Droplet electrocoupling between capillary electrophoresis and matrix assisted laser desorption/ionization-time of flight-mass spectroscopy and its application.

A droplet of an electroconductive solution was put on the sample plate of a matrix-assisted laser desorption/ionization-time of flight-mass spectroscope (MALDI-TOF-MS) and the outlet terminal of a capillary Electrophoresis (CE) capillary was put into this droplet in order to make an electro-connection and to apply high voltage between the metallic sample plate and the counter pole of the CE. This procedure was simple and gave much more stable interfacing than that of the electrospray method. Furthermore, the separated component was collected and concentrated in a droplet. By mixing each separated sample spot with the MALDI matrix on the sample plate, the spots were analyzed in separated sequences to make three-dimensional mass chromatograms, or applied to the enzyme digestion analysis for peptide sequencing.

Conversion of nitroxide radicals by phenolic and thiol antioxidants.

Nitrone/nitroso spin traps are often used for detection of unstable hydroxyl radical giving stable nitroxide radicals with characteristic electron spin resonance (ESR) signals. This technique may be useful only when the nitroxide radicals are kept stable in the reaction system. The aim of the present study is to clarify whether the nitroxide radicals are kept stable in the presence of the hydroxyl radical scavengers. Effect of hydroxyl radical scavengers on the ESR signals of nitroxide radicals, 2,2,6,6-tetramethyl-piperidine- N-oxyl (TEMPO) and the spin adduct (DMPO-OH) of 5,5-dimethyl-1-pyrroline N-oxide (DMPO) and hydroxyl radical, was examined. Although the ESR signals of TEMPO and the DMPO-OH spin adduct were unchanged on treatment with ethanol and dimethyl sulfoxide, their intensities were effectively decreased on treatment with 6-hydroxy-2,5,7, 8-tetra-methylchroman-2-carboxylic acid (Trolox), cysteine, glutathione, 2-mercaptoethanol and metallothionein. Hence, the results of the detection of hydroxyl radical in the presence of phenolic and thiol antioxidants by the ESR technique using nitrone/nitroso spin traps may be unreliable.

DNA single strand breaks by aromatic nitroso compounds in the presence of thiols.

Aromatic nitroso compounds, nitrosobenzene (NB), N,N-dimethyl-4-nitrosoaniline (DMNA) and 3,5-dibromo-4-nitrosobenzene sulfonate (DBNBS), caused DNA single strand breaks in the presence of thiol compounds. The strand breaking was inhibited completely by free radical scavenger ethanol. Electron spin resonance (ESR) studies showed that hydronitroxyl (or sulfur-substituted nitroxyl) radicals were generated in the early stage of the interactions. Formation of these radicals was not inhibited by ethanol, indicating that these radicals did not directly contribute to the strand breaking. The DNA strand breaking was inhibited partially by superoxide dismutase and catalase under the limited conditions, but not by removal of oxygen from or addition of metal chelators to the reaction mixture. By ESR-spin trapping technique using 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), the DMPO-OH spin adduct was detected. Formation of the spin adduct was inhibited by superoxide dismutase and catalase. The hydronitroxyl (or the sulfur-substituted nitroxyl) radicals may reduce oxygen into active oxygen species and also transformed by themselves into other unidentified free radical species to cause the DNA strand breaks.

Molecular cloning and cold-inducible gene expression of ferritin H subunit isoforms in rainbow trout cells.

Cold acclimation of rainbow trout cells is considered to be mediated by alterations in the mRNAs and proteins present in cold-treated cells. A subtracted cDNA library from cold-treated rainbow trout RTG-2 cells was constructed and screened to isolate cDNA induced in the cold-treated cells in order to elucidate which genes are induced by cold acclimation. A set of cDNA clones encoding three members of ferritin H isoforms was isolated as cold-inducible genes. Northern blot analysis and nuclear run-on transcription assay showed that the transcription and accumulation of the ferritin H isoforms mRNA were enhanced by cold acclimation. Furthermore, the ferritin level in the trout cells increased on cold acclimation in response to a temperature shift from 22 degrees C to 4 degrees C. When the trout cells were subjected to 4 degrees C under the condition of a decreased ferritin H level obtained by the addition of an antisense oligonucleotide, cell growth was apparently inhibited. These findings indicate an association between the induction of ferritin H and cellular mechanisms during cold acclimation of trout cells.

Effect of plant phenolics on the formation of the spin-adduct of hydroxyl radical and the DNA strand breaking by hydroxyl radical.

The effect of plant phenolics, including flavonoids and green tea polyphenolics, on hydroxyl radical was examined by a common method using an electron spin resonance (ESR) technique with 5,5-dimethyl-1-pyrroline N-oxide (DMPO) as a spin trapping agent. The intensity of the ESR signals of DMPO-OH adduct formed by the interaction of DMPO with Fenton reagent was reduced in the presence of each phenolic in a dose-dependent manner. However, the decrease in the intensity of the signals was due partly to the enhanced disappearance of the spin adduct by the phenolics, as has been previously shown. This spin trapping method was unreliable for evaluation of the effect of the phenolics against hydroxyl radical. Hydroxyl radical induced-DNA single-strand breaks may be a better index for evaluation of the activity of the phenolics regarding hydroxyl radical. The effect of the phenolics on DNA single-strand breaks induced by Fenton reagent was examined. While sesamol and esculetin were inhibitory, most polyphenolics, especially (-)-epigallocatechin (EGC) and (-)-epigallocatechin gallate (EGCG), were rather stimulatory. The results indicate that sesamol and esculetin scavenged hydroxyl radical, and EGC and EGCG generated hydroxyl radical under the conditions where hydroxyl radical was generating.

Induction of proteins in response to cold acclimation of rainbow trout cells.

The in vitro translation of poly(A)-RNA isolated from control and cold-treated cells showed that low temperatures induced changes in the population of translatable mRNAs. When cellular proteins extracted from cold-treated cells were subjected to two-dimensional gel electrophoresis, the 70 kDa protein was found to be synthesized during the cold treatment. N-terminal sequence analysis showed that the 70 kDa cold-inducible protein was a homolog of the mammalian valosin-containing protein and yeast CDC48p. The changes in mRNA and protein contents during cold acclimation may result from the expression of genes involved in the adjustment of cellular metabolism to low temperature or the induced proteins may be directly involved in the cold acclimation.