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.

Effect of Melatonin Implants during the Non-Breeding Season on the Onset of Ovarian Activity and the Plasma Prolactin in Dromedary Camel.

To examine a possible control of reproductive seasonality by melatonin, continual-release subcutaneous melatonin implants were inserted 4.5 months before the natural breeding season (October-April) into female camels (Melatonin-treated group). The animals were exposed to an artificial long photoperiod (16L:8D) for 41 days prior to implant placement to facilitate receptivity to the short-day signal that is expected with melatonin implants. The treated and control groups (untreated females) were maintained separately under outdoor natural conditions. Ovarian follicular development was monitored in both groups by transrectal ultrasonography and by plasma estradiol-17ß concentrations performed weekly for 8 weeks and then for 14 weeks following implant insertion. Plasma prolactin concentrations were determined at 45 and 15 days before and 0, 14, 28, 56, and 98 days after implant insertion. Plasma melatonin concentration was determined to validate response to the artificial long photoperiod and to verify the pattern of release from the implants. Results showed that the artificial long photoperiod induced a melatonin secretion peak of significantly (P < 0.05) shorter duration (about 2.5 h). Melatonin release from the implants resulted in higher circulating plasma melatonin levels during daytime and nighttime which persisted for more than 12 weeks following implants insertion. Treatment with melatonin implants advanced the onset of follicular growth activity by 3.5 months compared to untreated animals. Plasma estradiol-17ß increased gradually from the second week after the beginning of treatment to reach significantly (P < 0.01) higher concentrations (39.2 ± 6.2 to 46.4 ± 4.5 pg/ml) between the third and the fifth week post insertion of melatonin implants. Treatment with melatonin implants also induced a moderate, but significant (P < 0.05) suppressive effect on plasma prolactin concentration on the 28th day. These results demonstrate that photoperiod appears to be involved in dromedary reproductive seasonality. Melatonin implants may be a useful tool to manipulate seasonality and to improve reproductive performance in this species. Administration of subcutaneous melatonin implants during the transition period to the breeding season following an artificial signal of long photoperiod have the potential to advance the breeding season in camels by about 2.5 months.

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.

Immunoaffinity purification and characterization of mitochondrial membrane-bound D-3-hydroxybutyrate dehydrogenase from Jaculus orientalis.

BACKGROUND: The interconversion of two important energy metabolites, 3-hydroxybutyrate and acetoacetate (the major ketone bodies), is catalyzed by D-3-hydroxybutyrate dehydrogenase (BDH1: EC 1.1.1.30), a NAD+-dependent enzyme. The eukaryotic enzyme is bound to the mitochondrial inner membrane and harbors a unique lecithin-dependent activity. Here, we report an advanced purification method of the mammalian BDH applied to the liver enzyme from jerboa (Jaculus orientalis), a hibernating rodent adapted to extreme diet and environmental conditions. RESULTS: Purifying BDH from jerboa liver overcomes its low specific activity in mitochondria for further biochemical characterization of the enzyme. This new procedure is based on the use of polyclonal antibodies raised against BDH from bacterial Pseudomonas aeruginosa. This study improves the procedure for purification of both soluble microbial and mammalian membrane-bound BDH. Even though the Jaculus orientalis genome has not yet been sequenced, for the first time a D-3-hydroxybutyrate dehydrogenase cDNA from jerboa was cloned and sequenced. CONCLUSION: This study applies immunoaffinity chromatography to purify BDH, the membrane-bound and lipid-dependent enzyme, as a 31 kDa single polypeptide chain. In addition, bacterial BDH isolation was achieved in a two-step purification procedure, improving the knowledge of an enzyme involved in the lipid metabolism of a unique hibernating mammal. Sequence alignment revealed conserved putative amino acids for possible NAD+ interaction.

Ionophoric properties of atropine: complexation and transport of Na+, K+, Mg2+ and Ca2+ ions across a liquid membrane.

The activity of atropine on the complexation and transport of Na(+), K(+), Mg(2+) and Ca(2+) ions across a liquid membrane was investigated using a spectrophotometric method. Atropine is a natural drug that blocks muscarinic receptors. It is a competitive antagonist of the action of acetylcholine and other muscarinic agonists. Atropine is shown to extract Na(+), K(+), Mg(2+) and Ca(2+) ions from an aqueous phase into an organic one with a preference for Ca(2+) ions. According to a kinetic study, divalent cations (Mg(2+) and Ca(2+)) are more rapidly transported than monovalent ones (Na(+) and K(+)). In both complexation and transport, the flux of the ions increases with the increase of atropine concentration. Atropine might act on the membrane permeability; its complexation and ionophoric properties shed new lights on its therapeutic properties.

Purification and characterization of glyceraldehyde-3-phosphate dehydrogenase from European pilchard Sardina pilchardus.

The NAD+-dependent cytosolic glyceralehyde-3-phosphate dehydrogenase (GAPDH; EC 1.2.1.12) was purified from the skeletal muscle of European pilchard Sardina pilchardus and its physicochemical and kinetic properties were investigated. The purification method consisted of two steps, ammonium sulfate fractionation followed by Blue Sepharose CL-6B chromatography, resulting in an approximately 78-fold increase in specific activity and a final yield of approximately 25%. The Michaelis constants (K(m)) for NAD+ and D-glyceraldehyde-3-phosphate were 92.0 microM and 73.4 microM, respectively. The maximal velocity (V(max)) of the purified enzyme was estimated to be 37.6 U/mg. Under the assay conditions, the optimum pH and temperature were 8.0 and 30 degrees C. The molecular weight of the purified enzyme was 37 kDa determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Non-denaturing polyacrylamide gels yielding a molecular weight of 154 kDa suggested that the enzyme is a homotetramer. Polyclonal antibodies against the purified enzyme were used to recognize the enzyme in different sardine tissues by Western blot analysis. The isoelectric point, obtained by an isoelectric focusing system in polyacrylamide slab gels, revealed only one GAPDH isoform (pI 7.9).

Decolorization of azo dyes with Enterobacter agglomerans immobilized in different supports by using fluidized bed bioreactor.

Immobilized cells of Enterobacter agglomerans, able to reduce azo dyes enzymatically, were used as a biocatalyst for the decolorization of synthetic medium containing the toxic azo dye methyl red (MR). This bacterial strain exhibits high ability to completely decolorize 100 mg/L of MR after only 6 h of incubation under aerobic conditions. Cells of E. agglomerans were immobilized in calcium alginate, polyacylamide, cooper beech, and vermiculite, and were used for the decolorization of MR from synthetic water by using a fluidized bed bioreactor. The highest specific decolorization rate was obtained when E. agglomerans was entrapped in calcium alginate beads and was of about 3.04 mg MR/g cell/h with a 50% conversion time ( t(1/2)) of about 1.6 h. Moreover, immobilized cells in calcium alginate continuously decolorized MR even after seven repeated experiments without significant loss of activity, while polyacrylamide-, cooper beech-, and vermiculite-immobilized cells retained only 62, 15, and 13% of their original activity, respectively.

Biosorption of mercury from aqueous solution by Ulva lactuca biomass.

The mercury biosorption onto non-living protonated biomass of Ulva lactuca, as an alternative method for mercury removal from aqueous solutions, was investigated. Batch equilibrium tests showed that at pH 3.5, 5.5 and 7 the maxima of mercury uptake values, according to Langmuir adsorption isotherm, were 27.24, 84.74 and 149.25 mg/g, respectively. The ability of Ulva lactuca biomass to adsorb mercury in fixed-bed column, was investigated as well. The influence of column bed height, flow rate and effluent initial concentration of metal was studied. The adsorbed metal ions were easily desorbed from the algal biomass with 0.3 N H(2)SO(4) solution. After acid desorption and regeneration with distilled water, the biomass could be reused for other biosorption assays with similar performances.

Purification and partial characterization of azoreductase from Enterobacter agglomerans.

Azoreductase, an enzyme catalyzing the reductive cleavage of the azo bond of methyl red (MR) and related dyes, was purified to electrophoretic homogeneity from Enterobacter agglomerans. This bacterial strain, isolated from dye-contaminated sludge, has a higher ability to grow, under aerobic conditions, on culture medium containing 100mg/L of MR. The enzyme was purified approximately 90-fold with 20% yield by ammonium sulfate precipitation, followed by three steps of column chromatography (gel-filtration, anion-exchange, and dye-affinity). The purified enzyme is a monomer with a molecular weight of 28,000 Da. The maximal azoreductase activity was observed at pH 7.0 and at 35 degrees C. This activity was NADH dependent. The K(m) values for both NADH and MR were 58.9 and 29.4 microM, respectively. The maximal velocity (V(max)) was 9.2 micromol of NADH min(-1)mg(-1). The purified enzyme is inhibited by several metal ions including Fe(2+) and Cd(2+).