Pearl L. Bergad

Glutathione peroxidase from bovine lens: a selenoenzyme

Abstract Glutathione peroxidase was purified 12 000-fold to homogeneity from the bovine lens. Purification utilized ammonium sulfate, acetic acid and zinc sulfate precipitations, and covalent chromatography on activated Thiolsepharose 4B. The specific activity of the purified enzyme is 1600 and its estimated molecular weight is 140 000. It is a selenoenzyme, containing 4 mol of selenium per mol of enzyme. Its pH maximum is 7·9. The apparent K m s for glutathione, tert-butylhydroperoxide, cumene hydroperoxide and H 2 O 2 are 2·9, 0·54, 0·65 and 0·045 m m , respectively. Iodoacetic acid irreversibly inhibits both the reduced and oxidized forms of the enzyme, whereas p -chloromercuriphenyl sulfonic acid only inhibits the oxidized form and N -ethyl maleimide, the reduced form.

Inhibition of Na, K-ATPase by sodium selenite and reversal by glutathione

Sodium selenite has been shown to inhibit Na,K-ATPase. Glutathione, at sufficient excess, is able to prevent or reverse the inhibition. Dithiothreitol can also reverse much of the inhibition, but KCN cannot. Selenomethionine does not inhibit Na,K-ATPase. The interactions of sodium selenite with Na,K-ATPase and glutathione may aid in understanding the early events in selenium cataractogenesis.

Regulation of Spi 2.1 and 2.2 gene expression after turpentine inflammation: discordant responses to IL-6

The rat serine protease inhibitor (Spi) 2 gene family includes both positive (Spi 2.2) and negative (Spi 2.1) acute phase reactants, facilitating modeling of regulation of hepatic acute phase response (APR). To examine the role of signal transducer and activation of transcription (STAT) proteins in the divergent regulation of these model genes after induction of APR, we evaluated the proximal promoters of the genes, focusing on STAT binding sites contained in these promoter elements. Induction of APR by turpentine injection includes activation of a STAT3 complex that can bind to a gamma-activated sequence (GAS) in the Spi 2.2 gene promoter, although the Spi 2.2 GAS site can bind STAT1 or STAT5 as well. To create an in vitro model of APR, primary hepatocytes were treated with combinations of cytokines and hormones to mimic the hormonal milieu of the whole animal after APR induction. Incubation of primary rat hepatocytes with interleukin (IL)-6, a critical APR cytokine, leads to activation of STAT3 and a 28-fold induction of a chloramphenicol acetyltransferase reporter construct containing the -319 to +85 region of the Spi 2.2 promoter. This suggests the turpentine-induced increase of Spi 2.2 is mediated primarily by IL-6. In contrast, although turpentine treatment reduces Spi 2.1 mRNA in vivo and IL-6 does not increase Spi 2.1 mRNA in primary rat hepatocytes, treatment of hepatocytes with IL-6 results in a 5. 4-fold induction of Spi 2.1 promoter activity mediated through the paired GAS elements in this promoter. Differential regulation of Spi 2.1 and 2.2 genes is due in part to differences in the promoters of these genes at the GAS sites. IL-6 alone fails to reproduce the pattern of rat Spi 2 gene expression that results from turpentine-induced inflammation.The rat serine protease inhibitor (Spi) 2 gene family includes both positive (Spi 2.2) and negative (Spi 2.1) acute phase reactants, facilitating modeling of regulation of hepatic acute phase response (APR). To examine the role of signal transducer and activation of transcription (STAT) proteins in the divergent regulation of these model genes after induction of APR, we evaluated the proximal promoters of the genes, focusing on STAT binding sites contained in these promoter elements. Induction of APR by turpentine injection includes activation of a STAT3 complex that can bind to a γ-activated sequence (GAS) in the Spi 2.2 gene promoter, although the Spi 2.2 GAS site can bind STAT1 or STAT5 as well. To create an in vitro model of APR, primary hepatocytes were treated with combinations of cytokines and hormones to mimic the hormonal milieu of the whole animal after APR induction. Incubation of primary rat hepatocytes with interleukin (IL)-6, a critical APR cytokine, leads to activation of STAT3 and a 28-fold induction of a chloramphenicol acetyltransferase reporter construct containing the -319 to +85 region of the Spi 2.2 promoter. This suggests the turpentine-induced increase of Spi 2.2 is mediated primarily by IL-6. In contrast, although turpentine treatment reduces Spi 2.1 mRNA in vivo and IL-6 does not increase Spi 2.1 mRNA in primary rat hepatocytes, treatment of hepatocytes with IL-6 results in a 5.4-fold induction of Spi 2.1 promoter activity mediated through the paired GAS elements in this promoter. Differential regulation of Spi 2.1 and 2.2 genes is due in part to differences in the promoters of these genes at the GAS sites. IL-6 alone fails to reproduce the pattern of rat Spi 2 gene expression that results from turpentine-induced inflammation.

High Performance Liquid Chromatography Determination of Cystathionase Activity in Human and Rabbit Lenses

Cystathionase activity has been demonstrated in human and rabbit lenses, using L-homoserine as substrate. The amount of activity found was quite low, 0.05 and 0.08 nmol of alpha-ketobutyrate formed/h/mg protein in human and rabbit lenses, respectively. The human lens cystathionase was found to have a pH maximum of 8.5 and a Km of 23.8 mM for L-homoserine. A sensitive assay, involving the use of high performance liquid chromatography, was developed to measure the small amounts of alpha-ketobutyrate produced.

Growth hormone action in hypothyroid infant rats

In neonatal rats, expression of serine protease inhibitors 2.1 and 2.3 mRNA peaks on d 2 of life and declines shortly thereafter, coinciding with levels of circulating GH. To evaluate the role of GH in this increase and to test the hypothesis that GH is active in perinatal life, we studied GH action in a model of GH deficiency. Maternal/neonatal hypothyroidism with consequent GH deficiency was induced by methimazole administration to pregnant dams. The resultant hypothyroid neonates were treated at d 2 or 7 of age with GH or saline for 1 h before exsanguination. In d-7 neonates, but not at d 2, GH administration resulted in significant serine protease inhibitors 2.1 and 2.3 mRNA induction. This treatment did not result in increased production of either GH receptor or IGF-I mRNA at either age. There was a slight GH-independent increase in GH receptor and IGF-I mRNA expression by d 7. Electromobility shift assays using hepatic nuclear extracts from these neonates and the GH response element from the serine protease inhibitor 2.1 promoter showed signal transducer and activator of transcription 5 (Stat5) binding in response to GH in extracts from d-7 rats only. Immunoblots of these extracts showed twice as much Stat5 in the nuclei of d-7 treated neonates compared with d-2 treated neonates. We conclude that there is apparent insensitivity to GH treatment in d-2 neonates that remits by d 7 and that this remission correlates with increased abundance of GH receptor and Stat5.

ONTOGENY OF THE RESPONSE TO GROWTH HORMONE † 374

Growth hormone (GH) is essential for postnatal growth and regulation of fuel homeostasis. Its role in fetal and neonatal growth is less certain. The time at which GH begins to regulate perinatal growth in developing mammals is not established. Some models of GH deficiency show very early effects on growth. We have shown that the GH-responsive mRNAs, Spi 2.1 and 2.3, increase around birth in rat pups while GHR mRNA is stable at ≈10% of adult levels(A J Physiol 264:E973). To test the hypothesis that GH is active in perinatal life in promoting hepatic GH-responsive gene expression, we assessed GH action by measurement of hepatic Spi 2.1/2.3 gene expression in a model of neonatal GH deficiency. To determine if GH-responsive gene expression in the perinatal period is diminished by induction of GH deficiency and restored by GH administration, maternal/neonatal hypothyroidism (and consequent GH deficiency) was induced by administration of methimazole to timed pregnant female rats beginning at d 14 of gestation. Pups at days 2 and 7 of age were treated with GH 1.5 ug/g sq (GH-deficient pups injected with an equal vol of saline.) GH response was evaluated by measurement of hepatic Spi 2.1/2.3 mRNA, as expression of these genes is significantly increased by GH administration in adult animals. RNA levels were measured using Northern blots hybridized simultaneously to Spi 2.1/2.3 and GAPDH cDNAs. Intensity of hybridization was quantitated by phosphorimaging. Spi 2.1/2.3 values were normalized to GAPDH values. There was no significant difference between GH-deficient (def) and GH-treated (Rx) pups at d 2 (mean d 2 GH-def 0.40 +/- 0.01 (SEM) OD units, mean d 2 GH-Rx 0.44 +/- 0.04 OD units) but at d 7 GH administration resulted in significant (95% by Fisher PLSD) Spi 2.1/2.3 induction (mean d 7 GH-def 0.21 +/- 0.04 (SEM) OD units, mean d 7 GH-Rx 0.82 +/- 0.12 OD units). We conclude that the response to GH administration changes between days 2 and 7 of neonatal rat life, with relative insensitivity to GH administration at d 2 but clear response to GH by d 7. We speculate that this may be due to changes in post receptor responses to GH.