Sequence-specific detection using a universal probe system based on the formation of a four-way junction structure.

We have developed a novel hybridization detection system using a universal probe based on the formation of a four-way junction (4WJ) structure. This methodology employs a combination of two sequence-specific probes and a universal quenching probe, and the same universal probe can be used for any target gene, allowing cost-effective assays. This 4WJ detection is ideal for extensive parallel identification of nucleic acids such as in multiplex polymerase chain reaction (PCR) systems. Compared with gel electrophoresis, this detection procedure is not only sensitive and rapid but also free of hazardous chemicals such as ethidium bromide. In addition, the 4WJ hybridization technology is more specific as an identifier than the size of a band on an agarose gel. We used a model multiplex PCR method that detected eight different virulence genes in Escherichia coli isolates, demonstrating that our 4WJ detection system is rapid, sensitive, and specific.

High levels of oxidative stress exist in the brain than serum or kidneys in stroke-prone spontaneously hypertensive rats at ten weeks of age.

In the present study, we examined levels of oxidative stress in the serum, brain and kidneys of normotensive Wistar Kyoto rats (WKY) and stroke prone spontaneously hypertensive rats (SHRSP) at 10 weeks of age. Levels of advanced oxidation protein products (AOPP), oxidized albumin and oxidized proteins, markers of oxidative stress, were significantly decreased in serum among SHRSP as compared with WKY. Levels of oxidized proteins determined by immunoblotting were significantly increased in the brain, but not kidney, of SHRSP. The mRNA level of super oxide dismutase (SOD) determined by real time polymerase chain reaction (PCR) and the protein level of catalase assessed by immunoblotting were significantly increased in the brain of SHRSP. From these results, it was suggested that levels of oxidative stress were higher in the brain than serum or kidneys of SHRSP at 10 weeks of age, but are not caused by decreases in the expression of SOD and catalase.

Tissue distribution of mevalonate pyrophosphate decarboxylase in guinea pig.

We previously reported that mevalonate pyrophosphate decarboxylase (MPD) is located in the cytosol and that MPD level in the liver is higher than in other rat tissues. In the present study, we further investigated the tissue distribution of MPD in guinea pigs by immunoblotting using anti-rat MPD antiserum. When immunoblot analysis was carried out using guinea pig brain, the antiserum reacted with 46-kDa protein as well as a substance with the same molecular weight of MPD in mice. Protein of 46-kDa detected in guinea pig liver treated with 0.1% pravastatin, a 3-hydroxy-3-methylglutaryl-CoA reductase inhibitor indicating a liver-specific effect, was increased 3-fold as compared with nontreated guinea pigs; however, 46-kDa protein in the brain treated with pravastatin was similar to that treated without pravastatin. When the subcellular distribution of MPD in the brain, liver, kidney, and testis, was examined by cell fractionation, MPD was mostly detected in the cytosol fraction of all tissues. From these data, the 46-kDa protein was identified as MPD. Next, when the tissue distribution of MPD was examined, MPD in the liver was higher than in other tissues. The relative amount of MPD in guinea pig kidney was higher than in rats and similar to in mice, as MPD in the liver of the same species was taken as 1. Furthermore, the correlation coefficient between guinea pigs and rats or mice in the tissue distribution of MPD was 0.69 or 0.72, respectively. These data indicate a relationship in tissue distribution between guinea pigs and rats or mice, although the tissue-specific regulator of MPD between species somewhat differed.