Abstract
A mouse kidney cDNA isolated by differential screening was found to be highly homologous to rat, human, and bovine plasma glutathione peroxidase (GPx) sequences. Analysis of the full-length coding region sequence demonstrated an in-frame selenocysteine-encoding opal codon and putative signal sequence, suggesting that the sequence represents the mouse homolog of plasma GPx. The level of expression of plasma GPx in various mouse tissues and during development was investigated by Northern blot analysis. Plasma GPx mRNA was observed to be very abundant in kidney compared with placenta, epididymis, intestine, lung, heart, testis, ovary, salivary gland, spleen, thymus, stomach, brain, and fetal kidney and could not be detected in pancreas or in liver except from pregnant mice. In addition, plasma GPx mRNA levels were shown to increase during postnatal development of the kidney. In situ hybridization localized plasma GPx mRNA to proximal tubules, while primary cell culture demonstrated that plasma GPx is synthesized and secreted by proximal tubular epithelial cells. The relative abundance of plasma GPx mRNA in mouse kidney suggests that proximal tubules may be the primary source of the enzyme detectable in plasma and further suggests that plasma GPx has an important function in protecting the kidney from oxidative damage.
Highlights
A mouse kidneycDNA isolated by differentia1 screen- phosphatidylcholine hydropero~de[9]
Isolation and DNA Sequence Analysis of Mouse Plasma GPx CDNA-Differential screening of a 3-week normal cpk mouse kidney cDNA library to identify differentially expressed mRNAs in normal and polycystic kidneys' led to the isolation, subcloning, and DNA sequencing of a 277-base pair cDNA clone, 25-11
GPx-related mRNAs such as phospholipid hydroperoxide GPx, which is expressed in rodent testis, heart, and liver [50]; gas
Summary
90-95% confluence.Following the final mediumcollection,proximal tubule cells were lysedin 20 mM Tris,pH 7.5, 5 mM EDTA, and 0.1 mM phenylmethylsulfonylfluoride. Aliquotsof both the lysed cellsand the conditioned medium (one 24-h collectionw)ere concentrated (-50-fold) using Centriprep-10 concentrators (Amicon), electrophoresed in SDS-. After washing in tTBS, the immunoblotswere incubated with the appropriate secondary antibodies, either rabbit antichicken IgG alkaline phosphatase conjugate (for plasma Gpx) or goat anti-rabbit IgG alkaline phosphatase conjugate(for RdInsTP) (Sigma), for 2 hat room temperature, washed in tTBS, and developed at pH. 9.5 with nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate as substrates
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