Habitual exercise induced resistance to oxidative stress

We investigated whether habitual exercise (HE) modulates levels of oxidative DNA damage and responsiveness to oxidative stress induced by renal carcinogen Fe-nitrilotriacetic acid (Fe-NTA). During a ten week protocol, two groups of rats either remained sedentary or underwent swimming for 15-60 min per day, 5 days per week, with or without a weight equivalent to 5% of their body weight. Then we injected Fe-NTA and sacrificed the rats 1 h after the injection. We determined the activity of superoxide dismutase (SOD) in diaphragm and kidney, evaluated levels of 8-hydroxydeoxyguanosine (8OHdG), catalase, and glutathione peroxidase, and assayed OGG1 protein levels in kidney. SOD activity in the diaphragm and kidney was increased in HE rats. By itself, HE had no effect on the level of 8OHdG, but it did significantly suppress induction of 8OHdG by Fe-NTA, and the amount of suppression correlated with intensity of exercise. These results suggest that HE induces resistance to oxidative stress and, at least at the initiation stage, inhibits carcinogenesis.     

Molecular mechanism of the vitamin D antagonistic actions of (23S)-25-dehydro-1alpha-hydroxyvitamin D3-26,23-lactone depends on the primary structure of the carboxyl-terminal region of the vitamin d receptor

We reported that (23S)-25-dehydro-1alpha-hydroxyvitamin D(3)-26,23-lactone (TEI-9647) antagonizes vitamin D receptor (VDR)-mediated genomic actions of 1alpha,25-dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)] in human cells but is agonistic in rodent cells. Human and rat VDR ligand-binding domains are similar, but differences in the C-terminal region are important for ligand binding and transactivation and might determine the agonistic/antagonistic effects of TEI-9647. We tested TEI-9647 on 1alpha,25(OH)(2)D(3) transactivation using SaOS-2 cells (human osteosarcoma) or ROS 24/1 cells (rat osteosarcoma) cotransfected with human or rodent VDR and a reporter. In both cell lines, TEI-9647 was antagonistic with wild-type human (h)VDR, but agonistic with overexpressed wild-type rat (r)VDR. VDR chimeras substituting the hVDR C-terminal region (activation function 2 domain) with corresponding rVDR residues diminished antagonism and increased agonism of TEI-9647. However, substitution of 25 C-terminal rVDR residues with corresponding hVDR residues diminished agonism and increased antagonism of TEI-9647. hVDR mutants (C403S, C410N) demonstrated that Cys403 and/or 410 was necessary for TEI-9647 antagonism of 1alpha,25(OH)(2)D(3) transactivation. These results suggest that species specificity of VDR, especially in the C-terminal region, determines the agonistic/antagonistic effects of TEI-9647 that determine, in part, VDR interactions with coactivators and emphasize the critical interaction between TEI-9647 and the two C-terminal hVDR Cys residues to mediate the antagonistic effect of TEI-9647.     

Thioredoxin affinity chromatography: a useful method for further understanding the thioredoxin network

Thioredoxin affinity chromatography can be used to recognize the target proteins of thioredoxin or thioredoxin-related proteins in whole cells or certain cellular compartments. In the last couple of years, many potential target proteins have been identified from various organelles and organisms by this method. Based on the information on the target proteins provided by these studies, the complete thioredoxin-related redox networks can now be efficiently described.     

Transgenic mouse lines expressing synaptopHluorin in hippocampus and cerebellar cortex

We generated six transgenic mouse lines in which synaptopHluorin (SpH), one of green fluorescent protein-based sensors of vesicular exocytosis, was expressed under the control of neuron-specific Thy-1.2 promoter. In situ hybridization study revealed that SpH mRNA was expressed in a broad spectrum of brain regions in four of them, whereas in others it was expressed in the specific regions of the hippocampus. In one particular line, SpH immunoreactivity was specifically observed in the mossy fiber presynaptic terminals of both hippocampus and cerebellar cortex. The fluorescence intensity of these presynaptic terminals was somewhat decreased by acidic buffer superfusion and greatly increased by vesicular neutralization of pH, indicating that the SpH molecules are mainly distributed in the synaptic vesicles. The exocytosis-dependent fluorescence increment was measured upon activation of a single presynaptic terminal. These transgenic lines are expected to facilitate morphological and physiological studies of presynaptic terminals in a variety of regions of the brain.     

Determination of lipid-bound sulfate by ion chromatography and its application to quantification of sulfolipids from kidneys of various mammalian species

A variety of procedures have been developed for determining the sulfate ester content of various biomolecules. Ion chromatography (IC), that is, quantitation of ionic substances by ion conductimetry after separation by anion-exchange chromatography, has been increasingly utilized for the determination of inorganic sulfate in clinical and environmental samples. We adopted suppressed-mode IC to the determination of lipid- or glycolipid-bound sulfate released by acid hydrolysis and found that it has the advantage of increased precision for wide concentration ranges (30 pmol to approximately micromol) and lack of interference from other lipids. To minimize deterioration of the separation column, the lipophilic constituents in the acid hydrolysate were removed by a two-phase partition system of chloroform-methanol-water. The inorganic sulfate was quantitatively extracted into the aqueous phase by replacing water with an alkaline buffer. By this method, the concentration of sulfolipids was determined in the kidney of mammals with various body mass. Sulfolipids were more concentrated in the kidney of smaller animals, which have higher maximum urine concentrating activity per gram of body mass, supporting the hypothesis of the function of sulfolipids as an ion barrier on the luminal surface of renal tubules.     

A method for accurate detection of translocation junctions in ewing family of tumors

The Ewing family of tumors (ET) generally contain translocations involving the EWS gene and the FLI or ERG genes. Identification of the translocation confirms the diagnosis of ET. Currently, diagnosis of the translocation is made by several methods. In general, these methods require different primer sets for amplifying different translocations and subsequent efforts to identify the amplified product. The need to employ different sets of primers to amplify different translocation junctions presents some limitations. We have developed a method based on PCR with consensus primers followed by direct automated sequencing of the amplified product. With this method we have correctly determined known as well as unknown ET-associated EWS-FLI and EWS-ERG translocations in appropriate specimens. Use of our consensus primers eliminates the need for separate PCRs to amplify EWS-FLI and EWS-ERG translocation junctions, and because direct sequencing is used for confirming the identity of the amplification product, the accuracy of detection becomes 100%. The method might also accurately diagnose ET-associated translocations other than EWS-FLI and EWS-ERG translocations.     

Primary structure and catalytic properties of a cold-active esterase from a psychrotroph,Acinetobacter sp. strain No. 6. isolated from Siberian soil

We cloned a gene coding for a cold-active esterase from a genomic library of Acinetobacter sp. strain No. 6, a psychrotroph isolated from Siberian soil. The gene, aest, encoded a protein of 301 amino acid residues, the deduced sequence of which had less than 17% identity to sequences of known esterases and lipases. However, the esterase seemed to belong to the alpha/beta hydrolase superfamily, because it contained a sequence, Gly-Xaa-Ser-Xaa-Gly (with Xaa an arbitrary amino acid residue), found in most serine hydrolases of this superfamily. Sequence comparison earlier suggested a weak phylogenetic relationship of gene product AEST to the EST group of the esterase-lipase family, which has been found only in eukaryotes. The aest gene was expressed in Escherichia coli BL21(DE3) cells under the control of the T7 promoter, and the expression product was purified to homogeneity and characterized. It catalyzed the hydrolysis of esters with short-chain acyl groups and had lower activation energy and lower thermostability than do mesophilic enzymes, as expected from the cold-adapted nature of this enzyme.