Reconstitution of the Brain Mixed Function Oxidase System: Purification of NADPH-Cytochrome P450 Reductase and Partial Purification of Cytochrome P450 from Whole Rat Brain

NADPH-cytochrome P450 reductase was purified to apparent homogeneity and cytochrome P450 partially purified from whole rat brain. Purified reductase from brain was identical to liver P450 reductase by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blot techniques. Kinetic studies using cerebral P450 reductase reveal Km values in close agreement with those determined with enzyme purified from rat liver. Moreover, the brain P450 reductase was able to function successfully in a reconstituted microsomal system with partially purified brain cytochrome P450 and with purified hepatic P450c (P450IA1) as measured by 7-ethoxycoumarin and 7-ethoxyresorufin O-deethylation. Our results indicate that the reductase and P450 components may interact to form a competent drug metabolism system in brain tissue.     

Antioxidant Enzymes of Larvae of the Cabbage Looper Moth,Trzchoplusza Ni: Subcellular Distribution and Activities of Superoxide Dismutase,Catalase and Glutathione Reductase

In the mid-fifth instar larvae of the cabbage looper moth, Trichoplusia ni, the subcellular distribution of total superoxide dismutase was as follows: 3.05 units (70.0%), 0.97 units (22.3%), and 0.33 units (7.6%) mg^?1 protein in the mitochondrial, cytosolic and nuclear fractions, respectively. No superoxide dismutase activity was detected in the microsomal fraction. Catalase activity was unusually high and as follows: 283.4 units (47.3%), 150.1 units (25.1%). 142.3 units (23.8%), and 22.9 units (3.8%) mg^?1 protein in the mitochondrial, cytosolic, microsomal (containing peroxisomes), and nuclear fractions. No glutathione peroxidase activity was found, but appreciable glutathione reductase activity was detected with broad subcellular distribution as follows: 3.86 units (36.1%), 3.68 units (34.0%). 2.46 units (23.0%). and 0.70 units (6.5%) mg^?1 protein in the nuclear, mitochondrial, and cytosolic fractions, respectively. The unusually wide intracellular distribution of catalase in this phytophagous insect is apparently an evolutionary adaptation to the absence of glutathione peroxidase; hence, lack of a glutathione peroxidase-glutathione reductase role in alleviating stress from lipid peroxidation. Catalase working sequentially to superoxide dismutase, may nearly completely prevent the formation of the lipid peroxidizing OH radical from all intracellular compartments by the destruction of H₂O₂ which together with O^?₂ is a precursor of OH.     

Inactivation of Rabbit Liver Carbonyl Reductase by Phenylglyoxal and 2,3,4-Trinitrobenzenesulfonate Sodium

The chemical modifications of rabbit liver carbonyl reductase (RLCR) with phenylglyoxal (PGO) and 2,3,4-trinitrobenzenesulfonate sodium (TNBS), which are respective chemical modifiers of arginine and lysine residues, were examined. RLCR was rapidly inactivated by these modifiers. Kinetic data for the inactivation demonstrated that each one of arginine and lysine residues is essential for catalytic activity of the enzyme. Furthermore, based on the protective effects of NADP +, NAD + and their constituents against the inactivation of RLCR by PGO and TNBS, we propose the possibility that the functional arginine and lysine residues are located in the coenzyme-binding domain of RLCR and interact with the 2′-phosphate group of NADPH.     

The Use of New Methylene Blue in Pseudomonas aeruginosa Biofilm Destruction

A Pseudomonas aeruginosa biofilm was produced in a model system using the bacterial strain NCIMB 8295, grown on silicone tubing (bore size 0.75 cm). Destruction of the biofilm was attempted using either ampicillin or a combination of white light (light dose=7.2 J cm^{m 2}) and the phenothiazinium photosensitiser new methylene blue, and damage, both to extra-cellular polymeric substance (EPS) and to the organism, was monitored. It was found that although little damage to the EPS occurred with ampicillin, NMB caused both cell death and breakdown of the EPS, suggesting the use of photodynamic antimicrobial chemotherapy (PACT) in the disinfection of pathogenic biofilms, e.g. at external catheter surfaces.     

VKORC1L1, an Enzyme Rescuing the Vitamin K 2,3-Epoxide Reductase Activity in Some Extrahepatic Tissues during Anticoagulation Therapy

Vitamin K is involved in the γ-carboxylation of the vitamin K-dependent proteins, and vitamin K epoxide is a by-product of this reaction. Due to the limited intake of vitamin K, its regeneration is necessary and involves vitamin K 2,3-epoxide reductase (VKOR) activity. This activity is known to be supported by VKORC1 protein, but recently a second gene, VKORC1L1, appears to be able to support this activity when the encoded protein is expressed in HEK293T cells. Nevertheless, this protein was described as being responsible for driving the vitamin K-mediated antioxidation pathways. In this paper we precisely analyzed the catalytic properties of VKORC1L1 when expressed in Pichia pastoris and more particularly its susceptibility to vitamin K antagonists. Vitamin K antagonists are also inhibitors of VKORC1L1, but this enzyme appears to be 50-fold more resistant to vitamin K antagonists than VKORC1. The expression of Vkorc1l1 mRNA was observed in all tissues assayed, i.e. in C57BL/6 wild type and VKORC1-deficient mouse liver, lung, and testis and rat liver, lung, brain, kidney, testis, and osteoblastic cells. The characterization of VKOR activity in extrahepatic tissues demonstrated that a part of the VKOR activity, more or less important according to the tissue, may be supported by VKORC1L1 enzyme especially in testis, lung, and osteoblasts. Therefore, the involvement of VKORC1L1 in VKOR activity partly explains the low susceptibility of some extrahepatic tissues to vitamin K antagonists and the lack of effects of vitamin K antagonists on the functionality of the vitamin K-dependent protein produced by extrahepatic tissues such as matrix Gla protein or osteocalcin.     

Design and characterization of electrochemical dopamine–aptamer as convenient and integrated sensing platform

Here, an ultrasensitive label-free electrochemical aptasensor was developed for dopamine (DA) detection. Construction of the aptasensor was carried out by electrodeposition of gold–platinum nanoparticles (Au–PtNPs) on glassy carbon (GC) electrode modified with acid-oxidized carbon nanotubes (CNTs–COOH). A designed complementary amine-capped capture probe (ssDNA1) was immobilized at the surface of PtNPs/CNTs–COOH/GC electrode through the covalent amide bonds formed by the carboxyl groups on the nanotubes and the amino groups on the oligonucleotides. DA-specific aptamer was attached onto the electrode surface through hybridization with the ssDNA1. Methylene blue (MB) was used as an electrochemical indicator that was intercalated into the aptamer through the specific interaction with its guanine bases. In the presence of DA, the interaction between aptamer and DA displaced the MB from the electrode surface, rendering a lowered electrochemical signal attributed to a decreased amount of adsorbed MB. This phenomenon can be applied for DA detection. The peak current of probe (MB) linearly decreased over a DA concentration range of 1–30 nM with a detection limit of 0.22 nM.     

Tetrahydrofolate and 5-methyltetrahydrofolate are folates with high antioxidant activity. Identification of the antioxidant pharmacophore

The presumed protective effect of folic acid on the pathogenesis of cardiovascular, hematological and neurological diseases and cancer has been associated with the antioxidant activity of folic acid. Peroxynitrite (PON) scavenging activity and inhibition of lipid peroxidation (LPO) of the physiological forms of folate and of structurally related compounds were tested. It was found that the fully reduced forms of folate, i.e. tetrahydrofolate (THF) and 5-methyltetrahydrofolate (5-MTHF), had the most prominent antioxidant activity. It appeared that their protection against LPO is less pronounced than their PON scavenging activity. The antioxidant activity of these forms of folic acid resides in the pterin core, the antioxidant pharmacophore is 4-hydroxy-2,5,6-triaminopyrimidine. It is suggested that an electron donating effect of the 5-amino group is of major importance for the antioxidant activity of 4-hydroxy-2,5,6-triaminopyrimidine. A similar electron donating effect is probably important for the antioxidant activity of THF and 5-MTHF.     

Physiological role of soluble fumarate reductase in redox balancing during anaerobiosis in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, there are two isoenzymes of fumarate reductase (FRDS1 and FRDS2), encoded by the FRDS and OSM1 genes, respectively. Simultaneous disruption of these two genes results in a growth defect of the yeast under anaerobic conditions, while disruption of the OSM1 gene causes slow growth. However, the metabolic role of these isoenzymes has been unclear until now. In the present study, we found that the anaerobic growth of the strain disrupted for both the FRDS and OSM1 genes was fully restored by adding the oxidized form of methylene blue or phenazine methosulfate, which non-enzymatically oxidize cellular NADH to NAD(+). When methylene blue was added at growth-limiting concentrations, growth was completely arrested after exhaustion of oxidized methylene blue. In the double-disrupted strain, the accumulation of succinate in the supernatant was markedly decreased during anaerobic growth in the presence of methylene blue. These results suggest that fumarate reductase isoenzymes are required for the reoxidation of intracellular NADH under anaerobic conditions, but not aerobic conditions.     

Characterization of bromadiolone resistance in a danish strain of Norway rats, Rattus norvegicus, by hepatic gene expression profiling of genes involved in vitamin K-dependent gamma-carboxylation

The present study characterizes the anticoagulant resistance mechanism in a Danish bromadiolone-resistant strain of Norway rats (Rattus norvegicus), with a Y139C VKORC1 mutation. We compared liver expression of the VKORC1 gene, which encodes a protein of the vitamin K 2,3-epoxide reductase complex, the NQO1 gene, which encodes a NAD(P)H quinone dehydrogenase and the Calumenin gene between bromadiolone-resistant and anticoagulant-susceptible rats upon saline and bromadiolone administration. Additionally, we established the effect of bromadiolone on the gene expression in the resistant and susceptible phenotype. Bromadiolone had no effect on VKORC1 and NQO1 expression in resistant rats, but induced significantly Calumenin expression in the susceptible rats. Calumenin expression was similar between the resistant and the susceptible rats upon saline administration but twofold lower in resistant rats after bromadiolone treatment. These results indicate that Danish bromadiolone resistance does not involve an overexpression of calumenin. Independent of the treatment, we observed a low VKORC1 expression in resistant rats, which in conjugation with the Y139C polymorphism most likely explains the low VKOR activity and the enhanced need for vitamin K observed in Danish resistant rats. Furthermore the bromadiolone resistance was found to be associated with a low expression of the NQO1 gene.