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Polymerization of 3′,5′ cyclic guanosine monophosphate (3′,5′ cGMP) was previously reported to afford short RNA sequences from a plausible mildly activated prebiotic precursor in a non‐enzymatic and template‐free manner. In the current paper we analyze the reaction with PAGE as well as by infrared micro‐ and nanospectroscopy on time scales not considered before. We provide evidence that a preliminary oligomerization step in dry state allows for a continued synthesis of RNA oligomers even in the presence of subsequently added water. On a longer time scale, the oligomerization yield oscillates in an apparently chaotic fashion hinting at the importance of some non‐reversible phase‐separation processes on the experimentally observed outcome of the polymerization/degradation chemistries. The long‐lasting (hundreds of hours) intrinsic sustainability and resilience of the process gives a robust prebiotic potential to this unique oligomerization reaction, which could lead to the very first oligonucleotide sequences on the primordial Earth. [ABSTRACT FROM AUTHOR]

Eukaryofic DNA topoisomerase I (Top1) is a monomeric protein clamp that functions in DNA replication, transcription, and recombination. Opposable "lip" domains form a salt bridge to complete Top1 protein clamping of duplex DNA. Changes in DNA topology are catalyzed by the formation of a transient phosphotyrosyl linkage between the active-site Tyr-723 and a single DNA strand. Substantial protein domain movements are required for DNA binding, whereas the tight packing of DNA within the covalent Topl-DNA complex necessitates some DNA distortion to allow rotation. To investigate the effects of Topi-clamp closure on enzyme catalysis, molecular modeling was used to design a disulfide bond between residues Gly-365 and 5er-534, to crossiink protein loops more proximal to the active-site tyrosine than the protein loops held by the Lys-369-Glu-497 salt bridge. In reducing environments, Topi-Clamp was catalytically acfive. However, contrary to crosslinking the salt-bridge loops [Carey, J. F., Schultz, S. J., Sission, L., Fazzio, T. G. & Champoux, J. J. (2003) Proc. Natl. Acad. Sci. USA 100, 5640-5645], crosslinking the active-site proximal loops inhibited DNA rotation. Apparently, subtle alterations in Top1 clamp flexibility impact enzyme catalysis in vitro. Yet, the catalytically active Topi-Clamp was cytotoxic, even in the reducing environment of yeast cells. Remarkably, a shift in redox potential in glrlA cells converted the catalytically inactive Top1Y723F mutant clamp into a cellular toxin, which failed to induce an S-phase terminal phenotype. This cytotoxic mechanism is distinct from that of camptothecin chemotherapeutics, which stabilize covalent Topl-DNA complexes, and it suggests that the development of novel therapeutics that promote Topi-clamp closure is possible. [ABSTRACT FROM AUTHOR]

Eukaryotic DNA topoisomerase I catalyzes the relaxation of positively and negatively supercoiled DNA and plays a critical role in processes involving DNA, such as DNA replication, transcription and recombination. The enzyme is encoded by the TOP1 gene and is highly conserved in its amino acid sequence and sensitivity to the anti-neoplatic agent camptothecin. This plant alkaloid specifically targets DNA topoisomerase I by reversibly stabilizing the covalent enzyme-DNA intermediate. Presumably, it is the interaction of these drug-stabilized adducts with other cellular components, such as replication forks, that actually produces the DNA lesions leading to cell death. A conservation of the mechanism(s) of camptothecin-induced cell killing is also implicit in studies of the yeast Saccharomyces cerevisiae, where the camptothecin sensitivity of Δ TOP1 yeast cells can be restored by plasmids expressing either yeast or human TOP1 sequences. This genetically tractable system is currently being exploited to describe the specific molecular interactions required for the cytotoxic action of camptothecin. The results of mutational analyses of yeast and human DNA topoisomerase I are presented, as well as a genetic screen designed to identify genes, other than TOP1, that are required for the cytotoxic activity of camptothecin. [ABSTRACT FROM AUTHOR]

Abstract: The aim of this work is to investigate the possibility of producing ethanol by glucose fermentation under high pressure of carbon dioxide up to 48bar, in order to exploit both ethanol and denser CO2 as a by-product of the process. The fermentation is carried out using Ethanol Red™-Lesaffre Saccharomyces cerevisiae yeast strain, which is commonly applied in industrial bioethanol production. The experiments were performed in six small reactors (2mL of volume each) connected in parallel, to investigate the effect of the process variables at the same conditions of temperature and pressure, and in one pilot reactor (1L of volume) to confirm the results obtained at the lower scale. The influence of operative variables, such as carbon dioxide pressure (0–48bar), temperature (32 and 36°C), glucose (150–250g/L), inositol (0–400mg/L) and biomass concentration (OD 2 and 3.5), was measured in terms of ethanol concentration (by gas chromatography) and ethanol productivity (expressed as grams of ethanol per CFU of yeast). Both of these parameters were found to be strongly dependent on glucose concentration and CO2 pressure, which negatively affects the fermentation. Nevertheless, also at 50bar it is possible to produce appreciable amounts of ethanol. [Copyright &y& Elsevier]

Emerging evidence shows that G protein-coupled receptors can form homo- and heteromers. These include adenosine A2A receptor–dopamine D2 receptor heteromers, which are most probably localized in the dendritic spines of the striatopallidal GABAergic neurons, where they are in a position to modulate glutamatergic neurotransmission. The discovery of A2A receptor–dopamine D2 receptor heteromers gives a frame for the well-known antagonistic interaction between both receptors, which is the bases for a new therapeutic approach for neuro-psychiatric disorders, such as Parkinson''s disease and schizoprenia. The present review deals mainly with the biochemical and molecular aspects of A2A receptor–dopamine D2 receptor interactions. Recent results at the molecular level show that A2A receptor–dopamine D2 receptor heteromers represent the first example of epitope–epitope electrostatic interaction underlying receptor heteromerization. Most probably A2A receptor–D2 receptor heteromerization is not static, but subject to a dynamic regulation, related to the phosphorylation dependence of the A2A receptor epitope and to the ability of the D2 receptor epitope to bind different partners. Finding out the mechanisms involved in this dynamic regulation can have important implications for the treatment of basal ganglia disorders, schizophrenia and drug addiction. [Copyright &y& Elsevier]

There is evidence for strong functional antagonistic interactions between adenosine A[sub 2A] receptors (A[sub 2A]Rs) and dopamine D[sub 2] receptors (D[sub 2]Rs). Although a close physical interaction between both receptors has recently been shown using co-immunoprecipitation and co-localization assays, the existence of a A[sub 2A]R-D[sub 2]R protein-protein interaction still had to be demonstrated in intact living cells. In the present work, fluorescence resonance energy transfer (FRET) and bioluminescence resonance energy transfer (BRET) techniques were used to confirm the occurrence of A[sub 2A]R-D[sub 2]R interactions in cotransfected cells. The degree of A[sub 2A]R-D[sub 2]R heteromerization, measured by BRET, did not vary after receptor activation with selective agonists, alone or in combination. BRET competition experiments were performed using a chimeric D[sub 2]R-D[sub 1]R in which helices 5 and 6, the third intracellular loop (I3), and the third extracellular loop (E3) of the D[sub 2]R were replaced by those of the dopamine D[sub 1] receptor (D[sub 1]R). Although the wild type D[sub 2]R was able to decrease the BRET signal, the chimera failed to achieve any effect. This suggests that the helix 5-I3-helix 6-E3 portion of D[sub 2]R holds the site(s) for interaction with A[sub 2A]R. Modeling of A[sub 2A]R and D[sub 2]R using a modified rhodopsin template followed by molecular dynamics and docking simulations gave essentially two different possible modes of interaction between D[sub 2]R and A[sub 2A]R. In the most probable one, helix 5 and/or helix 6 and the N-terminal portion of I3 from D[sub 2]R approached helix 4 and the C-terminal portion of the C-tail from the A[sub 2A]R, respectively. [ABSTRACT FROM AUTHOR]