Isolation and characterization of violacein from an Antarctic Iodobacter: a non-pathogenic psychrotolerant microorganism.

Violacein is an intensely purple pigment synthesized by various genera of bacteria that has been discovered to have a wide range of interesting biological activities which range from anticarcinogenic to antibacterial. One of the hindrances for its real-life application is that the first microorganisms found to produce the compound may act as opportunistic pathogens. Here, we report the isolation and characterization of violacein from a non-pathogenic Antarctic Iodobacter strain. Its anti-microbial properties were also tested. The method proposed here for the purification of violacein shows high yields, indicating that this Antarctic microorganism could be a valuable source for this important pigment. This is the first characterization of violacein from an Antarctic Iodobacter strain and here we also present a viable method to obtain this pigment for potential biotechnological applications.

Photophysics and photochemical studies of the vitamin B6 group and related derivatives.

The photophysics and photochemical properties of vitamin B6 constituents and analogs were studied as function of pH and solvent. The pK of the phenolic oxygen and the pyridine ring nitrogen depends on the electron donor-acceptor ability of the 4-substituent, and agrees with the calculated proton affinity. For all studied compounds, the fluorescence properties showed that the phenolic oxygen is 8 units more acidic in the lowest singlet excited state than in the ground state. The pyridine N-atom is slightly more basic in the excited state. At pH of biological significance, pH 6-8, pyridoxamine and 4-pyridoxic acid are the more efficient chromophores with higher fluorescence yield and longer lifetime. Spectroscopic studies showed that the tautomeric equilibrium depends on the nature of the 4-substituent. The quenching of the singlet excited state of pyridoxamine and 4-pyridoxic acid by amino acids, free or in a peptide, and DNA bases at pH 7 was studied by time-resolved fluorescence techniques. The quenching rate constants are well correlated with the redox properties of the pyridoxinic compound and amino acids, and are related to the free energy change in the electron transfer process. Guanosine and pyrimidine bases also are efficient quenchers, involving an electron transfer reaction.

Structure and medium effects on the photochemical behavior of nonfluorinated quinolone antibiotics.

The photophysical behavior of the quinolone antibiotics, oxolinic (OX), cinoxacin (CNX) and pipemidic (PM) acids was studied as a function of pH and solvent properties. The ground state of these compounds exhibits different protonated forms, which also exist in the first excited states. Theoretical calculations of the Fukui indexes allowed to assigning the different protonation equilibria. The pK values indicate that the acidity of the 3-carboxylic and 4-carbonyl groups increases with the N-atom at position 2 in CNX. It has been found that fluorescence properties are strongly affected by pH, the more fluorescent species is that with protonated carboxylic acid, protonated species at the carbonyl group and the totally deprotonated form present very low fluorescence. The fluorescence behavior also depends on the chemical structure of the quinolone and on the solvent properties. The analysis of the solvent effect on the maximum and the width of the fluorescence band of OX, using the linear solvent-energy relation solvatochromic equation, indicates that the polarizability and hydrogen bond donor ability are the parameters that condition the spectral changes. The hydrogen bond acceptor ability of the solvents also contributes to the spectral shifts of CNX. The compound bearing the piperazinyl group at the position 7, PM only is fluorescent in high protic solvents. These results are discussed in terms of the competition between the intra- and intermolecular hydrogen bonds. The irradiation of OX, CNX and PM using 300 nm UV light led to a very low photodecomposition rate. Under the same conditions the nalidixic acid (NA), a structurally related quinolone, photodecomposes two orders of magnitude faster.

Photophysics and photochemical studies of 1,4-dihydropyridine derivatives.

The absorption and fluorescence properties of nifedipine (NPDHP), felodipine (CPDHP) and a series of structurally related 1,4-dihydropyridines were studied in aqueous solution and organic solvents of different properties. The absorption and fluorescence spectra were found to depend on the chemical nature of the substituents at the position 4 of the 1,4-dihydropyridine ring (DHP) and on solvent properties. In aqueous solution, the fluorescence spectra of 4-phenyl substituted compounds are blue-shifted with respect to the alkyl substituted compounds. The more fluorescent compound is CPDHP. Nifedipine is not fluorescent. All compounds, with the exception of CPDHP, present monoexponential fluorescence decay with very short lifetime (0.2-0.4 ns). CPDHP showed a biexponential emission decay with a long-lived component of 1.7 ns; this behavior is explained in terms of different conformers because of the hindered rotation of the phenyl group by the ortho-substitution. Analysis of the solvent effect on the maximum of the absorption spectrum by using the linear solvent-energy relation solvato-chromic equation indicates the redshifts are influenced by the polarizability, hydrogen bonding ability and the hydrogen bond acceptance of the solvent. Whereas, the fluorescence characteristics (spectra, quantum yields and lifetimes) are sensitive to the polarizabilty and hydrogen bond ability of the solvents. Photo-decomposition of nifedipine is dependent on the solvent properties. Faster decomposition rates were obtained in nonprotic solvents. The 4-carboxylic derivative goes to decarboxylation. Under similar conditions, the other DHP compounds did not show appreciable photodecomposition.

Photophysics and photochemistry of nalidixic acid.

The photophysics and photochemistry of nalidixic acid (NA) were studied as function of pH and solvent properties. The ground state of NA exhibits different protonated forms in the range of pH 1.8-10.0. Fluorescence studies showed that the same species exist at the lowest singlet excited state. Absorption experiments were carried out with NA and with the methylated analog of nalidixic acid (MNE) in different organic solvents and water pH 3, where the main species corresponds to that protonated at the carboxylic group. These studies and the DFT calculation of torsional potential energy profiles suggest that the most stable conformation of the NA in nonprotic solvents corresponds to a closed structure caused by the existence of intramolecular hydrogen bond. Absorption and fluorescence spectra were studied in sulfuric acid solution. The pK value (Ho -1.0) found in these conditions was attributed to the protonation of the 4' keto oxygen atom of the heterocyclic ring. Theoretical calculations (DFT/B3LYP/6-311G*) of the energies of the different monoprotonated forms of the NA and Fukui indexes (f(x)-) showed that the species with the proton attached to 4' keto oxygen atom is the most stable of all the cationic forms. MNE and enoxacin also showed the protonation of the 4' keto oxygen atom with similar pK values. The photodecomposition of NA is dependent on the medium properties. Faster decomposition rates were obtained in strong acid solution. In nonprotic solvents, a very slow decomposition rate was observed.

Normas de prevención para neumonias nosocomiales en las unidades de terapia intensiva Hospital Obrero N° 1, Instituto Nacional del Tórax y Clínica Caja Petrolera de Salud de la ciudad de La Paz- Gestión 2003 / Norms of prevention for pneumonias nosocomiales in the units of therapy intensive Hospital Obrero N° 1, National Institute of the Thorax and Clinical Oil of Health of the city of La Paz - Administration 2003

Las neumonías nosocomiales, son la principal causa de mortalidad y están dentro de las tre primeras causas de morbilidad de las infecciones nosocomiales, por lo tanto la importancia de las medidias de prevención es vital. Estas medidas involucran al personal médico y paramédico que intervienen en el manejo, diagnóstico y terapéutica que pueden alterar los mecanismos de defensa respiratoria del paciente. El conocimiento y aplicación de las medidias de prevención de la Neumonía Nosocomial harán disminuir la tasa de morbilidad producida por estas. El método empleado para el estudio fue de tipo descriptivo, retrospectivo, transversal y no experimental. Con los resultados obtenidos, se observa que existe una baja prevalencia de Neumonías nosocomiales, encontrándose en el Hospital Obrero N° 1 con el 1,1 por ciento, luego la Caja Petrolera de Salud con 1,2 por ciento y por último el Intituto Nacional del Tórax con una prevalencia del 2,1 por ciento de casos de Neumonía Nosocomial en las Unidades de Terapia Intensiva, durante la gestión 2003. En conclusiones los resultados obtenidos nos demuestran que no difieren mucho en relación a otros países, lo que demuestra que nuestros Centros Hospitalarios están capacitados para brindar una atención de calidad, con personal eficiente con permanente actualización.

Anaerobiospirillum succiniciproducens phosphoenolpyruvate carboxykinase: mutagenesis at metal site 2.

Phosphoenolpyruvate (PEP) carboxykinases harbor two divalent metal-binding sites. One cation interacts with the enzyme (metal binding site 1) to elicit activation, while a second cation (metal binding site 2) interacts with the nucleotide to serve as the metal nucleotide substrate. Mutants of Anaerobiospirillum succiniciproducens PEP carboxykinase have been constructed where Thr249 and Asp262, two residues of metal binding site 2 of the enzyme, were altered. Binding of the 3'(2')-O-(N-methylantraniloyl) derivative of ADP provides a test of the structural integrity of these mutants. The conservative mutation (Asp262Glu) retains a significant proportion of the wild type enzymatic activity. Meanwhile, removal of the OH group of Thr249 in the Thr249Ala mutant causes a decrease in V(max) by a factor of 1.1 x 10(4). Molecular modeling of wild type and mutant enzymes suggests that the lower catalytic efficiency of the Thr249Ala enzyme could be explained by a movement of the lateral chain of Lys248, a critical catalytic residue, away from the reaction center.

Evaluation by site-directed mutagenesis of active site amino acid residues of Anaerobiospirillum succiniciproducens phosphoenolpyruvate carboxykinase.

Anaerobiospirillum succiniciproducens phosphoenolpyruvate (PEP) carboxykinase catalyzes the reversible formation of oxaloacetate and adenosine triphosphate from PEP, adenosine diphosphate, and carbon dioxide, and uses Mn2+ as the activating metal ion. The enzyme is a monomer and presents 68% identity with Escherichia coli PEP carboxykinase. Comparison with the crystalline structure of homologous E. coli PEP carboxykinase [Tari, L. W., Matte, A., Goldie, H., and Delbaere, L. T. J. (1997). Nature Struct. Biol. 4, 990-994] suggests that His225, Asp262, Asp263, and Thr249 are located in the active site of the protein, interacting with manganese ions. In this work, these residues were individually changed to Gln (His225) or Asn. The mutated enzymes present 3-6 orders of magnitude lower values of Vmax/Km, indicating high catalytic relevance for these residues. The His225Gln mutant showed increased Km values for Mn2+ and PEP as compared with wild-type enzyme, suggesting a role of His225 in Mn2+ and PEP binding. From 1.5-1.6 Kcal/mol lower affinity for the 3'(2')-O-(N-methylantraniloyl) derivative of adenosine diphosphate was observed for the His225Gln and Asp263Asn mutant A. succiniciproducens PEP carboxykinases, implying a role of His225 and Asp263 in nucleotide binding.

Ligand interactions and protein conformational changes of phosphopyridoxyl-labeled Escherichia coli phosphoenolpyruvate carboxykinase determined by fluorescence spectroscopy.

Escherichia coli phosphoenolpyruvate (PEP) carboxykinase catalyzes the decarboxylation of oxaloacetate and transfer of the gamma-phosphoryl group of ATP to yield PEP, ADP, and CO2. The interaction of the enzyme with the substrates originates important domain movements in the protein. In this work, the interaction of several substrates and ligands with E. coli PEP carboxykinase has been studied in the phosphopyridoxyl (P-pyridoxyl)-enzyme adduct. The derivatized enzyme retained the substrate-binding characteristics of the native protein, allowing the determination of several protein-ligand dissociation constants, as well as the role of Mg2+ and Mn2+ in substrate binding. The binding affinity of PEP to the enzyme-Mn2+ complex was -8.9 kcal.mol-1, which is 3.2 kcal.mol-1 more favorable than in the complex with Mg2+. For the substrate nucleotide-metal complexes, similar binding affinities (-6.0 to -6.2 kcal.mol-1) were found for either metal ion. The fluorescence decay of the P-pyridoxyl group fitted to two lifetimes of 5.15 ns (34%) and 1.2 ns. These lifetimes were markedly altered in the derivatized enzyme-PEP-Mn complexes, and smaller changes were obtained in the presence of other substrates. Molecular models of the P-pyridoxyl-E. coli PEP carboxykinase showed different degrees of solvent-exposed surfaces for the P-pyridoxyl group in the open (substrate-free) and closed (substrate-bound) forms, which are consistent with acrylamide quenching experiments, and suggest that the fluorescence changes reflect the domain movements of the protein in solution.