Oxidation-reduction potentials in the cecal contents of rats and mice.

The oxidation-reduction potential (ORP) in the cecal contents of conventional rats, germ-free mice, and mice with a Colonization Resistance Factor flora (CRF-mice) was investigated. By using animals that were anaesthetized for a longer period of time, we attempted to eliminate the disturbing influence of oxygen. In addition, measurements were made under anaerobic conditions. For the rats, the ORP values reached a more or less constant level after about 30 min following the insertion of the electrodes. The mean ORP at that time was -458 mV (SD = 45 mV). The mean ORP values for the mice showed a more gradual reduction than was found in the rats. The curves leveled off at about 100 min following the insertion of the electrodes. The mean ORP values 100 min after electrode insertion were: germ-free mice, + 3 mV (SD = 39 mV); CRF-mice, -554 mV (SD = 29 mV). In rats, the ORP value decreased after death; no decrease was observed in mice. No difference was found in the values obtained when measuring under anaerobic or aerobic conditions after death.     

Flavin Mononucleotide-Based Fluorescent Proteins Function in Mammalian Cells without Oxygen Requirement

Usage of the enhanced green fluorescent protein (eGFP) in living mammalian cells is limited to aerobic conditions due to requirement of oxygen during chromophore formation. Since many diseases or disease models are associated with acute or chronic hypoxia, eGFP-labeling of structures of interest in experimental studies might be unreliable leading to biased results. Thus, a chromophore yielding a stable fluorescence under hypoxic conditions is desirable. The fluorescence of flavin mononucleotide (FMN)-based fluorescent proteins (FbFPs) does not require molecular oxygen. Recently, the advantages of FbFPs for several bacterial strains and yeasts were described, specifically, their usage as a real time fluorescence marker in bacterial expression studies and their ability of chromophore formation under anaerobic conditions. Our objective was to verify if FbFPs also function in mammalian cells in order to potentially broaden the repertoire of chromophores with ones that can reliably be used in mammalian studies under hypoxic conditions. In the present study, we demonstrate for the first time, that FbFPs can be expressed in different mammalian cells, among them murine neural stem cells during proliferative and differentiated stages. Fluorescence intensities were comparable to eGFP. In contrast to eGFP, the FbFP fluorescence did not decrease when cells were exposed to defined hypoxic conditions neither in proliferating nor in differentiated cells. Thus, FbFPs can be regarded as an alternative to eGFP in studies that target cellular structures which are exposed to hypoxic conditions.     

A Stable Bacterial Peroxidase with Novel Halogenating Activity and an Autocatalytically Linked Heme Prosthetic Group

Reconstructing the phylogenetic relationships of the main evolutionary lines of the mammalian peroxidases lactoperoxidase and myeloperoxidase revealed the presence of novel bacterial heme peroxidase subfamilies. Here, for the first time, an ancestral bacterial heme peroxidase is shown to possess a very high bromide oxidation activity (besides conventional peroxidase activity). The recombinant protein allowed monitoring of the autocatalytic peroxide-driven formation of covalent heme to protein bonds. Thereby, the high spin ferric rhombic heme spectrum became similar to lactoperoxidase, the standard reduction potential of the Fe(III)/Fe(II) couple shifted to more positive values (−145 ± 10 mV at pH 7), and the conformational and thermal stability of the protein increased significantly. We discuss structure-function relationships of this new peroxidase in relation to its mammalian counterparts and ask for its putative physiological role.     

Changes in blood glutathione concentrations, and in erythrocyte glutathione reductase and glutathione S-transferase activity after running training and after participation in contests

Previously sedentary men (n = 23) and women (n = 18) were trained to run a half marathon contest after 40 weeks. Total blood glutathione had increased by 20 weeks of training and had returned to normal after 40 weeks. Erythrocyte glutathione reductase activity had increased by 20 weeks and remained elevated after 40 weeks. This effect was accompanied by decreases in glutathione reductase coefficients, which indicated that increases in the presence of riboflavin may have been responsible for the changes in reductase activity. Erythrocyte glutathione S-transferase activity had increased slightly after 20 weeks of training and a much more marked increase was found after 40 weeks. This may have been indicative of the occurrence of lipid peroxidation in this phase of training. The participants ran a 15-km race after the first 20 weeks of training and a half marathon after 40 weeks. Blood glutathione tended to decrease after the 15-km race and increased after the half marathon. In both cases it had returned to normal values 5 days after the race. Erythrocyte glutathione reductase was elevated 1 day after the races, and had returned to normal after 5 days. This could also have been explained from concurrent changes in the riboflavin content of the erythrocytes. Erythrocyte glutathione S-transferase activity decreased after both races, but was restored 5 days after the half marathon while such was not the case after the 15-km race.     

Structural requirements for transformation of substrates by the (S)-adenosyl-L-methionine:delta 24(25)-sterol methyl transferase

The membrane-bound enzyme of microsomes obtained from sunflower embryos that catalyzes the bi-substrate transfer reaction whereby the methyl group of (S)-adenosyl-L-methionine is transferred to C-24 of the sterol side chain has been investigated. Optimal incubation conditions for assay of the microsomal (S)-adenosyl-L-methionine:sterol delta 24-methyl transferase (SMT) have been established for the first time. The microsomal preparation was found to catalyze the formation of a delta 24(28)-sterol and to be free of contaminating methyl transferase enzymes, e.g. those which form delta 23-24 methyl sterols (cyclosadol) and delta 25-24 beta-methyl sterols (cyclolaudenol) and other sterolic enzymes which might transform the acceptor molecule to metabolites which could compete in the assay with the test substrate. From a series of incubations with 27 sterol and sterol-like (triterpenoids) substrates of which 23 compounds possessed a 24,25-double bond, we observed a marked dependence on precise structural features and three-dimensional shape of the acceptor molecule in its ability to be transformed by the SMT. In contrast to the yeast SMT where cycloartenol fails to bind to the SMT and zymosterol is the best substrate for methylation, the sunflower SMT studied here utilizes cycloartenol preferentially to zymosterol and the other substrates. Of the chemical groups which distinguishes cycloartenol, a free 3 beta-OH,9 beta,19-cyclopropyl group, trimethylated saturated nucleus, and delta 24-double bond, only the nucleophilic centers at C-3 and C-24 were obligatory for substrate binding and methylation. Of the bent or flat conformations which cycloartenol may orient in the enzyme-substrate complex, our results indicate a selection for acceptor molecules which possess the shape that closely resembles the crystal state and solution orientation of cycloartenol which is now known to be flat rather than bent (Nes, W. D., Benson, M., Lundin, R. E., and Le, P. H. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 5759-5763).     

Optical coherence tomography to detect acute esophageal radiation‐induced damage in mice: A validation study

Radiation therapy for patients with non‐small‐cell lung cancer is hampered by acute radiation‐induced toxicity in the esophagus. This study aims to validate that optical coherence tomography (OCT), a minimally invasive imaging technique with high resolution (~10 μm), is able to visualize and monitor acute radiation‐induced esophageal damage (ARIED) in mice. We compare our findings with histopathology as the gold standard. Irradiated mice receive a single dose of 40 Gy at proximal and distal spots of the esophagus of 10.0 mm in diameter. We scan mice using OCT at two, three, and seven days post‐irradiation. In OCT analysis, we define ARIED as a presence of distorted esophageal layering, change in backscattering signal properties, or change in the esophageal wall thickness. The average esophageal wall thickness is 0.53 mm larger on OCT when ARIED is present based on histopathology. The overall sensitivity and specificity of OCT to detect ARIED compared to histopathology are 94% and 47%, respectively. However, the overall sensitivity of OCT to assess ARIED is 100% seven days post‐irradiation. We validate the capability of OCT to detect ARIED induced by high doses in mice. Nevertheless, clinical studies are required to assess the potential role of OCT to visualize ARIED in humans.