cAMP mediates transepithelial K+ and Na+ transport in a strial marginal cell line

The recently described gastrointestinal glutathione peroxidase (GI-GPx) is the fourth member of the family of the selenoenzymes glutathione peroxidases (GPx). In contrast to the more uniform distribution of, for example, the classical glutathione peroxidase (cGPx), it is expressed exclusively in the gastrointestinal tract and has, therefore, been suggested to function as a primary barrier against alimentary hydroperoxides. In order to get an idea of its relative importance we investigated its position in the hierarchy of selenoprotein expression. The selenium-dependent expression of GI-GPx was analyzed in comparison with that of other GPx types at the level of mRNA and protein in HepG2 and CaCo-2 cells. Furthermore, the selenocysteine insertion sequence (SECIS) efficiencies of GI-GPx, phospholipid hydroperoxide glutathione peroxidase (PHGPx) and cGPx in response to selenium were determined by a reporter-gene assay in human hepatoma cells and baby hamster kidney cells. GI-GPx mRNA levels increased during selenium deficiency, whereas cGPx mRNA levels decreased and PHGPx mRNA levels remained almost unaffected. In cells grown in selenium-poor media, all GPx-types were low in both activity and immunochemical reactivity. Upon selenium repletion immunoreactive GI-GPx protein reached a plateau after 10 h, whereas cGPx started to be expressed at 24 h and did not reach its maximum level before 3 days. SECIS efficiencies decreased in the order PHGPx > cGPx > GI-GPx. The augmentation of SECIS efficiencies by selenium was highest for cGPx and intermediate for PHGPx, whereas it was marginal for GI-GPx. The high mRNA stability under selenium restriction, the speed of biosynthesis upon selenium repletion and the marginal effect of selenium on the SECIS efficiency indicate that of the GPx isotypes, GI-GPx ranks highest in the hierarchy of selenoproteins and point to a vital role of GI-GPx in the gastrointestinal tract.     

The cloning and characterization of a new stress response protein. A mammalian member of a family of theta class glutathione s-transferase-like proteins

Using differential display, a cDNA fragment was identified as being overexpressed in a mouse lymphoma cell line that had gained resistance to cell death after exposure to a variety of agents used in cancer therapy. The full-length cDNA of 1.1 kb that was cloned contained an open reading frame coding for a previously unidentified 28-kDa mammalian protein, p28. p28 showed significant homologies to a large family of stress response proteins that contain a glutathione S-transferase (GST) domain. In correspondence with the sequence homology, p28 was found to bind glutathione; however, GST or glutathione peroxidase activity could not be demonstrated. Northern analysis of the mRNA of this protein showed abundant expression in mouse heart and liver tissues, whereas anti-p28 antibody binding identified p28 expression in mouse 3T3 cells and early passage mouse embryo fibroblasts. Subcellular protein fractionation revealed p28 localization in the cytoplasm, but with thermal stress p28 relocated to the nuclear fraction of cellular proteins. Based on sequence homology and protein activity we conclude that p28 acts as a small stress response protein, likely involved in cellular redox homeostasis, and belongs to a family of GST-like proteins related to class theta GSTs.     

PHGPx and phospholipase A2/GPx: comparative importance on the reduction of hydroperoxides in rat liver mitochondria

The comparative importance of phospholipid hydroperoxide glutathione peroxidase (PHGPx) and of "classic" glutathione peroxidase (GPx) in the reduction of phospholipid hydroperoxides is unclear. Although GPx activity is 500-fold higher than that of PHGPx in rat liver, the reduction of phospholipid hydroperoxides by glutathione (GSH) through GPx may be strongly limited by a low PLA2 activity. We address this issue using a moderately detailed kinetic model of mitochondrial lipid peroxidation in rat liver. The model was based on published data and was subjected to validation as reported in the references. It is analysed by computer simulation and sensitivity analysis. Results suggest that in rat liver mitochondria PHGPx is responsible for almost all phospholipid hydroperoxide reduction. Under physiological conditions, the estimated flux of phospholipid hydroperoxides reduction through PHGPx is about four orders of magnitude higher than the estimated hydrolysis flux through PLA2. On the other hand, virtually all hydrogen peroxide is reduced through GPx. Therefore, a functional complementarity between PHGPx and GPx is suggested. Because the results are qualitatively robust to changes of several orders of magnitude in PLA2 and PHGPx levels, the conclusions may not be limited to mitochondria.     

A novel glutathione peroxidase in bovine eye. Sequence analysis, mRNA level, and translation

Bovine ciliary body contains a selenium-independent glutathione peroxidase (GPX) with a molecular mass of about 100 kDa that is composed of four identical subunits and exhibits no glutathione S-transferase activity. In this study, we isolated cDNA clones and determined the nucleotide sequence to deduce the primary structure of the enzyme. The cDNA contained 672 base pairs encoding a polypeptide with an estimated molecular mass of 25,064 Da. Translation of bovine ciliary mRNA produced a protein which was immunologically indistinguishable from GPX and showed high enzyme activity. The encoded amino acid sequence of the protein was 95% identical with that of a human keratinocyte gene product expressed in response to keratinocyte growth factor. It also showed sequence identity to bacterial alkyl hydroperoxide reductases and thiol specific antioxidant enzymes. GPX mRNA level was highest in the ciliary body, followed by the retina and iris. In various rat organs, the level of GPX mRNA was highest in the lung, followed by the muscle, liver, eye, heart, testis, thymus, kidney, and spleen. A very low level of mRNA was detected in the brain. Enzyme-linked immunosorbent assay with an antibody raised against the NH2-terminal sequence of GPX detected GPX protein in all rat tissues examined.     

Human geranylgeranyl diphosphate synthase: isolation of the cDNA, chromosomal mapping and tissue expression

We report the nucleotide sequence of human geranylgeranyl diphosphate (GGPP) synthase cDNA isolated from a fetal heart library. The 2.5 kb cDNA encodes a protein of 34 kDa. The protein contains six domains that have been identified previously in many other prenyltransferases. Recombinant, purified histidine-tagged protein exhibited the enzymatic properties associated with GGPP synthase, namely the synthesis of GGPP from farnesyl diphosphate and isopentenyl diphosphate. Transient transfection of mammalian cells with a plasmid encoding the putative GGPP synthase resulted in a 55-fold increase in GGPP synthase activity. Taken together, these results establish that the cDNA encodes the mammalian GGPP synthase protein. The mRNA for GGPP synthase was expressed ubiquitously. Of the 16 human tissues examined, the highest expression of the mRNA was in testis. The mRNA levels in cultured HeLa cells were unaffected by alterations in cellular sterol levels and contrasted with the significant regulation of isopentenyl diphosphate synthase mRNA under these same conditions. Fluorescent in situ hybridization was used to map the single gene encoding human GGPP synthase to chromosome 1q43.     

Cis-acting elements are required for selenium regulation of glutathione peroxidase-1 mRNA levels

Classical glutathione peroxidase (GPX1) mRNA levels can decrease to less than 10% in selenium (Se)-deficient rat liver. The cis-acting nucleic acid sequence requirements for Se regulation of GPX1 mRNA levels were studied by transfecting Chinese hamster ovary (CHO) cells with GPX1 DNA constructs in which specific regions of the GPX1 gene were mutated, deleted, or replaced by comparable regions from unregulated genes such as phospholipid hydroperoxide glutathione peroxidase (GPX4). For each construct, stable transfectants were pooled two weeks after transfection, divided into Se-deficient (2 nM Se) or Se-adequate (200 nM Se) medium, and grown for an additional four days. On day of harvest, Se-deficient GPX1 and GPX4 activities averaged 13 +/- 2% and 15 +/- 2% of Se adequate levels, confirming that cellular Se status was dramatically altered by Se supplementation. RNA was isolated from replicate plates of cells and transfected mRNA levels were specifically determined by RNase protection assay. Analysis of chimeric GPX1/GPX4 constructs showed that the GPX4 3'-UTR can completely replace the GPX1 3'-UTR in Se regulation of GPX1 mRNA. We did not find any GPX1 coding regions that could be replaced by the corresponding GPX4 coding regions without diminishing or eliminating Se regulation of the transfected GPX1 mRNA. Further analysis of the GPX1 coding region demonstrated that the GPX1 Sec codon (UGA) and the GPX1 intron sequences are required for full Se regulation of transfected GPX1 mRNA levels. Mutations that moved the GPX1 Sec codon to three different positions within the GPX1 coding region suggest that the mechanism for Se regulation of GPX1 mRNA requires a Sec codon within exon 1. Lastly, we found that addition of the GPX1 3'-UTR to beta-globin mRNA can convey significant Se regulation to beta-globin mRNA levels when a UGA codon is placed within exon 1. We conclude that Se regulation of GPX1 mRNA requires a functional selenocysteine insertion sequence (SECIS) in the 3'-UTR and a Sec codon followed by an intron.     

Expression, characterization, and tissue distribution of a new cellular selenium-dependent glutathione peroxidase, GSHPx-GI

We have characterized a new selenium-dependent glutathione peroxidase, GSHPx-GI, by expressing a GSHPx-GI cDNA isolated from human hepatoma HepG2 cells in human mammary carcinoma MCF-7 cells, which have virtually undetectable expression of either the classical cellular enzyme, GSHPx-1, or GSHPx-GI at the protein level. One of the G418-resistant clones, neo-D1, expresses the transfected GSHPx-GI cDNA. This is based on 1) the presence of an additional GSHPx-GI DNA restriction fragment detected by Southern analysis; 2) the presence of a 1.9-kilobase (kb) GSHPx-GI mRNA in addition to the 1.0-kb endogenous mRNA by Northern analysis; and 3) the appearance of a 22-kDa 75Se-labeled protein which is absent in parental MCF-7 cells revealed by SDS-polyacrylamide gel electrophoresis. GSHPx-GI expressed in neo-D1 is a tetrameric protein localized in cytosol. GSHPx-GI does not cross-react with antisera against human GSHPx-1 or human plasma glutathione peroxidase (GSHPx-P). Similar substrate specificities are found for GSHPx-1 and GSHPx-GI; they both catalyze the reduction of H2O2, tert-butyl hydroperoxide, cumene hydroperoxide, and linoleic acid hydroperoxide with glutathione, but not of phosphatidylcholine hydroperoxide. GSHPx-GI mRNA was readily detected in human liver and colon, and occasionally in human breast samples, but not other human tissues including kidney, heart, lung, placenta, or uterus. In rodent tissues, GSHPx-GI mRNA is only detected in the gastrointestinal tract, and not in other tissues including liver. In fact, GSHPx-GI appears to be the major glutathione-dependent peroxidase activity in rodent GI tract. This finding suggests that GSHPx-GI could play a major role in protecting mammals from the toxicity of ingested lipid hydroperoxides. In conclusion, we have demonstrated that GSHPx-GI is the fourth member in the selenium-dependent glutathione peroxidase family, in addition to GSHPx-1, GSHPx-P, and phospholipid hydroperoxide glutathione peroxidase (PHGPX).     

Molecular cloning of a full-length cDNA for human type 3 adenylyl cyclase and its expression in human islets

The GK (Goto-Kakizaki) rat is a lean model of type 2 diabetes in which the diabetic state was spontaneously induced. We recently demonstrated the presence in GK rats of two functional point mutations in the promoter region of the type 3 adenylyl cyclase (AC3) gene that resulted in overexpression of AC3 mRNA associated with increased cAMP generation. The AC3 gene promoter mutations are the first molecular changes to be described in any specific gene in the GK rat. Here we report cloning of a full-length cDNA encoding human AC3 from a human fetal brain cDNA library using a PCR-based screening method. This 4142-bp cDNA predicts an open reading frame encoding 1144 amino acids containing putative 12 transmembrane-spanning domains which are typically found in other mammalian AC isoforms. Comparison of the translated amino acid sequence of the AC3 gene between human and rat shows 95% homology. Using RT-PCR, clear AC3 expression was detected in isolated human islets as well as a cDNA panel containing templates from eight different tissues (brain, heart, kidney, liver, lung, pancreas, placenta, and skeletal muscle). This wide distribution of AC3 expression may involve a number of physiological and pathophysiological metabolic processes.     

Calcium sensitization in perfused beating guinea pig heart by a positive inotropic agent MCI-154

An in vitro import system was used to characterize the mechanism of import of phospholipid hydroperoxide glutathione peroxidase (PHGPx) into mitochondria. Mitochondria were isolated from rat liver and incubated at 25 degrees C with [35S]methionine-labeled products of the in vitro translation of mRNA that encoded 23-kDa and 20-kDa PHGPx. 23-kDa PHGPx was imported into mitochondria in a time-dependent manner and was processed to yield the 20-kDa form of PHGPx. The 20-kDa form of PHGPx, without a leader sequence, associated weakly with mitochondria but was not imported. An analysis with an uncoupler of oxidative phosphorylation showed that a membrane potential in the mitochondria was also required for the import of PHGPx. It appears, therefore, that the leader sequence in the precursor to PHGPx is the signal for import into the mitochondria. This is the first report to indicate that the precursor to PHGPx is imported into the mitochondria via the action of a leader sequence.     

Cloning and expression analysis of a novel salicylate suppressible gene, Hs-CUL-3, a member of cullin/Cdc53 family

By using a mRNA differential display technique to search for salicylate suppressible genes, we identified a cDNA in human foreskin fibroblasts, which by GenBankTM DNA data base search shows sequence homology to the recently reported cullin/Cdc53 (CUL) family genes, especially CUL-3. We have cloned the full-length human CUL-3 (Hs-CUL-3) cDNA. It encodes a 768-amino acid polypeptide and has a predicted molecular weight of 88,939. The amino acid sequence of Hs-CUL-3 shows 46% homology to that of its Caenorhabditis elegans ortholog, Ce-CUL-3, and 27 and 23% to that of Hs-CUL-1 and Hs-CUL-2, respectively. Northern blot analysis showed that phorbol 12-myristate 13-acetate increased the expression of Hs-CUL-3 mRNA in a concentration- and time-dependent manner, and this increase was inhibited by sodium salicylate. Hs-CUL-3 widely expressed in human tissues and its expression in cultured COLO205 colon cancer cells was increased when compared with that in normal colon cells. It is likely that Hs-CUL-3 is involved in cell proliferation control.     

cDNA cloning and characterization of a rat spermatogenesis-associated protein RSP29

RSP29, a protein secreted by rat round spermatids, stimulates the secretory function of Sertoli cells in the testis. By making use of the N-terminal sequence homology of RSP29 and a human protein hDP1 that we had previously isolated, we cloned the full length cDNA sequence that encodes RSP29. The entire amino acid sequence of RSP29 showed significant homology with that of hDP1, which was later identified as glyoxalase II. Southern analysis showed that the RSP29 protein sequence is highly conserved in eukaryotes and possibly in prokaryotes. The RSP29 mRNA is expressed in many tissues but has an extremely high abundance in testis. These data suggest that RSP29 may have an important function in most tissues of enkaryotic organisms. The high expression of RSP29 in testis and its stimulatory effects on Sertoli cells suggest that RSP29 could be especially important in the regulation of spermatogenesis.     

Differential regulation of leucine-rich primary response gene 1 (LRPR1) mRNA expression in rat testis and ovary

In immature rat Sertoli cells, leucine-rich primary response gene 1 (LRPR1) represents a follicle stimulating hormone (FSH)-responsive gene; the function of the encoded protein is not yet known. LRPR1 mRNA expression is up-regulated very rapidly and specifically by FSH, both in cultured Sertoli cells and in vivo in regulation in more detail, in testis and ovary of fetal, immature, and adult rats. In addition, we have studied the expression of FSH receptor (FSHR) mRNA in relation to LRPR1 mRNA expression. In rat testis, LRPR1 mRNA and FSHR mRNA followed a similar expression pattern, during postnatal development and also at different stages of the spermatogenic cycle in the adult rat. Furthermore, after short-term challenge of the FSH signal transduction pathway in intact immature rats by injection with a relatively high dose of FSH, an inverse relationship between LRPR1 mRNA (up-regulation) and FSHR mRNA expression (down-regulation) was observed. Similar studies in the ovary provided completely different results. LRPR1 mRNA in the postnatal ovary is present well before expression of FSHR mRNA can be first detected. In addition, incubation of ovaries of immature rats with FSH or dibutyryl cyclic AMP (dbcAMP) did not result in up-regulation of LRPR1 mRNA expression. During fetal development, the LRPR1 mRNA expression pattern involved many more tissues, in contrast to the relatively tissue-specific expression of LRPR1 mRNA in gonads of 21 day old and adult rats. Moreover, LRPR1 mRNA expression could be detected as early as 12.5 days post-coitum, whereas FSHR mRNA is absent at this stage of fetal development. We concluded that the pronounced regulation of LRPR1 by FSH observed in the immature rat testis does not occur in the ovary. Furthermore, in the ovary LRPR1 mRNA expression does not appear to be dependent on FSH action. Finally, the LRPR1 gene product may play a general role during fetal development.     

A Na(+)-dependent creatine transporter in rabbit brain, muscle, heart, and kidney. cDNA cloning and functional expression

A cDNA has been cloned from rabbit brain that is a new member of the emerging family of Na(+)-dependent plasma membrane transporters for several neurotransmitters and structurally related compounds. Functional expression of this cDNA in COS-7 cells identifies its product as a Na(+)- and Cl(-)-dependent creatine transporter with a Km of approximately 35 microM. Its creatine transporter activity is efficiently antagonized by 3-guanidinopropionate, a well characterized alternative substrate of creatine transport in several tissues, and by 4-guanidinobutyrate. More distant structural analogues of creatine are much less efficient or inactive as antagonists, indicating a high substrate specificity of the transporter. Northern blot hybridization detects the expression of its mRNA in most tissues tested, most prominently in kidney, heart, and muscle, but not in liver and intestine. A full-length cDNA clone was also isolated from a muscle cDNA library and found to contain the same coding sequence. Substrate affinity and specificity of the cloned transporter are very similar to the endogenous creatine transporter of the COS-7 cells and to the creatine transport activities of several cell types described in the literature. Moreover, its mRNA is most abundant in the tissues known to possess high creatine uptake capacity.