that induces EX1 exchange in proteins. The calculations also indicate a shift from the EX2 to the EX1 mechanism for less protected groups within the same protein. This should be borne in mind when interpreting exchange data as a measure of local stability in less protected region

Activating transcription factor 3 is crucial for antitumor activity and to strengthen the antiviral properties of onconase

Onconase is a ribonuclease that presents both antitumor and antiviral properties linked to its ribonucleolytic activity and represents a new class of RNA-damaging drugs. It has reached clinical trials for the treatment of several cancers and human papilloma virus warts. Onconase targets different RNAs in the cell cytosol but Onconase-treated cells present features that are different from a simple arrest of protein synthesis. We have used microarray-derived transcriptional profiling to identify Onconase-regulated genes in two ovarian cancer cell lines (NCI/ADR-RES and OVCAR-8). RT-qPCR analyses have confirmed the microarray findings. We have identified a network of up-regulated genes implicated in different signaling pathways that may explain the cytotoxic effects exerted by Onconase. Among these genes, activating transcription factor 3 (ATF3) plays a central role in the key events triggered by Onconase in treated cancer cells that finally lead to apoptosis. This mechanism, mediated by ATF3, is cell-type independent. Up-regulation of ATF3 may also explain the antiviral properties of this ribonuclease because this factor is involved in halting viral genome replication, keeping virus latency or preventing viral oncogenesis. Finally, Onconase-regulated genes are different from those affected by nuclear-directed ribonuclease

A nuclear-directed human pancreatic ribonuclease (PE5) targets the metabolic phenotype of cancer cells

Ribonucleases represent a new class of antitumor RNA-damaging drugs. However, many wild-type members of the vertebrate secreted ribonuclease family are not cytotoxic because they are not able to evade the cytosolic ribonuclease inhibitor. We previously engineered the human pancreatic ribonuclease to direct it to the cell nucleus where the inhibitor is not present. The best characterized variant is PE5 that kills cancer cells through apoptosis mediated by the p21WAF1/CIP1 induction and the inactivation of JNK. Here, we have used microarray-derived transcriptional profiling to identify PE5 regulated genes on the NCI/ADR-RES ovarian cancer cell line. RT-qPCR analyses have confirmed the expression microarray findings. The results show that PE5 cause pleiotropic effects. Among them, it is remarkable the down-regulation of multiple genes that code for enzymes involved in deregulated metabolic pathways in cancer cellsThis work has been supported by grants BFU2009-06935 and BIO2013-43517 from MINECO (Spain) and SING12/0 from UdG (Spain)

Transcriptional profiling of NCI/ADR-RES cells unveils a complex network of signaling pathways and molecular mechanisms of drug resistance

Ovarian cancer has the highest mortality rate among all the gynecological cancers. This is mostly due to the resistance of ovarian cancer to current chemotherapy regimens. Therefore, it is of crucial importance to identify the molecular mechanisms associated with chemoresistance. Methods: NCI/ADR-RES is a multidrug-resistant cell line that is a model for the study of drug resistance in ovarian cancer. We carried out a microarray-derived transcriptional profiling analysis of NCI/ADR-RES to identify differentially expressed genes relative to its parental OVCAR-8. Results: Gene-expression profiling has allowed the identification of genes and pathways that may be important for the development of drug resistance in ovarian cancer. The NCI/ADR-RES cell line has differential expression of genes involved in drug extrusion, inactivation, and efficacy, as well as genes involved in the architectural and functional reorganization of the extracellular matrix. These genes are controlled through different signaling pathways, including MAPK–Akt, Wnt, and Notch. Conclusion: Our findings highlight the importance of using orthogonal therapies that target completely independent pathways to overcome mechanisms of resistance to both classical chemotherapeutic agents and molecularly targeted drug

A human ribonuclease induces apoptosis associated with p21WAF1/CIP1 induction and JNK inactivation

Ribonucleases are promising agents for use in anticancer therapy. Among the different ribonucleases described to be cytotoxic, a paradigmatic example is onconase which manifests cytotoxic and cytostatic effects, presents synergism with several kinds of anticancer drugs and is currently in phase II/III of its clinical trial as an anticancer drug against different types of cancer. The mechanism of cytotoxicity of PE5, a variant of human pancreatic ribonuclease carrying a nuclear localization signal, has been investigated and compared to that of onconase. Methods: Cytotoxicity was measured by the MTT method and by the tripan blue exclusion assay. Apoptosis was assessed by flow cytometry, caspase enzymatic detection and confocal microscopy. Cell cycle phase analysis was performed by flow cytometry. The expression of different proteins was analyzed by western blot.n Results: We show that the cytotoxicity of PE5 is produced through apoptosis, that it does not require the proapoptotic activity of p53 and is not prevented by the multiple drug resistance phenotype. We also show that PE5 and onconase induce cell death at the same extent although the latter is also able to arrest the cell growth. We have compared the cytotoxic effects of both ribonucleases in the NCI/ADR-RES cell line by measuring their effects on the cell cycle, on the activation of different caspases and on the expression of different apoptosis- and cell cycle-related proteins. PE5 increases the number of cells in S and G2/M cell cycle phases, which is accompanied by the increased expression of cyclin E and p21WAF1/CIP1 together with the underphosphorylation of p46 forms of JNK. Citotoxicity of onconase in this cell line does not alter the cell cycle phase distribution and it is accompanied by a decreased expression of XIAP. Conclusions: We conclude that PE5 kills the cells through apoptosis associated with the p21WAF1/CIP1 induction and the inactivation of JNK. This mechanism is significantly different from that found for onconas

A human ribonuclease induces apoptosis associated with p21WAF1/CIP1 induction and JNK inactivation

Background: Ribonucleases are promising agents for use in anticancer therapy. Among the different ribonucleases described to be cytotoxic, a paradigmatic example is onconase which manifests cytotoxic and cytostatic effects, presents synergism with several kinds of anticancer drugs and is currently in phase II/III of its clinical trial as an anticancer drug against different types of cancer. The mechanism of cytotoxicity of PE5, a variant of human pancreatic ribonuclease carrying a nuclear localization signal, has been investigated and compared to that of onconase. Methods: Cytotoxicity was measured by the MTT method and by the tripan blue exclusion assay. Apoptosis was assessed by flow cytometry, caspase enzymatic detection and confocal microscopy. Cell cycle phase analysis was performed by flow cytometry. The expression of different proteins was analyzed by western blot. Results: We show that the cytotoxicity of PE5 is produced through apoptosis, that it does not require the proapoptotic activity of p53 and is not prevented by the multiple drug resistance phenotype. We also show that PE5 and onconase induce cell death at the same extent although the latter is also able to arrest the cell growth. We have compared the cytotoxic effects of both ribonucleases in the NCI/ADR-RES cell line by measuring their effects on the cell cycle, on the activation of different caspases and on the expression of different apoptosis- and cell cycle-related proteins. PE5 increases the number of cells in S and G2/M cell cycle phases, which is accompanied by the increased expression of cyclin E and p21WAF1/CIP1 together with the underphosphorylation of p46 forms of JNK. Citotoxicity of onconase in this cell line does not alter the cell cycle phase distribution and it is accompanied by a decreased expression of XIAP. Conclusions: We conclude that PE5 kills the cells through apoptosis associated with the p21WAF1/CIP1 induction and the inactivation of JNK. This mechanism is significantly different from that found for onconase

Métodos analíticos y equipos automáticos para la determinación cuantitativa de beta-galactosidasa

Métodos analíticos y equipos automáticos para la determinación cuantitativa de beta-galactosidasa. un primer método consiste en analizar la concentración de b-galactosidasa mediante análisis por inyección en flujo (fia), y se realiza automáticamente mediante un ordenador. Un segundo método aplicado en procesos fermentativos consiste en analizar la concentracion de b-galactosidasa mediante analisis por inyeccion en flujo (fia), y se caracteriza por un tratamiento previo de la muestra mediante sonicación. Inyectando una concentración y volumen de reactivo conocido en una corriente de muestra sonicada portadora, estando presente otro portador: tampón que permite realizar diluciones y homogeneizar la muestra. El caudal de la muestra es variable en función de la concentración de b-galactosidasa y es controlado por ordenador en función de los valores obtenidos y de parámetros programados de antemano. Comprende los respectivos equipos para la realización de los métodos citados. También se refiere a la utilización del método aplicado en procesos fermentativos para la determinación de biomasa, se reduce el tiempo de análisis

A family of manganese complexes containing heterocyclic-based ligands with cytotoxic properties

We describe the synthesis of three new manganese (II) complexes containing the bidentate ligands 2-(1-methyl-3-pyrazolyl)pyridine (pypz-Me) and ethyl 2-(3-(pyridine-2-yl)-1H–pyrazol-1-yl)acetate (pypz-CH2COOEt), with formula [MnX2(pypz-Me)2] (X = Cl−1, CF3SO3−2) and [Mn(CF3SO3)2(pypz-CH2COOEt)2] 3. Complexes 1–3 have been characterized through analytical, spectroscopic and electrochemical techniques, as well as by monocrystal X-ray diffraction analysis. The complexes show a six-coordinated Mn(II) ion though different stereoisomers have been isolated for the three compounds. Complexes 1–3, together with the previously described compounds [MnCl2(pypz-H)2] 4, [Mn(CF3SO3)2(pypz-H)2] 5, [Mn(NO3)2(pypz-H)2] 6, [MnCl2(H2O)2(pypz-H)2] 7 (pypz-H = 2-(3-pyrazolyl)pyridine) and ([Mn(CF3SO3)2((−)-L)2] 8, ((−)-L = (−)-pinene[5,6]bipyridine), were tested in vitro for cytotoxic activity against NCI-H460 and OVCAR-8 cancer cell lines. The geometry of a specific compound does not seem to influence its activity in a significant extent. However, among the tested compounds those that display hydrophobic substituents on the pyrazole ring and triflate ions as labile ligands show the best antiproliferative properties. Specifically, compound 8 containing the pinene-bipyridine ligand presents an antiproliferative activity similar to that of cisplatin and higher than that of carboplatin, and displays selectivity for tumour cells. Its antiproliferative effect is due to the generation of ROS species that allow the compound to interact with DNAThis research has been financed by Ministerio de Economia y Competitividad (MINECO) Spain, through projects CTQ2015-66143-P and BIO2013-43517, by Universitat de Girona through projects MPCUdG2016-18 and MPCUdG2016/048 and by DGU, Generalitat de Catalunya through projects SGR2014-70 and SGR2014-14

Proteína híbrida para utilizar en la liberación dirigida de un compuesto a una célula humana o animal

Molécula hibrida para utilizar en la liberación dirigida de un compuesto a una célula humana o animal. La invención se refiere a una molécula híbrida para utilizar en la liberación dirigida de un compuesto a una célula humana o animal. La molécula híbrida de la invención proporciona la posibilidad de liberar de manera dirigida un compuesto a una célula humana o animal sin utilizar la totalidad de las partículas de un virus infeccioso. Es especialmente ventajosa para el tratamiento médico de humanos y animale

Aprenentatge basat en objectius aplicat a un mòdul docent de continguts preclínics del grau de Medicina

L’experiència que es presenta s’ha dut a terme en el mòdul “Morfologia, estructura i funció del cos humà I” de primer curs del Grau de Medicina per tal d’adaptar-se a l’Espai Europeu d’Educació Superior. Particularment es va tenir en compte el fort debat existent en els darrers anys sobre els ensenyaments de pre-grau en àmbits de Ciències de la Salut [1-5] Els objectius principals plantejats en aquest projecte van ser dos: 1) convertir un ensenyament fragmentat de tres matèries afins en un ensenyament integrat i focalitzat per a estudiants de medicina; 2) fer protagonista directe del seu aprenentatge a aquests estudiants procedents d’un ensenyament molt dirigit (ESO i Batxillerat). El mòdul conté coneixements teòrics i pràctics de Biologia Cel·lular, Genètica i Bioquímica, amb la finalitat de donar una visió integrada de l’estructura i funció cel·lular i per tant de l’homeòstasi del cos humà a nivell molecular i cel·lular. La metodologia docent s’ha basat en aprenentatge per objectius. El mòdul s’ha dividit en cinc períodes anomenats Unitats d’Aprenentatge per Objectius (UAOs). S’han dissenyat diferents eines d’ajuda per a aquest autoaprenentatge i a més el seguiment d’aquest procés s’ha realitzat utilitzant diferents tipus de tutories. El contingut del mòdul es completa amb unes sessions pràctiques al laboratori que es fan de forma intensiva amb la finalitat de familiaritzar els estudiants amb les tècniques bàsiques d’un laboratori de recerca biomèdic