Protective Effect of Some Essential Oils Against Gamma-Radiation Damages in Tetrahymena pyriformis Exposed to Cobalt-60 Source.

The main purpose of this study was to investigate the protective effect of Rosmarinus officinalis, Origanum compactum, Lavandula angustifolia, and Eucalyptus globulus essential oils (EOs) against gamma-radiation-induced damages on Tetrahymena pyriformis growing in presence of cobalt-60 source. The chemical composition of the 4 EOs was analyzed by gas chromatography-mass spectrometry. The protective effects of EOs on growth, on morphology, and on some metabolic enzymes and antioxidant markers have been evaluated. Thus, addition of EOs significantly improves the growth parameters (generation number and time) in irradiating conditions. All EOs allowed restoring growth parameters over more than 90% compared to the controls. The morphological analysis indicated that T. pyriformis cells growing in irradiating conditions were able to regain their normal form in presence of the different EOs. Our results indicate that the 4 EOs also have protective effects on some metabolic enzymes. They allowed recovering totally or partially the glyceraldehyde 3-phosphate dehydrogenase and the succinate dehydrogenase activities compared to the controls. Moreover, the addition of EOs reduced the lipid peroxidation level and decreased the activities of catalase and superoxide dismutase induced by the gamma-radiation exposure. A more pronounced protective effect was found for O. compactum and L. angustifolia EOs compared to R. officinalis and E. globulus EOs. These results suggest that the studied EOs are efficient natural antioxidants that could offer protection against gamma-radiation-induced damages and can therefore be useful in clinical medicine.

Changes in Growth, Morphology, and Physiology of Tetrahymena pyriformis Exposed to Continuous Cesium-137 and Cobalt-60 Gamma-Radiation.

This study investigated the effects of gamma-radiation on Tetrahymena pyriformis. The experimental approach consists of exposing T. pyriformis growing in presence of Cesium-137 (137Cs) at dose rates of 1, 2, 4, and 6 cGy h-1 and Cobalt-60 (60Co) at dose rates of 8, 10, 15, and 20 cGy h-1. The radiation doses effects on growth, morphology, some metabolic enzymes, and reactive oxygen species (ROS) markers have been evaluated. When cells were growing in irradiating conditions at dose rates beyond 4 cGy h-1, a decreasing of cells and generation numbers with a prolongation of generation time and a change of morphological aspect with rounding-off of cells were observed compared to the control. The 50%-inhibitory dose (ID50) for radiation was estimated at 1568.72 ± 158.45 cGy. The gamma-radiation at dose rates more than 6 cGy h-1, affected both glyceraldehyde 3-phosphate dehydrogenase and succinate dehydrogenase by inhibiting their activities. All of these effects were more pronounced when cells were irradiated at the dose rate of 20 cGy h-1 using 60Co source. For ROS markers generated by gamma-radiation in T. pyriformis, the results showed an increase of the lipid peroxidation in cells grown in presence of gamma-radiation at dose rates more than 6 cGy h-1 and an enhancement in catalase and superoxide dismutase activities from the dose rate of 1 cGy h-1. These encouraging results suggested the use of T. pyriformis as a unicellular model cell to investigate other aspects of the response to ionizing radiation.

Effects of the Cobalt-60 gamma radiation on Pichia pastoris glyceraldehyde-3-phosphate dehydrogenase.

PURPOSE: Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a key enzyme of the glycolytic pathway, can play a physiological regulatory role and vital other roles in metabolism. This study investigated the effects of gamma radiation generated by Cobalt-60 source on GAPDH activity and protein levels in Pichia pastoris as an eukaryotic organism model. MATERIALS AND METHODS: After purification of the GAPDH from P. pastoris, in vitro effects of irradiation to the dose of 2 Gy, using Cobalt-60 at the dose rate of 0.25 Gy/min, on activity and kinetic parameters were investigated. In vivo effects of gamma exposition (dose of 5 Gy) on P. pastoris GAPDH and on reactive oxygen species (ROS) markers were also explored. RESULTS AND CONCLUSIONS: The in vitro irradiation of the purified GAPDH reduces the specific activity and the maximum velocity (Vmax) without alteration of substrates binding (Km). No changes occurred in the specific activity and in kinetic parameters when P. pastoris cells were exposed to Cobalt-60 source. However, this in vivo irradiation of cells produced a significant increase of the GAPDH protein level. The changes of GAPDH activity and the increase of the enzyme population as a target for gamma radiation exposure will play a role in cells adaptation under stress conditions. On the other hand, the increase of malondialdehyde and carbonyl contents and the enhancement of catalase and superoxide dismutase in irradiated cells have been noticed. The antioxidant system can play an important role in the protection of P. pastoris GAPDH against the gamma induced-ROS damage. This is the first report of the P. pastoris GAPDH as a physiological target of gamma exposition.

Silica-coated calcium pectinate formulations for controlling carbendazim release: water and soil release studies.

This study aims to encapsulate the fungicide carbendazim using a biodegradable polymer (pectin). First, we have obtained calcium pectinate beads (CPG-Carb) by ionotropic gelation using calcium ions as a crosslinking agent. These beads were then coated with silica starting from tetraethoxysilane (TEOS), by a sol-gel process to form hybrid beads (CPG-Carb-SG). The morphology, composition and structure of both beads were characterized and the controlled release assays of the fungicide were studied in both water and soil columns. The encapsulation efficiency for CPG-Carb was slightly higher (75%) compared to CPG-Carb-SG (67%) due to carbendazim loss during the impregnation and condensation steps. The release rate in water and soil columns was about 4 times lower for CPG-Carb-SG than CPG-Carb demonstrating the efficiency of the silica coating to delay the release of carbendazim. Moreover, the release of CPG-Carb-SG is due to the erosion of the silica layer during the first two weeks. After this period, the silica layer was degraded, and the release is then controlled by the swelling of the organic part of the bead as observed for CPG-Carb. Finally, the biodegradability of the pectin, and the release profile make such systems promising candidates for sustained and economical pesticide delivery systems.

Lateral flow immunogold assay as a rapid detection tool for screening of congenital hypothyroidism.

Congenital hypothyroidism (CH) is one of the most common preventable causes of mental retardation. The majority of infants are diagnosed after detection through newborn screening programs using thyroid-stimulating hormone (TSH) test. A rapid immunochromatographic lateral flow assay based on monoclonal antibodies (MAbs) colloidal gold nanoparticles was developed in a sandwich format for the detection of TSH. Two MAbs binding distinct TSH epitopes are used; one is conjugated to the detection reagent while the other is immobilized at the test line on the membrane. The colloidal gold was prepared by the reduction of gold salt coupled with MAbs and this optimal concentration was determined by spectrophotometry method. The sensitivity of our developed lateral flow immunoassay was determined using 5, 10, 15, 25 and 50 µUI/mL of TSH. The color intensity of the test line was directly proportional to the TSH concentration and the visual limit of detection was 10 µUI/mL. Twenty samples of umbilical cord serum were analyzed by the developed strips and the intensity of the signal was in agreement with the results obtained by the conventional radioimmunoassay method. The results suggest that this rapid test can be used in initial screening for congenital hypothyroidism especially in rural areas.

Immunoaffinity purification and characterization of glyceraldehyde-3-phosphate dehydrogenase from human erythrocytes.

A new procedure utilizing immunoaffinity column chromatography has been used for the purification of glyceraldehyde-3-phosphate dehydrogenase (GAPDH, EC 1.2.1.12) from human erythrocytes. The comparison between this rapid method (one step) and the traditional procedure including ammonium sulfate fractionation followed by Blue Sepharose CL-6B chromatography shows that the new method gives a highest specific activity with a highest yield in a short time. The characterization of the purified GAPDH reveals that the native enzyme is a homotetramer of ~150 kDa with an absolute specificity for the oxidized form of nicotinamide adenine dinucleotide (NAD(+)). Western blot analysis using purified monospecific polyclonal antibodies raised against the purified GAPDH showed a single 36 kDa band corresponding to the enzyme subunit. Studies on the effect of temperature and pH on enzyme activity revealed optimal values of about 43 degrees C and 8.5, respectively. The kinetic parameters were also calculated: the Vmax was 4.3 U/mg and the Km values against G3P and NAD(+) were 20.7 and 17.8 muM, respectively. The new protocol described represents a simple, economic, and reproducible tool for the purification of GAPDH and can be used for other proteins.

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

Previous 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 H(2)O(2) treatment. Analytical chromatofocusing showed a single basic isoform (pI 8.8) in enzyme preparations from control and H(2)O(2)-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 H(2)O(2) and NO, mainly due to a decrease in its V(max) 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 H(2)O(2)-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.

Cloning, gene expression and characterization of a novel bacterial NAD-dependent non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase from Neisseria meningitidis strain Z2491.

Alignment of the amino acid sequence of some archaeal, bacterial and eukaryotic non-phosphorylating glyceraldehydes-3-phosphate dehydrogenases (GAPNs) and aldehyde dehydrogenases (ALDHs) with the sequence of a putative GAPN present in the genome of the Gram-negative bacterium Neisseria meningitidis strain Z2491 demonstrated the conservation of residues involved in the catalytic activity. The predicted coding sequence of the N. meningitidis gapN gene was cloned in Escherichia coli XL1-blue under the expression of an inducible promoter. The IPTG-induced GAPN was purified ca. 48-fold from E. coli cells using a procedure that sequentially employed conventional ammonium sulfate fractionation as well as anion-exchange and affinity chromatography. The purified recombinant enzyme was thoroughly characterized. The protein is a homotetramer with a 50-kDa subunit, exhibiting absolute specificity for NAD and a broad spectrum of aldehyde substrates. Isoelectric focusing analysis with the purified fraction showed the presence of an acidic polypeptide with an isoelectric point of 6.3. The optimum pH of the purified enzyme was between 9 and 10. Studies on the effect of increasing temperatures on the enzyme activity revealed an optimal value ca. 64 degrees C. Molecular phylogenetic data suggest that N. meningitidis GAPN has a closer relationship with archaeal GAPNs and glyceraldehyde dehydrogenases than with the typical NADP-specific GAPNs from Gram-positive bacteria and photosynthetic eukaryotes.

Purification and characterization of cytosolic glyceraldehyde-3-phosphate dehydrogenase from the dromedary camel.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (EC 1.2.1.12), a key enzyme of carbon metabolism, was purified and characterized to homogeneity from skeletal muscle of Camelus dromedarius. The protein was purified approximately 26.8 folds by conventional ammonium sulphate fractionation followed by Blue Sepharose CL-6B chromatography, and its physical and kinetic properties were investigated. The native protein is a homotetramer with an apparent molecular weight of approximately 146 kDa. Isoelectric focusing analysis showed the presence of only one GAPDH isoform with an isoelectric point of 7.2. The optimum pH of the purified enzyme was 7.8. Studies on the effect of temperature on enzyme activity revealed an optimal value of approximately 28-32 degrees with activation energy of 4.9 kcal/mol. The apparent K(m) values for NAD(+) and DL-glyceraldehyde-3-phophate were estimated to be 0.025+/-0.040 mM and 0.21+/-0.08 mM, respectively. The V(max) of the purified protein was estimated to be 52.7+/-5.9 U/mg. These kinetic parameter values were different from those described previously, reflecting protein differences between species.

Widespread occurrence of non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase among gram-positive bacteria / 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 (AU)

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 (AU)

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.

Different thermostability of skeletal muscle glyceraldehyde-3-phosphate dehydrogenase from hibernating and euthermic jerboa (Jaculus orientalis).

In previous study, we demonstrated that the specific activity of D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH, EC 1.2.1.12) in skeletal muscle of induced hibernating jerboa (hibernating GAPDH) was 3 4 folds lower than that of the one in the skeletal muscle of the euthermic jerboa (euthermic GAPDH). A significant decrease in both GAPDH protein and GapC mRNA levels occurs when hibernating, but the purified hibernating GAPDH is less active than the euthermic GAPDH. To investigate the physico-chemical basis of this lower activity, the behaviour during thermal inactivation of skeletal muscle GAPDH from hibernating and euthermic tissues was examined by a variety of spectroscopic techniques, including fluorescence emission, circular dichroism and ultraviolet absorption. A clear resistance to thermal denaturation was observed in the hibernating GAPDH compared with the euthermic GAPDH. The different temperature of denaturation found in these proteins by both fluorimetry and circular dichroism indicates that there might exist conformational changes of GAPDH upon hibernation that could affect the stability of this enzyme.

Purification of recombinant non-phosphorylating NADP-dependent glyceraldehyde-3-phosphate dehydrogenase from Streptococcus pyogenes expressed in E. coli.

Streprococcus pyogenes gapN was cloned and expressed by functional complementation of the Escherichia gap mutant W3CG. The IPTG-induced NADP non-phosphorylating GAPDH (GAPN) has been purified about 75.4 fold from E. coli cells, using a procedure involving conventional ammonium sulfate fractionation, anion-exchange chromatography, hydrophobic chromatography and hydroxyapatite chromatography. The purified protein was characterised: it's an homotetrameric structure with a native molecular mass of 224 kDa, have an acid pI of 4.9 and optimum pH of 8.5. Studies on the effect of assay temperature on enzyme activity revealed an optimal value of about 60 degrees C with activation energy of 51 KJ mole(-1). The apparent Km values for NADP and D-G3P or DL-G3P were estimated to be 0.385 +/- 0.05 and 0.666 +/- 0.1 mM, respectively and the Vmax of the purified protein was estimated to be 162.5 U mg(-1). The S. pyogenes GAPN was markedly inhibited by sulfydryl-modifying reagent iodoacetamide, these results suggest the participation of essential sulfydryl groups in the catalytic activity.

Expression, purification, and characterization of recombinant nonphosphorylating NADP-dependent glyceraldehyde-3-phosphate dehydrogenase from Clostridium acetobutylicum.

Clostridium acetobutylicum gapN was cloned and expressed in Escherichia coli BL-21. The IPTG-induced nonphosphorylating NADP-dependent GAPDH (GAPN) has been purified about 34-fold from E. coli cells and its physical and kinetic properties were investigated. The purification method consisted of a rapid and straightforward procedure involving anion-exchange and hydroxyapatite chromatographies. The purified protein is an homotetrameric of 204kDa exhibiting absolute specificity for NADP. Chromatofocusing analysis showed the presence of only one acidic GAPN isoform with an acid isoelectric point of 4.2. The optimum pH of purified enzyme was 8.2. Studies on the effect of assay temperature on enzyme activity revealed an optimal value of about 65 degrees C with activation energy of 18KJmol(-1). The apparent K(m) values for NADP and D-glyceraldehyde-3-phosphate (D-G3P) or DL-G3P were estimated to be 0.200+/-0.05 and 0.545+/-0.1 mM, respectively. No inhibition was observed with L-D3P. The V(max) of the purified protein was estimated to be 78.8 U mg(-1). The Cl. acetobutylicum GAPN was markedly inhibited by sulfhydryl-modifying reagent iodoacetamide, these results suggest the participation of essential sulfhydryl groups in the catalytic activity.

A phosphate-stimulated NAD(P)+-dependent glyceraldehyde-3-phosphate dehydrogenase in Bacillus cereus.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a key enzyme of central carbon metabolism, was studied in a Bacillus cereus strain isolated from the phosphate layer from Morocco. Enzymatic assays with cell extracts demonstrated that when grown on Luria-Bertani (LB) medium, B. cereus contains a major NAD+-dependent GAPDH activity and only traces of NADP+-dependent activity, but in cells grown on Pi-supplemented LB medium a strong increase of the NADP+-dependent activity, that became predominant, occurs concurrently with a GAPDH protein increase. Our results show that B. cereus possesses two GAPDH activities, namely NAD+- and NADP+-dependent, catalyzed by two enzymes with distinct coenzyme specificity and different phosphate regulation patterns. The finding of a phosphate-stimulated NADP+-dependent GAPDH in B. cereus indicates that this bacterium can modulate its primary carbon metabolism according to phosphate availability.

Glyceraldehyde-3-phosphate dehydrogenase from the newt Pleurodeles waltl. Protein purification and characterization of a GapC gene.

The NAD(+)-dependent cytosolic glyceraldehyde-3-phosphate dehydrogenase (GAPDH, EC 1.2.1.12) has been purified to homogeneity from skeletal muscle of the newt Pleurodeles waltl (Amphibia, Urodela). The purification procedure including ammonium sulfate fractionation followed by Blue Sepharose CL-6B chromatography resulted in a 24-fold increase in specific activity and a final yield of approximately 46%. The native protein exhibited an apparent molecular weight of approximately 146 kDa with absolute specificity for NAD(+). Only one GAPDH isoform (pI 7.57) was obtained by chromatofocusing. The enzyme is an homotetrameric protein composed of identical subunits with an apparent molecular weight of approximately 37 kDa. Monospecific polyclonal antibodies raised in rabbits against the purified newt GAPDH immunostained a single 37-kDa GAPDH band in extracts from different tissues blotted onto nitrocellulose. A 510-bp cDNA fragment that corresponds to an internal region of a GapC gene was obtained by RT-PCR amplification using degenerate primers. The deduced amino acid sequence has been used to establish the phylogenetic relationships of the Pleurodeles enzyme--the first GAPDH from an amphibian of the Caudata group studied so far--with other GAPDHs of major vertebrate phyla.