Nitrate reductases (NR) belong to the DMSO reductase family of Mo-containing enzymes and perform key roles in the metabolism of the nitrogen cycle, reducing nitrate to nitrite. Due to variable cell location, structure and function, they have been divided into periplasmic (Nap), cytoplasmic, and membrane-bound (Nar) nitrate reductases. The first crystal structure obtained for a NR was that of the monomeric NapA from Desulfovibrio desulfuricans in 1999. Since then several new crystal structures were solved providing novel insights that led to the revision of the commonly accepted reaction mechanism for periplasmic nitrate reductases. The two crystal structures available for the NarGHI protein are from the same organism (Escherichia coli) and the combination with electrochemical and spectroscopic studies also lead to the proposal of a reaction mechanism for this group of enzymes. Here we present an overview on the current advances in structural and functional aspects of bacterial nitrate reductases, focusing on the mechanistic implications drawn from the crystallographic data.

Several copper complexes have been assessed as anti-tumor agents against cancer cells. In this work, a copper compound [Cu(H2O){OS(CH3)2}L](NO3)2 incorporating the ligand 4′-phenyl-terpyridine antiproliferative activity against human colorectal, hepatocellular carcinomas and breast adenocarcinoma cell lines was determined, demonstrating high cytotoxicity. The compound is able to induce apoptosis and a slight delay in cancer cell cycle progression, probably by its interaction with DNA and induction of double-strand pDNA cleavage, which is enhanced by oxidative mechanisms. Moreover, proteomic studies indicate that the compound induces alterations in proteins involved in cytoskeleton maintenance, cell cycle progression and apoptosis, corroborating its antiproliferative potential.

The developments in the use of plasmid DNA (pDNA) in gene therapy and vaccines have motivated the search and improvement of optimized purification processes. In this context, dipeptides l-tyrosine-l-tyrosine and l-tyrosine-l-arginine are synthetized to explore their application as affinity ligands for supercoiled (sc) plasmid DNA (pDNA) purification. The synthesis is based on the protection of N-Boc-l-tyrosine, followed by condensation with l-tyrosine or l-arginine methyl esters in the presence of dicyclohexylcarbodiimide (DCC), which after hydrolysis and acidification give the afforded dipeptides. The supports are then obtained by coupling l-tyrosine, l-tyrosine-l-tyrosine and l-tyrosine-l-arginine to epoxy-activated Sepharose and are characterized by high resolution magic angle spinning (HR-MAS) NMR and Fourier transform infrared spectroscopy (FTIR). Surface plasmon resonance (SPR) biosensor is used to establish the promising ligand to be used in the chromatographic experiments and ascertain experimental conditions. Sc isoform showed the highest affinity to the dipeptides, followed by linear (ln) pDNA, being the open circular (oc) the one that promoted the lowest affinity to l-tyrosine-l-arginine. Saturation transfer difference (STD)-NMR experiments show that the interaction is mainly hydrophobic with the majority of the 5'-mononucleotides, except for 5'-GMP with l-tyrosine-l-arginine Sepharose that is mainly electrostatic. The support l-tyrosine Sepharose used in chromatographic experiments promotes the separation of native pVAX1-LacZ and pcDNA3-FLAG-p53 samples (oc+sc) by decreasing the salt concentration. The results suggest that it is possible to purify different plasmids with the l-tyrosine Sepharose, with slight adjustments in the gradient conditions.

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The use of electrospun polyvinylpyrrolidone (PVP) nanofibers containing silver, copper, and zinc nanoparticles was studied to prepare antimicrobial mats using silver and copper nitrates and zinc acetate as precursors. Silver became reduced during electrospinning and formed nanoparticles of several tens of nanometers. Silver nanoparticles and the insoluble forms of copper and zinc were dispersed using low molecular weight PVP as capping agent. High molecular weight PVP formed uniform fibers with a narrow distribution of diameters around 500 nm. The fibers were converted into an insoluble network using ultraviolet irradiation crosslinking. The efficiency of metal-loaded mats against the bacteria Escherichia coli and Staphylococcus aureus was tested for different metal loadings by measuring the inhibition of colony forming units and the staining with fluorescent probes for metabolic viability and compromised membranes. The assays included the culture in contact with mats and the direct staining of surface attached microorganisms. The results indicated a strong inhibition for silver-loaded fibers and the absence of significant amounts of viable but non-culturable microorganisms. Copper and zinc-loaded mats also decreased the metabolic activity and cell viability, although in a lesser extent. Metal-loaded fibers allowed the slow release of the soluble forms of the three metals.