Structure and peroxidase activity of ferric Streptomyces clavuligerus orf10-encoded protein P450CLA: UV-visible, CD, MCD and EPR spectroscopic characterization

The present study reports the spectroscopic characterization by UV-visible absorption spectroscopy, magnetic circular dichroism (MCD) and electron paramagnetic resonance (EPR) of the recombinant orf10-encoded P450-camphor like protein (P450CLA)of Streptomyces clavuligerus expressed in Escherichia coli Rosetta in the native form and associated to external ligands containing the β-lactam, oxazole and alkylamine-derived (alcohol) moieties of the clavulamic acid. Considering the diversity of potential applications for the enzyme, the reactivity with tert-butylhydroperoxide (tert-BuOOH) was also characterized. P450CLA presents a covalently bound heme group and exhibited the UV-visible, CD and MCD spectral features of P450CAM including the fingerprint Soret band at 450 nm generated by the ferrous CO-complex. P450CLA was converted to high valence species by tert-BuOOH and promoted homolytic scission of the O-O bond. The radical profile of the reaction was tert-butyloxyl as primary and methyl and butylperoxyl as secondary radicals. The secondary methyl and butylperoxyl radicals resulted respectively from the β-scission of the alkoxyl radical and from the reaction of methyl radical with molecular oxygen.

Physico-chemical, spectroscopical and thermal characterization of biodiesel obtained by enzymatic route as a tool to select the most efficient immobilized lipase

Two microbial lipases from Burkholderia cepacia and Pseudomonas fluorescens were evaluated as catalysts for the enzymatic transesterification of beef tallow with ethanol and the most efficient lipase source was selected by taking into account the properties of the product to be used as fuel. Both lipases were immobilized on an epoxy silica-polyvinyl alcohol composite by covalent immobilization and used to perform the reactions under the following operational conditions: beef tallow-to-ethanol molar ratio of 1:9, 45ºC and 400 units of enzymatic activity per gram of fat. Products, characterized using Fourier Transform Infrared spectroscopy (FTIR), viscosimetry, thermogravimetry and ¹H NMR spectroscopy, suggested that the biodiesel sample obtained in the reaction catalyzed by Burkholderia cepacia lipase has the best set of properties for fuel usage.

Characterization of immobilized biomass by amplified rDNA restriction analysis (ARDRA) in an anaerobic sequencing-batch biofilm reactor (ASBBR) for the treatment of industrial wastewater

The performance of an anaerobic sequencing-batch biofilm reactor (ASBBR- laboratory scale- 14L )containing biomass immobilized on coal was evaluated for the removal of elevated concentrations of sulfate (between 200 and 3,000 mg SO4-2·L-1) from industrial wastewater effluents. The ASBBR was shown to be efficient for removal of organic material (between 90% and 45%) and sulfate (between 95% and 85%). The microbiota adhering to the support medium was analyzed by amplified ribosomal DNA restriction analysis (ARDRA). The ARDRA profiles for the Bacteria and Archaea domains proved to be sensitive for the determination of microbial diversity and were consistent with the physical-chemical monitoring analysis of the reactor. At 3,000 mg SO4-2·L-1, there was a reduction in the microbial diversity of both domains and also in the removal efficiencies of organic material and sulfate.

Biohydrogen production from food industry waste by suspended and immobilized thermophylic bacteria

This work describes hydrogen production by anaerobic digestion of glucose, molasses and milk whey by 4 thermophilic Thermotoga strains. In the attached-cell tests, the biofilm support characterized by the highest specific surface resulted in the best H2 rate. All the Thermotoga strains examined (T. neapolitana, T. maritima, T. naphtophila, T. petrophila) could produce H2 from glucose, molasses and milk whey, both in suspended- and attached-cell tests. With all the three substrates, the best performances were obtained with T. neapolitana. Some tests were conducted out to select the optimal carrier for the attached-cell conditions. 4 types of carrier were tested: 3 sintered glass carriers and a ceramic one; the chosen carrier was Biomax.

Glutathione peroxidase overexpression causes aberrant ERK activation in neonatal mouse cortex after hypoxic preconditioning

Preconditioning of neonatal mice with nonlethal hypoxia (HPC) protects the brain from hypoxic-ischemic (HI) injury. Overexpression of human glutathione peroxidase 1 (GPx1), which normally protects the developing murine brain from HI injury, reverses HPC protection, suggesting that a certain threshold of hydrogen peroxide concentration is required for activation of HPC signaling.

Kinetics of the interaction of desAABB-fibrin monomer with immobilized fibrinogen

The soluble and stable fibrin monomer-fibrinogen complex (SF) is well known to be present in the circulating blood of healthy individuals and of patients with thrombotic diseases. However, its physiological role is not yet fully understood. To deepen our knowledge about this complex, a method for the quantitative analysis of interaction between soluble fibrin monomers and surface-immobilized fibrinogen has been established by means of resonant mirror (IAsys) and surface plasmon resonance (BIAcore) biosensors. The protocols have been optimized and validated by choosing appropriate immobilization procedures with regeneration steps and suitable fibrin concentrations. The highly specific binding of fibrin monomers to immobilized fibrin(ogen), or vice versa, was characterized by an affinity constant of approximately 10(-8)M, which accords better with the direct dissociation of fibrin triads (KD approximately 10(-8) -10(-9) M) (J. R. Shainoff and B. N. Dardik, Annals of the New York Academy of Science, 1983, Vol. 27, pp. 254-268) than with earlier estimations of the KD for the fibrin-fibrinogen complex (KD approximately 10(-6) M) (J. L. Usero, C. Izquierdo, F. J. Burguillo, M. G. Roig, A. del Arco, and M. A. Herraez, International Journal of Biochemistry, 1981, Vol. 13, pp. 1191-1196).

Hypoxic preconditioning reverses protection after neonatal hypoxia-ischemia in glutathione peroxidase transgenic murine brain

The effect of hypoxic preconditioning (PC) on hypoxic-ischemic (HI) injury was explored in glutathione peroxidase (GPx)-overexpressing mice (human GPx-transgenic [hGPx-tg]) mice. Six-day-old hGPx-tg mice and wild-type (Wt) littermates were pre-conditioned with hypoxia for 30 min and subjected to the Vannucci procedure of HI 24 h after the PC stimulus. Histopathological injury was determined 5 d later (P12). Additional animals were killed 2 h or 24 h after HI and ipsilateral cerebral cortices assayed for GPx activity, glutathione (GSH), and hydrogen peroxide (H2O2). In line with previous studies, hypoxic PC reduced injury in the Wt brain. Preconditioned Wt brain had increased GPx activity, but reduced GSH, relative to naive 24 h after HI. Hypoxic PC did not reduce injury to hGPx-tg brain and even reversed the protection previously reported in the hGPx-tg. GPx activity and GSH in hGPx-tg cortices did not change. Without PC, hGPx-tg cortex had less H2O2 accumulation than Wt at both 2 h and 24 h. With PC, H2O2 remained low in hGPx-tg compared with Wt at 2 h, but at 24 h, there was no longer a difference between hGPx-tg and Wt cortices. Accumulation of H2O2 may be a mediator of injury, but may also induce protective mechanisms.

Pseudovacuoles--immobilized by high-pressure freezing--are associated with blebbing in walker carcinosarcoma cells

By applying high pressure freezing and freeze-substitution, we observed large inclusions of homogeneous appearance in the front of locomoting Walker carcinosarcoma cells that have not been described earlier. Live cell imaging revealed that these inclusions were poor in lipids and nucleic acids but had a high lysine (and hence protein) content. Usually one such structure 2-5 mum in size was present at the front of motile Walker cells, predominantly in the immediate vicinity of newly forming blebs. By correlating the lysine-rich areas in fixed and embedded cells with electron microscopic pictures, inclusions could be assigned to confined, faintly stained cytoplasmic areas that lacked a surrounding membrane; they were therefore called pseudovacuoles. After high-pressure freezing and freeze substitution, pseudovacuoles appeared to be filled with 20 nm large electron-transparent patches surrounded by 12 and 15 nm large particles. The heat shock protein Hsp90 was identified by peptide sequencing as a major fluorescent band on SDS-PAGE of lysine-labelled Walker cell extracts. By immunofluorescence, Hsp90 was found to be enriched in pseudovacuoles. Colocalization of the lysine with a potassium-specific dye in living cells revealed that pseudovacuoles act as K+ stores in the vicinity of forming blebs. We propose that pseudovacuoles might support blebbing by locally regulating the intracellular hydrostatic pressure.

Post-translational tyrosine nitration of eosinophil granule toxins mediated by eosinophil peroxidase

Nitration of tyrosine residues has been observed during various acute and chronic inflammatory diseases. However, the mechanism of tyrosine nitration and the nature of the proteins that become tyrosine nitrated during inflammation remain unclear. Here we show that eosinophils but not other cell types including neutrophils contain nitrotyrosine-positive proteins in specific granules. Furthermore, we demonstrate that the human eosinophil toxins, eosinophil peroxidase (EPO), major basic protein, eosinophil-derived neurotoxin (EDN) and eosinophil cationic protein (ECP), and the respective murine toxins, are post-translationally modified by nitration at tyrosine residues during cell maturation. High resolution affinity-mass spectrometry identified specific single nitration sites at Tyr349 in EPO and Tyr33 in both ECP and EDN. ECP and EDN crystal structures revealed and EPO structure modeling suggested that the nitrated tyrosine residues in the toxins are surface exposed. Studies in EPO(-/-), gp91phox(-/-), and NOS(-/-) mice revealed that tyrosine nitration of these toxins is mediated by EPO in the presence of hydrogen peroxide and minute amounts of NOx. Tyrosine nitration of eosinophil granule toxins occurs during maturation of eosinophils, independent of inflammation. These results provide evidence that post-translational tyrosine nitration is unique to eosinophils.

THE EFFECTS OF STREPTOZOTOCIN DIABETES AND DIETARY IRON INTAKE ON CATALASE, GLUTATHIONE PEROXIDASE, SUPEROXIDE DISMUTASE AND LIPID PEROXIDATION IN CARDIAC AND SKELETAL MUSCLES OF RATS

Catalase, glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) prevent oxygen free radical mediated tissue damage. Diabetes increases and a low dietary intake of iron decreases catalase activity in muscle. Therefore, the combined effects of diabetes and iron deficiency on the free radical scavenging enzyme system and lipid peroxidation were studied. Male, weanling rats were injected with streptozotocin (65 mg/kg, IV) and fed diets containing either 35 ppm iron (Db + Fe) or 8 ppm iron (Db $-$ Fe). Sham injected animals served as iron adequate (C + Fe) or iron deficient (C $-$ Fe) controls. Heart, gastrocnemius (GT), soleus and tibialis anterior (TA) muscles were dissected, weighted and analyzed for catalase, GSH-Px and SOD activities after 3, 6 or 9 weeks on the respective diets. The TBA assay was used to assess lipid peroxidation in the GT muscle. Diabetes elevated catalase activity in all muscles while it had a slight lowering effect on SOD and GSH-Px activities in the GT and TA muscles. In the C $-$ Fe rats, catalase activity declined and remained depressed in all muscles except the heart. There was an elevation in GSH-Px and SOD in the GT muscles of these animals after 6 weeks but not after 9 weeks of consuming the low iron diet. The Db $-$ Fe animals were unable to respond to the diabetic state with catalase activity as high as observed in the Db + Fe rats. Treatment with insulin or iron returned catalase to control levels. The C $-$ Fe animals had significantly lower levels of lipid peroxidation than the other groups at 6 and 9 weeks. Refeeding an iron adequate diet resulted in an increase in lipid peroxidation levels. These studies indicate that skeletal muscle free radical scavenging enzymes are sensitive to metabolic states and that dietary iron influences lipid peroxidation in this tissue. ^

Probing the Electrocatalytic Oxygen Reduction Reaction Reactivity of Immobilized Multicopper Oxidase CueO

The bioelectrocatalytic (oxygen reduction reaction, ORR) properties of the multicopper oxidase CueO immobilized on gold electrodes were investigated. Macroscopic electrochemical techniques were combined with in situ scanning tunneling microscopy (STM) and surface-enhanced Raman spectroscopy at the ensemble and at the single-molecule level. Self-assembled monolayer of mercaptopropionic acid, cysteamine, and p-aminothiophenol were chosen as redox mediators. The highest ORR activity was observed for the protein attached to amino-terminated adlayers. In situ STM experiments revealed that the presence of oxygen causes distinct structure and electronic changes in the metallic centers of the enzyme, which determine the rate of intramolecular electron transfer and, consequently, affect the rate of electron tunneling through the protein. Complementary Raman spectroscopy experiments provided access for monitoring structural changes in the redox state of the type 1 copper center of the immobilized enzyme during the CueO-catalyzed oxygen reduction cycle. These results unequivocally demonstrate the existence of a direct electronic communication between the electrode substrate and the type 1 copper center.