Widespread occurrence of non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase among gram-positive bacteria

The non-phosphorylating glyceraldehyde 3-phosphate dehydrogenase (GAPDHN, NADP+-specific, EC 1.2.1.9) is present in green eukaryotes and some Streptococcus strains. The present report describes the results of activity and immunoblot analyses, which were used to generate the first survey of bacterial GAPDHN distribution in a number of Bacillus, Streptococcus and Clostridium strains. Putative gapN genes were identified after PCR amplification of partial 700-bp sequences using degenerate primers constructed from highly conserved protein regions. Alignment of the amino acid sequences of these fragments with those of known sequences from other eukaryotic and prokaryotic GAPDHNs, demonstrated the presence of conserved residues involved in catalytic activity that are not conserved in aldehyde dehydrogenases, a protein family closely linked to GAPDHNs. The results confirm that the basic structural features of the members of the GAPDHN family have been conserved throughout evolution and that no identity exists with phosphorylating GAPDHs. Furthermore, phylogenetic trees generated from multiple sequence alignments suggested a close relationship between plant and bacterial GAPDHN families. [Int Microbiol 2005; 8(4):251-258

Determination of carbendazim residues in Moroccan tomato samples using local enzyme-linked immunosorbent assay and comparison with liquid chromatography

The fungicide carbendazim (CBZ) is not approved for agricultural uses in some countries but is still used by many farmers due to its effectiveness. For this reason, in previous work of the same authors, they developed a competitive enzyme immunoassay (ELISA) using rabbit polyclonal antibodies to detect CBZ. This study aimed to validate this in-house ELISA after extraction with methanol for CBZ analysis in tomato samples, and the results were compared with the conventional high-performance liquid chromatography (HPLC) method after QuEChERS extraction. The results showed that both ELISA and HPLC methods have good repeatability, reproducibility and high precision with a good variation verified by principal components analysis (PCA). ANOVA tested the detection limit (LOD), and quantification limit (LOQ), and the values for ELISA (LOD = 0.026± 0.001 µg/L and LOQ = 0.083 ± 0.003 µg/L) were significantly lower than those obtained by HPLC (LOD = 0.61 ± 0.02 µg/L and LOQ = 1.85 ± 0.07 µg/L). ELISA and HPLC were used for analyzing CBZ in 100 Moroccan tomato samples. These two methods detected the presence of CBZ above the Maximum Residue Limit (MRL) level in 9 samples. However, the presence of the  CBZ was detected in the 79 samples by ELISA and quantified in 66 samples. In contrast, the presence of CBZ was detected in 57 and quantified in 35 samples by HPLC. These results showed that the ELISA system coupled with a simple methanol extraction is much more sensitive than HPLC after QuEChERS extraction

Correlation between circulating cortisol and indicators of stress and oxidant stress during the preslaughter operations in camels

In livestock, pre-slaughter stress begins at the farm or market, continues during transport and upon arrival at the slaughterhouse, ending at slaughter. In this investigation, a survey was conducted in the slaughterhouse of Casablanca in Morocco to record the duration of the preslaughter operations and the frequency of urination in camels. Two groups of camels were constituted, the least stressed animals (Group I, n= 12) and the most stressed animals (Group II, n= 12). Group I animals had a waiting time before loading ≤ 24 h, a loading time ≤ 15 min, an unloading time ≤ 5 min, a water and food deprivation time before slaughter ≤ 24 h, a duration of accompaniment to the slaughter room ≤ 11 min and a frequency of urination during this accompaniment < 3 times. Those in group II had higher duration and frequency values for the same parameters. In addition, serum stress [cortisol (COR)], oxidant stress biomarkers [malondialdehyde (MDA)] and activities of catalase (CAT) and superoxide dismutase (SOD) were analyzed in both groups, and correlations between these biomarkers and the durations of various preslaughter operations and the frequency of urination were established. The most stressed camels (G II) showed high serum concentrations of COR and MDA, and low CAT and SOD activities by comparison to the less stressed camels (G I) (P<0.05). Significant correlations were recorded between COR, MDA, CAT and SOD, and the durations of various preslaughter operations, and between COR and the frequency of urination

Amplia distribución de la gliceraldehído-3-fosfato deshidrogenasa no-fosforilante entre las bacterias gram-positivas

The non-phosphorylating glyceraldehyde 3-phosphate dehydrogenase (GAPDHN, NADP+ -specific, EC 1.2.1.9) is present in green eukaryotes and some Streptococcus strains. The present report describes the results of activity and immunoblot analyses, which were used to generate the first survey of bacterial GAPDHN distribution in a number of Bacillus, Streptococcus and Clostridium strains. Putative gapN genes were identified after PCR amplification of partial 700-bp sequences using degenerate primers constructed from highly conserved protein regions. Alignment of the amino acid sequences of these fragments with those of known sequences from other eukaryotic and prokaryotic GAPDHNs, demonstrated the presence of conserved residues involved in catalytic activity that are not conserved in aldehyde dehydrogenases, a protein family closely linked to GAPDHNs. The results confirm that the basic structural features of the members of the GAPDHN family have been conserved throughout evolution and that no identity exists with phosphorylating GAPDHs. Furthermore, phylogenetic trees generated from multiple sequence alignments suggested a close relationship between plant and bacterial GAPDHN families.La gliceraldehído-3-fosfato deshidrogenasa no-fosforilante (GAPDHN, NADP+ -específica, EC 1.2.1.9) está presente en organismos eucariotas fotosintéticos y en algunas cepas de Streptococcus y Clostridium. En este trabajo se presentan los resultados de los análisis de actividad e inmunotransferencia, que se utilizaron para la primera prospección de la distribución de GAPDHN bacteriana en diversas cepas de Bacillus, Streptococcus y Clostridium. Se han identificado genes putativos gapN mediante amplificación por PCR de secuencias parciales de 700 bp utilizando cebadores degenerados construidos a partir de regiones proteínicas muy conservadas. Las secuencias de aminoácidos de estos fragmentos se alinearon con las de otras secuencias conocidas de GAPDHN eucarióticas y procarióticas, lo que demuestra la presencia de residuos conservados que participan en la actividad catalítica y que no se han conservado en las aldehído deshidrogenasas, una familia de proteínas estrechamente relacionadas con las GAPDHN. Los resultados confirman que las características estructurales básicas de los miembros de la familia GAPDHN se han conservado durante la evolución y que no existe identidad con las GAPDH fosforilantes. Además, los árboles filogenéticos generados a partir de alineaciones de secuencia múltiples sugieren una estrecha relación entre las familias GAPDHN en plantas y bacterias.This work was supported by AECI (Spain) and Collaborative Grants from the Andalusian Government (Consejería de Presidencia, Junta de Andalucía, Ayuda de Cooperación al Desarrollo en el Ambito Universitario no. 52/02, 54/04, and PAI CVI-261 group) – Ministére d’Education et de la Recherche Scientifique and PARS (Morocco). FV was supported by an EU Marie Curie reintegration project no. 505303.Peer reviewe

Effects of oxidative and nitrosative stress on Tetrahymena pyriformis glyceraldehydes-3-phosphate dehydrogenase

Journal of Eukaryotic Microbiology,54(4) p.338-346Previous reports showed that hydrogen peroxide and the NO-generating reagent sodium nitroprusside (SNP)-modulated enzymatic activity of animal glyceraldehyde-3-phosphate dehydrogenase (GAPDH, EC 1.2.1.12). These modifications are suggested to have a physiological regulatory role. To gain further insight into this regulatory process the model ciliated protozoan Tetrahymena pyriformis was chosen. Both reagents inhibited growth of T. pyriformis cultures and produced a specific increase of GAPDH protein but only NO seemed to reduce GAPDH activity in cell-free extracts. Both specific activity and pI were found to be altered in the in vivo NO-treated purified enzyme, but no effect was detected by the in vivo H2O2 treatment. Analytical chromatofocusing showed a single basic isoform (pI 8.8) in enzyme preparations from control and H2O2-treated cells. In contrast to this, three more acidic isoforms (pIs, 8.6, 8.0 and 7.3) were resolved in purified fractions from SNP-treated cells, suggesting post-translational modification of the enzyme by NO. Nevertheless, a decrease of GAPDH activity by H2O2 and NO, mainly due to a decrease in its Vmax without apparent change in substrate affinity, was observed in vitro in the whole enzyme population. The increase of GAPDH protein level found in vivo suggests a cell response in order to compensate for the inhibitory effect on activity observed in the purified enzyme. This is the first report of NO- and H2O2-dependent effects on GAPDH of T. pyriformis, and identifies this key protein of central carbon metabolism as a physiological target of oxidative and nitrosative stress in this ciliated protozoan.Peer reviewe

Ampla distribuição da gliceraldeído-3-fosfato desidrogenase não fosforiladora entre as bactérias gram-positivas

The non-phosphorylating glyceraldehyde 3-phosphate dehydrogenase (GAPDHN, NADP+-specific, EC 1.2.1.9) is present in green eukaryotes and some Streptococcus strains. The present report describes the results of activity and immunoblot analyses, which were used to generate the first survey of bacterial GAPDHN distribution in a number of Bacillus, Streptococcus and Clostridium strains. Putative gapN genes were identified after PCR amplification of partial 700-bp sequences using degenerate primers constructed from highly conserved protein regions. Alignment of the amino acid sequences of these fragments with those of known sequences from other eukaryotic and prokaryotic GAPDHNs, demonstrated the presence of conserved residues involved in catalytic activity that are not conserved in aldehyde dehydrogenases, a protein family closely linked to GAPDHNs. The results confirm that the basic structural features of the members of the GAPDHN family have been conserved throughout evolution and that no identity exists with phosphorylating GAPDHs. Furthermore, phylogenetic trees generated from multiple sequence alignments suggested a close relationship between plant and bacterial GAPDHN families.La gliceraldehído-3-fosfato deshidrogenasa no-fosforilante (GAPDHN, NADP+-específica, EC 1.2.1.9) está presente en organismos eucariotas fotosintéticos y en algunas cepas de Streptococcus y Clostridium. En este trabajo se presentan los resultados de los análisis de actividad e inmunotransferencia, que se utilizaron para la primera prospección de la distribución de GAPDHN bacteriana en diversas cepas de Bacillus, Streptococcus y Clostridium. Se han identificado genes putativos gapN mediante amplificación por PCR de secuencias parciales de 700 bp utilizando cebadores degenerados construidos a partir de regiones proteínicas muy conservadas. Las secuencias de aminoácidos de estos fragmentos se alinearon con las de otras secuencias conocidas de GAPDHN eucarióticas y procarióticas, lo que demuestra la presencia de residuos conservados que participan en la actividad catalítica y que no se han conservado en las aldehído deshidrogenasas, una familia de proteínas estrechamente relacionadas con las GAPDHN. Los resultados confirman que las características estructurales básicas de los miembros de la familia GAPDHN se han conservado durante la evolución y que no existe identidad con las GAPDH fosforilantes. Además, los árboles filogenéticos generados a partir de alineaciones de secuencia múltiples sugieren una estrecha relación entre las familias GAPDHN en plantas y bacterias.Indicou-se a presença da gliceraldeído-3-fosfato desidrogenase não fosforiladora (GAPDHN, NADP+-específica, EC 1.2.1.9) em organismos eucariotas fotossintéticos e em algumas cepas de Streptococcus e Clostridium. Neste trabalho apresenta-se os resultados da atividade en imunotransferência, usados para a primeira prospecção da distribuição da GAPDHN bacteriana em diversas cepas de Bacillus, Streptococcus e Clostridium. Se identificaram genes putativos gapN mediante amplificação por PCR de seqüências parciais de 700 bp utilizando iniciadores degenerados construídos a partir de regiões proteicas altamente conservadas. As seqüências de aminoácidos destes fragmentos se alinharam com as de outras seqüências desconhecidas de GAPDHNs eucarióticas e procarióticas, o que demonstra a presença de resíduos conservados que participam da atividade catalítica que não estão conservados nas aldeído desidrogenases, uma família de proteínas estreitamente relacionados com as GAPDHN. Este trabalho confirma que as características estruturais básicas dos membros da família GAPDHN se conservaram durante a evolução e que não existe identidade com as GAPDH fosforilantes. Além disso, as árvores filogenéticas geradas a partir de alinhamentos de seqüência múltiplas indicam uma estreita relação entre as famílias de GAPDHN de plantas e bactérias.Junta de Andalucía 52/02 54/04 PAI CVI-261EU Marie Curie 50530