The uncharacterized Pseudomonas aeruginosa PA4189 is a novel and efficient aminoacetaldehyde dehydrogenase.

Neither the Pseudomonas aeruginosa aldehyde dehydrogenase encoded by the PA4189 gene nor its ortholog proteins have been biochemically or structurally characterized and their physiological function is unknown. We cloned the PA4189 gene, obtained the PA4189 recombinant protein, and studied its structure-function relationships. PA4189 is an NAD+-dependent aminoaldehyde dehydrogenase highly efficient with protonated aminoacetaldehyde and 3-aminopropionaldehyde, which are much more preferred to the non-protonated species as indicated by pH studies. Based on the higher activity with aminoacetaldehyde than with 3-aminopropionaldehyde, we propose that aminoacetaldehyde might be the PA4189 physiological substrate. Even though at the physiological pH of P. aeruginosa cells the non-protonated aminoacetaldehyde species will be predominant, and despite the competition of these species with the protonated ones, PA4189 would very efficiently oxidize ACTAL in vivo, producing glycine. To our knowledge, PA4189 is the first reported enzyme that might metabolize ACTAL, which is considered a dead-end metabolite because its consuming reactions are unknown. The PA4189 crystal structure reported here suggested that the charge and size of the active-site residue Glu457, which narrows the aldehyde-entrance tunnel, greatly define the specificity for small positively charged aldehydes, as confirmed by the kinetics of the E457G and E457Q variants. Glu457 and the residues that determine Glu457 conformation inside the active site are conserved in the PA4189 orthologs, which we only found in proteobacteria species. Also is conserved the PA4189 genomic neighborhood, which suggests that PA4189 participates in an uncharacterized metabolic pathway. Our results open the door to future efforts to characterize this pathway.

Relación entre los niveles basales de fructosamina y glucosa en adultos mayores / Relationship between basal levels of fructosamine and glucose in older adults

INTRODUCCIÓN: los cambios fisiológicos a los que están expuestos los adultos mayores, son muchas veces factores negativos en su calidad de vida, sobre todo en aquellos que se encuentran en residencias geriátricas, existen parámetros como la glicemia y hemoglobina glucosilada que podrían ser útiles en el control metabólico. OBJETIVO: relacionar los niveles basales de Fructosamina y Glucosa en adultos mayores institucionalizados en residencias geriátricas del municipio de Tiquipaya, septiembre 2019. METODOLOGÍA: estudio no experimental, observacional, prospectivo, transversal, con enfoque de análisis positivista cuantitativo, con un universo de 97 adultos mayores de 65 años, con una muestra de 79 que cumplen con los criterios de inclusión y exclusión, con un 4,77% de error máximo aceptable. RESULTADOS: el 71% (n=56) de los pacientes fueron mujeres. Los ancianos predominaron como grupo etario. Se evidenció que no hay una buena concordancia entre los niveles basales de Glicemia y Fructosamina, mediante el cálculo estadístico del índice de Kappa que fue de 0,023; Test de Wilcoxon 0.081; Test correlación Pearson r=0.281. Los niveles basales de Fructosamina tienen una sensibilidad y especificidad altas del 82,14% (L.I. 62,42% -L.S. 93,23%) y 56.92% (L.I. 47,95% - L.S. 65,48%), respectivamente. CONCLUSIONES: no existe relación entre los niveles basales de Glucosa y la Fructosamina puesto que son parámetros de evaluación metabólica en diferente tiempo y una no remplaza la otra, por lo tanto, se debería implementar adicionalmente a la Glucosa la determinación de la Fructosamina para monitorizar a los pacientes adultos a mediano plazo.

INTRODUCTION: the physiological changes to which older adults are exposed; are many times negative factors in their quality of life, especially in those who are in geriatric residences. Glycemia and glycosylated hemoglobin are useful as metabolic control parameters. OBJECTIVE: to relate the basal levels of Fructosamine and Glucose in institutionalized older adults in geriatric residences in the municipality of Tiquipaya, September 2019. METHODOLOGY: non-experimental, observational, prospective, cross-sectional study, with a quantitative positivist analysis approach, with a universe of 97 adults over 65 years of age, with a sample of 79 that met the inclusion and exclusion criteria, with a maximum acceptable error of 4.77%. RESULTS: 71% (n=56) of the patients were female. The elderly dominated as an age group. It was evidenced that there is not a good agreement between basal levels of Glycemia and Fructosamine, through the statistical calculation of the Kappa index which was 0.023; Wilcoxon test 0.081; Pearson correlation test r= 0.281. The basal levels of Fructosamine have a high sensitivity and specificity of 82.14% (L.I. 62.42% - L.S. 93.23%) and 56.92% (L.I.47.95% - L.S. 65.48%), respectively. CONCLUSIONS: there is no relationship between basal levels of Glucose and Fructosamine since they are parameters of metabolic evaluation in different time and one does not replace the other, therefore, the determination of Fructosamine should be implemented in addition to Glucose to monitor adult patients in the medium term.

Relación de los niveles plasmáticos de homocisteína y apolipoproteína b-100 con el riesgo cardiovascular / Relationship of plasma levels of homocysteine and apolipoprotein b-100 with cardiovascular risk

INTRODUCCION: las enfermedades cardiovasculares son responsables del 31% de la mortalidad mundial, existen parámetros como la homocisteína y la Apolipoproteína B-100 que podrían tener utilidad en la predicción del riesgo. OBJETIVO: relacionar los niveles plasmáticos de Homocisteína y Apolipoproteína B-100 con el riesgo cardiovascular en pacientes que acuden a consulta externa del Hospital Univalle, durante julio-agosto del 2018 METODOLOGIA: el presente estudio es no experimental observacional, tipo prospectivo, transversal, con un enfoque de análisis positivista cuantitativo, con un universo de (N=133) que se redujo a una unidad de análisis de 81, que cumplieron con los criterios de inclusión y exclusión con un 6.83% de error máximo aceptable. RESULTADOS: el 52% de los pacientes fueron mujeres. La edad media de fue de 49,8 (Rango 25 a 83), el grupo etario predominante fueron los adultos mayores. Según el IMC los sujetos de estudio presentan sobre peso (n=31) y grado de obesidad 1 (n=24) más frecuentemente. Los niveles plasmáticos elevados de Apolipoproteína B en ambos sexos no muestran una diferencia significativa, mientras en que los de homocisteína la diferencia fue de 8:1. Se constato que los niveles séricos de la Apolipoproteína B-100 tienen una sensibilidad y especificidad bajas del 19.40% y 28.42%, mientas los de la homocisteína fueron del 14.29% y 27.27% respectivamente en comparación con la técnica convencional. CONCLUSIONES: los niveles plasmáticos de homocisteína y Apolipoproteína B-100 no son parámetros predictores de padecer riesgo cardiovascular

INTRODUCTION: cardiovascular diseases are responsible for 31% of world mortality, there are parameters such as homocysteine and Apolipoprotein B-100 that could be useful in predicting risk. OBJECTIVE: to relate the plasma levels of Homocysteine and Apolipoprotein B-100 with cardiovascular risk in patients who attend the outpatient clinic of the Univalle Hospital, during July-August 2018. METHODOLOGY: this study is non-experimental, observational, prospective, cross-sectional, with a quantitative positivist analysis approach, with a universe of (N=133) that was reduced to an analysis unit of 81, who met the inclusion criteria. and exclusion with a 6.83% maximum acceptable error. RESULTS: 52% of the patients were women. The mean age was 49.8 (range 25 to 83), the predominant age group was the elderly. According to the BMI, the study subjects are overweight (n=31) and obesity grade 1 (n=24) more frequently. The elevated plasmatic levels of Apolipoprotein B in both sexes do not show a significant difference, while in those of homocysteine the difference was 8:1. It was found that the serum levels of Apolipoprotein B-100 have a low sensitivity and specificity of 19.40% and 28.42%, while those of homocysteine were 14.29% and 27.27% respectively compared to the conventional technique. CONCLUSIONS: plasma levels of homocysteine and Apolipoprotein B-100 are not predictors of cardiovascular risk.

Multiple conformations in solution of the maize C4-phosphoenolpyruvate carboxylase isozyme.

The photosynthetic phosphoenolpyruvate carboxylase isozyme from C4 plants (PEPC-C4) has a complex allosteric regulation, involving positive cooperativity in binding the substrate phosphoenolpyruvate as well as positive and negative allosteric effectors. Besides the proposed R- and T-states, previous kinetic results suggested functionally relevant different R-states of the maize enzyme (ZmPEPC-C4) elicited by PEP or its two kinds of activators, glucose 6-phosphate or glycine. To detect these different R-state conformations, we used as conformational probes the fluorescence of 8-anilino-1-naphthalene sulfonate (ANS), near-UV circular dichroism (CD) spectroscopy, and limited proteolysis by trypsin. Phosphoenolpyruvate and malate binding caused distinct concentration-dependent fluorescence changes of ZmPEPC-C4/ANS, suggesting that they elicited conformational states different from that of the free enzyme, while glucose 6-phosphate or glycine binding did not produce fluorescence changes. Differences were also observed in the near UV CD spectra of the enzyme, free or complexed with its substrate or allosteric effectors. Additionally, differences in the trypsin-digestion fragmentation patterns, as well as in the susceptibility of the free and complexed enzyme to digestion and digestion-provoked loss of activity, provided evidence of several ZmPEPC-C4 conformations in solution elicited by the substrate and the allosteric effectors. Using the already reported ZmPEPC-C4 crystal structures and bioinformatics methods, we predicted that the most probable trypsin-cleavage sites are located in superficial flexible regions, which seems relevant for the protein dynamics underlying the function and allosteric regulation of this enzyme. Together, our findings agree with previous kinetic results, shed light on this enzyme's complex allosteric regulation, and place ZmPEPC-C4 in the growing list of allosteric enzymes possessing an ensemble of closely related R-state conformations.

Site-directed mutagenesis of Bradyrhizobium diazoefficiens USDA 110 aroA improves bacterial growth and competitiveness for soybean nodulation in the presence of glyphosate.

The in vitro growth of Bradyrhizobium diazoefficiens USDA 110 strain is inhibited by glyphosate. The herbicide affects 5-enolpyruvylshikimic acid-3-phosphate synthase, a key enzyme for aromatic aminoacid synthesis. In this study, site-directed mutagenesis was used to change only two nucleotides of the coding region of phosphoenolpyruvate binding site. This change improved the in vitro growth of B. diazoefficiens USDA 110 in the presence of glyphosate, without affecting its normal growth in the absence of the herbicide. Plant co-inoculation experiments demonstrated a better competitiveness of the glyphosate-resistant strain for soybean nodulation in the presence of glyphosate.

Structural and biochemical evidence of the glucose 6-phosphate-allosteric site of maize C4-phosphoenolpyruvate carboxylase: its importance in the overall enzyme kinetics.

Activation of phosphoenolpyruvate carboxylase (PEPC) enzymes by glucose 6-phosphate (G6P) and other phospho-sugars is of major physiological relevance. Previous kinetic, site-directed mutagenesis and crystallographic results are consistent with allosteric activation, but the existence of a G6P-allosteric site was questioned and competitive activation-in which G6P would bind to the active site eliciting the same positive homotropic effect as the substrate phosphoenolpyruvate (PEP)-was proposed. Here, we report the crystal structure of the PEPC-C4 isozyme from Zea mays with G6P well bound into the previously proposed allosteric site, unambiguously confirming its existence. To test its functionality, Asp239-which participates in a web of interactions of the protein with G6P-was changed to alanine. The D239A variant was not activated by G6P but, on the contrary, inhibited. Inhibition was also observed in the wild-type enzyme at concentrations of G6P higher than those producing activation, and probably arises from G6P binding to the active site in competition with PEP. The lower activity and cooperativity for the substrate PEP, lower activation by glycine and diminished response to malate of the D239A variant suggest that the heterotropic allosteric activation effects of free-PEP are also abolished in this variant. Together, our findings are consistent with both the existence of the G6P-allosteric site and its essentiality for the activation of PEPC enzymes by phosphorylated compounds. Furthermore, our findings suggest a central role of the G6P-allosteric site in the overall kinetics of these enzymes even in the absence of G6P or other phospho-sugars, because of its involvement in activation by free-PEP.

Subtypes of Blastocystis sp. isolated in fecal samples from transplant candidates in São Paulo, Brazil.

Blastocystis sp. is an intestinal protozoan commonly found in fecal samples of many animal species, including humans, but poorly studied in transplant candidates. The aim of this study was to evaluate the occurrence and molecular identification of Blastocystis sp. in fecal samples from transplant candidates. A polymerase chain reaction was performed using specific primers for Blastocystis ribosomal DNA. The DNA sequences obtained were aligned and compared with other sequences from the GenBank and MLST databases. The analyzed samples showed a positivity of 16% (24 of 150) for Blastocystis sp. The highest occurrence was observed in renal transplant candidates (31.4%), followed by hepatic transplant candidates (10.4%) and candidates for bone marrow transplantation (5.9%). Subtype (ST) 3 (45.8%) was the most prevalent among the isolates, followed by ST1 (37.5%), ST2 (12.5%), and ST7 (4.2%). This is the first study of molecular identification Blastocystis sp. in transplant candidates. Our results confirmed that ST3 was the most common subtype in transplant candidates and reinforce the importance of new studies to investigate of Blastocystis sp. in these patients.

Diversity, abundance, and host relationships of avian malaria and related haemosporidians in New Mexico pine forests.

Avian malaria and related haemosporidian parasites (genera Haemoproteus, Plasmodium, and Leucocytozoon) affect bird demography, species range limits, and community structure, yet they remain unsurveyed in most bird communities and populations. We conducted a community-level survey of these vector-transmitted parasites in New Mexico, USA, to describe their diversity, abundance, and host associations. We focused on the breeding-bird community in the transition zone between piñon-juniper woodland and ponderosa pine forests (elevational range: 2,150-2,460 m). We screened 186 birds representing 49 species using both standard PCR and microscopy techniques to detect infections of all three avian haemosporidian genera. We detected infections in 68 out of 186 birds (36.6%), the highest proportion of which were infected with Haemoproteus (20.9%), followed by Leucocytozoon (13.4%), then Plasmodium (8.0%). We sequenced mtDNA for 77 infections representing 43 haplotypes (25 Haemoproteus, 12 Leucocytozoon, 6 Plasmodium). When compared to all previously known haplotypes in the MalAvi and GenBank databases, 63% (27) of the haplotypes we recovered were novel. We found evidence for host specificity at the avian clade and species level, but this specificity was variable among parasite genera, in that Haemoproteus and Leucocytozoon were each restricted to three avian groups (out of six), while Plasmodium occurred in all groups except non-passerines. We found striking variation in infection rate among host species, with nearly universal infection among vireos and no infection among nuthatches. Using rarefaction and extrapolation, we estimated the total avian haemosporidian diversity to be 70 haplotypes (95% CI [43-98]); thus, we may have already sampled ∼60% of the diversity of avian haemosporidians in New Mexico pine forests. It is possible that future studies will find higher diversity in microhabitats or host species that are under-sampled or unsampled in the present study. Fortunately, this study is fully extendable via voucher specimens, frozen tissues, blood smears, parasite images, and documentation provided in open-access databases (MalAvi, GenBank, and ARCTOS).

Reversible, partial inactivation of plant betaine aldehyde dehydrogenase by betaine aldehyde: mechanism and possible physiological implications.

In plants, the last step in the biosynthesis of the osmoprotectant glycine betaine (GB) is the NAD(+)-dependent oxidation of betaine aldehyde (BAL) catalysed by some aldehyde dehydrogenase (ALDH) 10 enzymes that exhibit betaine aldehyde dehydrogenase (BADH) activity. Given the irreversibility of the reaction, the short-term regulation of these enzymes is of great physiological relevance to avoid adverse decreases in the NAD(+):NADH ratio. In the present study, we report that the Spinacia oleracea BADH (SoBADH) is reversibly and partially inactivated by BAL in the absence of NAD(+)in a time- and concentration-dependent mode. Crystallographic evidence indicates that the non-essential Cys(450)(SoBADH numbering) forms a thiohemiacetal with BAL, totally blocking the productive binding of the aldehyde. It is of interest that, in contrast to Cys(450), the catalytic cysteine (Cys(291)) did not react with BAL in the absence of NAD(+) The trimethylammonium group of BAL binds in the same position in the inactivating or productive modes. Accordingly, BAL does not inactivate the C(450)SSoBADH mutant and the degree of inactivation of the A(441)I and A(441)C mutants corresponds to their very different abilities to bind the trimethylammonium group. Cys(450)and the neighbouring residues that participate in stabilizing the thiohemiacetal are strictly conserved in plant ALDH10 enzymes with proven or predicted BADH activity, suggesting that inactivation by BAL is their common feature. Under osmotic stress conditions, this novel partial and reversible covalent regulatory mechanism may contribute to preventing NAD(+)exhaustion, while still permitting the synthesis of high amounts of GB and avoiding the accumulation of the toxic BAL.

Residues that influence coenzyme preference in the aldehyde dehydrogenases.

To find out the residues that influence the coenzyme preference of aldehyde dehydrogenases (ALDHs), we reviewed, analyzed and correlated data from their known crystal structures and amino-acid sequences with their published kinetic parameters for NAD(P)(+). We found that the conformation of the Rossmann-fold loops participating in binding the adenosine ribose is very conserved among ALDHs, so that coenzyme specificity is mainly determined by the nature of the residue at position 195 (human ALDH2 numbering). Enzymes with glutamate or proline at 195 prefer NAD(+) because the side-chains of these residues electrostatically and/or sterically repel the 2'-phosphate group of NADP(+). But contrary to the conformational rigidity of proline, the conformational flexibility of glutamate may allow NADP(+)-binding in some enzymes by moving the carboxyl group away from the 2'-phosphate group, which is possible if a small neutral residue is located at position 224, and favored if the residue at position 53 interacts with Glu195 in a NADP(+)-compatible conformation. Of the residues found at position 195, only glutamate interacts with the NAD(+)-adenosine ribose; glutamine and histidine cannot since their side-chain points are opposite to the ribose, probably because the absence of the electrostatic attraction by the conserved nearby Lys192, or its electrostatic repulsion, respectively. The shorter side-chains of other residues-aspartate, serine, threonine, alanine, valine, leucine, or isoleucine-are distant from the ribose but leave room for binding the 2'-phosphate group. Generally, enzymes having a residue different from Glu bind NAD(+) with less affinity, but they can also bind NADP(+) even sometimes with higher affinity than NAD(+), as do enzymes containing Thr/Ser/Gln195. Coenzyme preference is a variable feature within many ALDH families, consistent with being mainly dependent on a single residue that apparently has no other structural or functional roles, and therefore can easily be changed through evolution and selected in response to physiological needs.

Potential monovalent cation-binding sites in aldehyde dehydrogenases.

Potassium ions are non-essential activators of several aldehyde dehydrogenases (ALDHs), whereas a few others require the cation for activity. Two kinds of cation-binding sites, which we named intra-subunit and inter-subunit, have been observed in crystal structures of ALDHs, and based on reported crystallographic data, we here propose the existence of a third kind located in the central cavity of some tetrameric ALDHs. Given the high structural similarity between these enzymes, cation-binding sites may be present in many other members of this superfamily. To explore the prevalence of these sites, we compared 37 known crystal structures from 13 different ALDH families and evaluated the possible existence of a cation on the basis of the number, distance and geometry of its potential interactions, as well as of B-factor values of modeled cations obtained in new refinements of some reported crystal structures. Also, by performing multiple alignments of 855 non-redundant amino acid sequences, we assessed the degree of conservation in their respective families of the amino acid residues putatively relevant for cation binding. Among the ALDH enzymes studied, and according to our analyses, potential intra-subunit cation-binding sites seem to be present in most members of ALDH2, ALDH1L, ALDH4, ALDH5, ALDH7, ALDH10, and ALDH25 families, as well as in the bacterial and fungal members of the ALDH9 family and in a few ALDH1, ALDH6, ALDH11 and ALDH26 enzymes; potential inter-subunit sites in members of ALDH1L, ALDH3, ALDH4 from bacillales, ALDH5, ALDH7, ALDH9, ALDH10, ALDH11 and ALDH25 families; and potential central-cavity sites only in some bacterial and animal ALDH9s and in most members of the ALDH1L family. Because potassium is the most abundant intracellular cation, we propose that these are potassium-binding sites, but the specific structural and/or functional roles of the cation bound to these different sites remain to be investigated.

Structural determinants of substrate specificity in aldehyde dehydrogenases.

Within the aldehyde dehydrogenase (ALDH) superfamily, proteins belonging to the ALDH9, ALDH10, ALDH25, ALDH26 and ALDH27 families display activity as ω-aminoaldehyde dehydrogenases (AMADHs). These enzymes participate in polyamine, choline and arginine catabolism, as well as in synthesis of several osmoprotectants and carnitine. Active site aromatic and acidic residues are involved in binding the ω-aminoaldehydes in plant ALDH10 enzymes. In order to ascertain the degree of conservation of these residues among AMADHs and to evaluate their possible relevance in determining the aminoaldehyde specificity, we compared the known amino acid sequences of every ALDH family that have at least one member with known crystal structure, as well as the electrostatic potential surface of the aldehyde binding sites of these structures. Our analyses showed that four or three aromatic residues form a similar "aromatic box" in the active site of the AMADH enzymes, being the equivalents to Phe170 and Trp177 (human ALDH2 numbering) strictly conserved in all of them, which supports their relevance in binding the aminoaldehyde by cation-π interactions. In addition, all AMADHs exhibit a negative electrostatic potential surface in the aldehyde-entrance tunnel, due to side-chain carboxyl and hydroxyl groups or main-chain carbonyl groups. In contrast, ALDHs that have non-polar or negatively charged substrates exhibit neutral or positive electrostatic potential surfaces, respectively. Finally, our comparative sequence analyses revealed that the residues equivalent to Asp121 and Phe170 are highly conserved in many ALDH families irrespective of their substrate specificity-suggesting that they perform a role in catalysis additional or different to binding of the substrate-and that the positions Met124, Cys301, and Cys303 are hot spots changed during evolution to confer aldehyde specificity to several ALDH families.

Amino acid residues critical for the specificity for betaine aldehyde of the plant ALDH10 isoenzyme involved in the synthesis of glycine betaine.

Plant Aldehyde Dehydrogenase10 (ALDH10) enzymes catalyze the oxidation of ω-primary or ω-quaternary aminoaldehydes, but, intriguingly, only some of them, such as the spinach (Spinacia oleracea) betaine aldehyde dehydrogenase (SoBADH), efficiently oxidize betaine aldehyde (BAL) forming the osmoprotectant glycine betaine (GB), which confers tolerance to osmotic stress. The crystal structure of SoBADH reported here shows tyrosine (Tyr)-160, tryptophan (Trp)-167, Trp-285, and Trp-456 in an arrangement suitable for cation-π interactions with the trimethylammonium group of BAL. Mutation of these residues to alanine (Ala) resulted in significant K(m)(BAL) increases and V(max)/K(m)(BAL) decreases, particularly in the Y160A mutant. Tyr-160 and Trp-456, strictly conserved in plant ALDH10s, form a pocket where the bulky trimethylammonium group binds. This space is reduced in ALDH10s with low BADH activity, because an isoleucine (Ile) pushes the Trp against the Tyr. Those with high BADH activity instead have Ala (Ala-441 in SoBADH) or cysteine, which allow enough room for binding of BAL. Accordingly, the mutation A441I decreased the V(max)/K(m)(BAL) of SoBADH approximately 200 times, while the mutation A441C had no effect. The kinetics with other ω-aminoaldehydes were not affected in the A441I or A441C mutant, demonstrating that the existence of an Ile in the second sphere of interaction of the aldehyde is critical for discriminating against BAL in some plant ALDH10s. A survey of the known sequences indicates that plants have two ALDH10 isoenzymes: those known to be GB accumulators have a high-BAL-affinity isoenzyme with Ala or cysteine in this critical position, while non GB accumulators have low-BAL-affinity isoenzymes containing Ile. Therefore, BADH activity appears to restrict GB synthesis in non-GB-accumulator plants.

Crystallographic evidence for active-site dynamics in the hydrolytic aldehyde dehydrogenases. Implications for the deacylation step of the catalyzed reaction.

The overall chemical mechanism of the reaction catalyzed by the hydrolytic aldehyde dehydrogenases (ALDHs) involves three main steps: (1) nucleophilic attack of the thiol group of the catalytic cysteine on the carbonyl carbon of the aldehyde substrate; (2) hydride transfer from the tetrahedral thiohemiacetal intermediate to the pyridine ring of NAD(P)(+); and (3) hydrolysis of the resulting thioester intermediate (deacylation). Crystal structures of different ALDHs from several organisms-determined in the absence and presence of bound NAD(P)(+), NAD(P)H, aldehydes, or acid products-showed specific details at the atomic level about the catalytic residues involved in each of the catalytic steps. These structures also showed the conformational flexibility of the nicotinamide half of the cofactor, and of the catalytic cysteinyl and glutamyl residues, the latter being the general base that activates the hydrolytic water molecule in the deacylation step. The architecture of the ALDH active site allows for this conformational flexibility, which, undoubtedly, is crucial for catalysis in these enzymes. Focusing in the deacylation step of the ALDH-catalyzed reaction, here we review and systematize the crystallographic evidence of the structural features responsible for the conformational flexibility of the catalytic glutamyl residue, and for the positioning of the hydrolytic water molecule inside the ALDH active site. Based on the analysis of the available crystallographic data and of energy-minimized models of the thioester reaction intermediate, as well as on the results of theoretical calculations of the pK(a) of the carboxyl group of the catalytic glutamic acid in its three different conformations, we discuss the role that the conformational flexibility of this residue plays in the activation of the hydrolytic water. We also propose a critical participation in the water activation process of the peptide bond to which the catalytic glutamic acid in the intermediate conformation is hydrogen bonded.

The crystal structure of a ternary complex of betaine aldehyde dehydrogenase from Pseudomonas aeruginosa Provides new insight into the reaction mechanism and shows a novel binding mode of the 2'-phosphate of NADP+ and a novel cation binding site.

In the human pathogen Pseudomonas aeruginosa, the NAD(P)(+)-dependent betaine aldehyde dehydrogenase (PaBADH) may play the dual role of assimilating carbon and nitrogen from choline or choline precursors--abundant at infection sites--and producing glycine betaine and NADPH, potentially protective against the high-osmolarity and oxidative stresses prevalent in the infected tissues. Disruption of the PaBADH gene negatively affects the growth of bacteria, suggesting that this enzyme could be a target for antibiotic design. PaBADH is one of the few ALDHs that efficiently use NADP(+) and one of the even fewer that require K(+) ions for stability. Crystals of PaBADH were obtained under aerobic conditions in the presence of 2-mercaptoethanol, glycerol, NADP(+) and K(+) ions. The three-dimensional structure was determined at 2.1-A resolution. The catalytic cysteine (C286, corresponding to C302 of ALDH2) is oxidized to sulfenic acid or forms a mixed disulfide with 2-mercaptoethanol. The glutamyl residue involved in the deacylation step (E252, corresponding to E268 of ALDH2) is in two conformations, suggesting a proton relay system formed by two well-conserved residues (E464 and K162, corresponding to E476 and K178, respectively, of ALDH2) that connects E252 with the bulk water. In some active sites, a bound glycerol molecule mimics the thiohemiacetal intermediate; its hydroxyl oxygen is hydrogen bonded to the nitrogen of the amide groups of the side chain of the conserved N153 (N169 of ALDH2) and those of the main chain of C286, which form the "oxyanion hole." The nicotinamide moiety of the nucleotide is not observed in the crystal, and the adenine moiety binds in the usual way. A salt bridge between E179 (E195 of ALDH2) and R40 (E53 of ALDH2) moves the carboxylate group of the former away from the 2'-phosphate of the NADP(+), thus avoiding steric clashes and/or electrostatic repulsion between the two groups. Finally, the crystal shows two K(+) binding sites per subunit. One is in an intrasubunit cavity that we found to be present in all known ALDH structures. The other--not described before for any ALDH but most likely present in most of them--is located in between the dimeric unit, helping structure a region involved in coenzyme binding and catalysis. This may explain the effects of K(+) ions on the activity and stability of PaBADH.

Development of low-cost metal oxide pH electrodes based on the polymeric precursor method.

In this work, the polymeric precursor method was used to prepare low-cost solid-state sensors for pH determination based on iridium oxide as the main pH sensitive material. The iridium content was reduced with addition of TiO(2), forming the binary system IrO(x)-TiO(2), whose electroanalytical properties were evaluated in comparison with a commercial glass pH electrode. The minimum iridium content which gave suitable results was 30mol%, and the electrode presented Nernstian and fast response in the pH range from 1 to 13, with no hysteresis effect observed. Besides, the electrode showed high selectivity in the presence of alkali ions as Li(+), Na(+) or K(+). The amount of iridium in the prepared electrodes was very small (<0.1mg), supporting the efficiency of this method on the simple preparation of functional low-cost pH electrodes.

Manifestações bucais em pacientes portadores de doenças sexualmente transmissíveis / Oral manifestations in subjects with sexually transmitted diseases

Introdução: a despeito dos avanços tecnológicos ocorridos na área médica nas últimas décadas, as DST persistem em nosso meio como importanteagravo de Saúde Pública, ancorado, quiçá, na falta de conhecimento dos seus mecanismos de transmissão. Particularmente a cavidade oral não tem sido objeto de muitos estudos aprofundados sobre o seu papel na transmissão e no desenvolvimento das DST. O presente estudo visa diminuir tal vácuo de informações em nosso município, assim como contribuir para o conhecimento da epidemiologia das DST em nosso meio. Objetivo: averiguar a prevalência de manifestações intraorais em pacientes portadores de doenças sexualmente transmissíveis (DST) na cidade de Manaus, Amazonas, no ano de 2008. Métodos: foram avaliados 157 portadores de doenças sexualmente transmissíveis em atendimento na Fundação Alfredo da Matta. As lesões orais foram detectadas por exame clínico oral, exames citopatológicos (raspados das lesões) e histopatológicos (biópsias). Resultados: em 24,2% dos participantesforam encontradas 46 (29,1%) lesões orais possivelmente relacionadas com as DST. Observou-se associação signifi cativa entre o nível educacional e o conhecimento acerca da relação entre lesões orais e DST, porém não se obteve associação entre a presença de lesões orais e práticas do sexo oral, nível de escolaridade ou grau de higienização bucal. As análises estatísticas foram feitas no SPSS (nível de confi ança 95%) e foram aplicados testes de qui-quadrado. Conclusão: este estudo evidenciou a existência de demanda reprimida de portadores de DST com relação ao diagnóstico de lesões em mucosa oral, assim como de tratamento e/ou encaminhamento para outros serviços especializados. A necessidade de implantação e oferta de atenção à saúde na área de estomatologia no Amazonas deve ser considerada

Introduction: despite the technological progresses that have taken place in the medical fi eld in the last decades, STD persist to be an aggravating factor in public health. This is perhaps due to the lack of knowledge of our transmission mechanisms. Particularly, the role of the oral cavity in the transmission and development of STD has not been the subject of many detailed studies. This study aims to reduce the information vacuum in our municipality, as well as contribute to the knowledge of epidemiology in STD in our . Objective: investigating the prevalence of oral manifestations of sexually transmitted diseases (STD) in infected individuals in Manaus, Amazon, in the year 2008. Methods: 157 people with confi rmed clinical and laboratorial STD diagnosis undergoing treatment at the Alfredo da Matta Foundation. The oral lesions were detected by oral clinical examination and microscopic techniques for cells and tissue studies. Results: in 24,2% participants 46 (29,1%) oral lesions were observed, which could possibly linked to STDs. Direct correlation was observed between educational level and knowledge of oral lesions and STD, but no correlation was found between oral lesions and oral sexual practice,educational level or oral hygiene. The statistical analysis was done on SPSS (95% confi dence level) and chi-square tests were applied. Conclusion: this study showed the existence of a hidden population of individuals with needs on precocious oral lesions diagnosis, treatment and referring for other specialized services, for example, the Oncology, considering the risk of malignancy of some lesions. The necessity to establish a stomatology service in the Amazon must be considered.

Complex, unusual conformational changes in kidney betaine aldehyde dehydrogenase suggested by chemical modification with disulfiram.

The NAD+-dependent animal betaine aldehyde dehydrogenases participate in the biosynthesis of glycine betaine and carnitine, as well as in polyamines catabolism. We studied the kinetics of inactivation of the porcine kidney enzyme (pkBADH) by the drug disulfiram, a thiol-reagent, with the double aim of exploring the enzyme dynamics and investigating whether it could be an in vivo target of disulfiram. Both inactivation by disulfiram and reactivation by reductants were biphasic processes with equal limiting amplitudes. Under certain conditions half of the enzyme activity became resistant to disulfiram inactivation. NAD+ protected almost 100% at 10 microM but only 50% at 5mM, and vice versa if the enzyme was pre-incubated with NAD+ before the chemical modification. NADH, betaine aldehyde, and glycine betaine also afforded greater protection after pre-incubation with the enzyme than without pre-incubation. Together, these findings suggest two kinds of active sites in this seemingly homotetrameric enzyme, and complex, unusual ligand-induced conformational changes. In addition, they indicate that, in vivo, pkBADH is most likely protected against disulfiram inactivation.

Characterization of the spleen B-cell compartment at the early and late blood-stage Plasmodium chabaudi malaria.

Polyclonal B-cell activation is a feature of the early spleen cell response to blood-stage Plasmodium chabaudi malaria. Immunity to blood-stage malaria is guaranteed by the generation of B cells able to produce parasite-specific antibodies mainly from the immunoglobulin (Ig)G2a isotype. In the present study, we characterized the spleen B-cell compartment during blood-stage P. chabaudi infection. The numbers of B220(+) and B220(LOW) CD138(+) (plasma) cells increased sharply between days 4 and 7 post-infection (p.i.). At this time B220(+) cells expressed surface (s)IgM, but nearly all B220(LOW) CD138(+) cells showed concomitantly intracellular (i)IgM and IgG2a. Both follicular and marginal zone B cells were activated expressing high amounts of CD69. At day 40 p.i., B220(LOW) CD138(+) cell population was still increased but, differently from acute infection, 61.1% of these cells were positive for iIgG2a while only 14.2% expressed iIgM. Moreover, at days 20 and 40 p.i., 29.2% and 13.0% of B220(+) cells expressed sIgG2a, respectively. According to cell size and expression of CD80, CD86, CD11b, CD44 and CD38, B220(+) sIgG2a(+) cells had a phenotype characteristic of activated/memory B cells. Furthermore, 14.1% of B220(+) sIgG2a(+) cells at day 30 p.i. expressed a marginal zone B-cell phenotype. Importantly, B cells from 40-day-infected mice were very efficient in presenting parasite antigens leading to proliferation of both CD4(+) and CD8(+) cells. Our results contribute for understanding the dynamics of B cells during P. chabaudi infection, underlying the mechanisms of antigen presentation and antibody production, which are essential for the acquisition of protective immunity against malaria.

Long term patency of the ductus arteriosus after stent placement: report of two cases and review of the literature

Stent placement in the patent ductus arteriosus (PDA) is becoming an alternative treatment to improve cardiac function and quality of life in patients with ductal dependent cardiac conditions. Although, today most of these cardiac defects can be surgically corrected without the need of maintaining PDA patency for a long period of time, some countries lack the resources, and in other cases, surgical intervention is preferably delayed due to medical reasons. This report presents 2 cases where stents were placed in the PDA.