Isolation and characterization of glutathione reductase from Physarum polycephalum and stage-specific expression of the enzyme in life-cycle stages with different oxidation-reduction levels

Physarum polycephalum has a life cycle with several distinct phases that have different oxidation-reduction requirements. To investigate the relationship between the life cycle and the oxidation-reduction state, we isolated glutathione reductase (GR; EC 1.6.4.2) from Physarum microplasmodia. The enzyme was found to be a homodimer with a subunit M(r) of 49,000, and K(m) values for oxidized glutathione and NADPH of 40 and 28.6 microM, respectively. We then constructed a cDNA library from microplasmodium mRNA and cloned GR cDNA from the library. The isolated cDNA consisted of 1,475 bp encoding a polypeptide of 452 amino acids. The amino acid sequence similarity was about 50% with GRs of other organisms, and several conserved sequence motifs thought to be necessary for activity are evident in the Physarum enzyme. Escherichia coli transformed with an expression vector containing the cDNA synthesized the active GR. Genomic Southern blot analysis indicated that the GR gene is present as a single copy in the Physarum genome. Immunoblot analysis and RT-PCR analysis detected GR mRNA expression in the microplasmodium, plasmodium, and sclerotium, but not in the spore or flagellate. GR activity was low in the spore and flagellate. These results suggest that the glutathione oxidation-reduction system relates to the Physarum life cycle.     

Impaired transactivation of the glucocorticoid receptor cloned from the Guyanese squirrel monkey

Squirrel monkeys are among a diverse group of New World primates that demonstrate unusually high levels of circulating corticosteroids and glucocorticoid receptor (GR) insensitivity. Recent evidence suggests that overexpression of an immunophilin impairs dexamethasone binding to GR in the Bolivian squirrel monkey (Saimiri boliviensis). Here we describe the cloning, expression, and functional characterization of GR from the closely related Guyanese squirrel monkey (S. sciureus). The cloned Guyanese squirrel monkey GR (gsmGR) cDNA closely resembles human GR (hGR) cDNA, and yields a high affinity dexamethasone binding receptor when expressed in COS-1 cells. Transactivation analysis of hGR and gsmGR expressed in CV-1 cells and cultured squirrel monkey kidney (SMK) cells indicates that: (1) SMK cells elaborate a functional high activity GR from human GR cDNA; (2) gsmGR is an order of magnitude less efficient than hGR at transactivation in CV-1 and SMK cells; and (3) maximal transactivation by gsmGR is attenuated in both cell lines. Glucocorticoid resistance in S. sciureus is at least partly attributable to a naturally occurring mutation in the GR gene that results in impaired GR transactivation.     

Simultaneous targeting of pea glutathione reductase and of a bacterial fusion protein to chloroplasts and mitochondria in transgenic tobacco

N-terminal presequences from cDNAs encoding mitochondrion- or chloroplast-specific proteins are able, with variable efficiencies, to target preproteins to their respective organelles. In the few cases studied in which a nuclear-encoded protein is found in both these organelles, each compartment-specific isoform is encoded by a separate gene. Glutathione reductase (GR) from peas is encoded by a single nuclear gene and yet GR is distributed between chloroplasts, mitochondria and the cytosol. Previous sequence analysis of a full-length GR cDNA revealed the presence of a putative plastid transit peptide. However, expression of this cDNA in transgenic tobacco resulted in substantially elevated GR activities in both chloroplasts and mitochondria in four independent lines examined. There was no effect on expression of the endogenous tobacco GR genes. Replacement of the GR presequence with presequences from pea rbcS (chloroplast) and Nicotiana plumbaginifolia Mn-SOD (mitochondrion) resulted in targeting of GR only into the appropriate organelle. Expression of a fusion protein between the amino terminal region of GR and phosphinothricin acetyl transferase resulted in targeting of the foreign protein to chloroplasts and mitochondria. Thus, the pea GR presequence is capable of co-targeting this enzyme or a foreign protein to chloroplasts and mitochondria in vivo . This is the first example of co-targeting by a higher plant preprotein.     

Cloning of the human glucocorticoid receptor cDNA.

We show that the human glucocorticoid receptor (GR), isolated from the breast cancer cell line MCF-7, has an apparent molecular weight identical to that of rat liver GR (94 kDa) and reacts with antibodies raised against the latter. These antibodies were used to clone cDNA sequences corresponding to the human GR from a lambda gt11 expression library constructed using MCF-7 poly(A)+ RNA. Three non-homologous cDNA clones with inserts of 125, 220 and 350 bp, which express epitopes recognised by the rat liver GR antibodies, were isolated. Rat liver GR antibodies, immunopurified using the immobilised purified beta-galactosidase fusion proteins, detect partially purified rat liver and human GRs on Western blots. In addition, these antibodies immuno-adsorb rat liver and human GRs affinity-labelled with [3H] triamcinolone acetonide. Northern blot analysis, using all three cDNA probes, reveals the presence of a major MCF-7 poly(A)+ RNA species of approximately 7 kb.     

Cloning and characterisation of glutathione reductase cDNAs and identification of two genes encoding the tobacco enzyme

We have isolated 4 cDNA clones (GRT1-4) encoding glutathione reductase (GR) from a tobacco (Nicotiana tabacum L.) leaf cDNA library. The cDNAs were almost identical: GRT1, GRT3 and GRT4 represented the same gene, differing only in that GRT4 contained an intron within the C-terminal part of the coding sequence. Failure to splice out this intron resulted in a substitution of the final 13 amino acids of the deduced amino acid sequence. A second gene was represented by GRT2. Southern blots indicated that there were two related GR genes in tobacco. The presence of multiple isoforms of GR in tobacco may be explained in part by the expression of a small gene family. In addition, alternative isoforms may result from translation of different mRNAs derived from the same gene by intron skipping during the splicing of nascent GR mRNAs.     

Identification and characterization of a plastid-localized Arabidopsis glyoxylate reductase isoform: comparison with a cytosolic isoform and implications for cellular redox homeostasis and aldehyde detoxification

Enzymes that reduce the aldehyde chemical grouping (i.e. H-C=O) to its corresponding alcohol could be crucial in maintaining plant health. Recently, recombinant expression of a cytosolic enzyme from Arabidopsis thaliana (L.) Heynh (designated as glyoxylate reductase 1 or AtGR1) revealed that it effectively catalyses the in vitro reduction of both glyoxylate and succinic semialdehyde (SSA). In this paper, web-based bioinformatics tools revealed a second putative GR cDNA (GenBank Accession No. AAP42747; designated herein as AtGR2) that is 57% identical on an amino acid basis to GR1. Sequence encoding a putative targeting signal (N-terminal 43 amino acids) was deleted from the full-length GR2 cDNA and the resulting truncated gene was co-expressed with the molecular chaperones GroES/EL in Escherichia coli, enabling production and purification of soluble recombinant protein. Kinetic analysis revealed that recombinant GR2 catalysed the conversion of glyoxylate to glycolate (K(m) glyoxylate=34 microM), and SSA to gamma-hydroxybutyrate (K(m) SSA=8.96 mM) via an essentially irreversible, NADPH-based mechanism. GR2 had a 350-fold higher preference for glyoxylate than SSA, based on the performance constants (k(cat)/K(m)). Fluorescence microscopic analysis of tobacco (Nicotiana tabacum L.) suspension cells transiently transformed with GR1 linked to the green fluorescent protein (GFP) revealed that GR1 was localized to the cytosol, whereas GR2-GFP was localized to plastids via targeting information contained within its N-terminal 45 amino acids. The identification and characterization of distinct plastidial and cytosolic glyoxylate reductase isoforms is discussed with respect to aldehyde detoxification and the plant stress response.     

逆境胁迫对油菜谷胱甘肽还原酶基因表达及其酶活性的影响

利用cDNA末端快速分离(RACE)技术从陇油6号油菜中克隆得到一个新的谷胱甘肽还原酶基因GR2,全长2073 bp,开放阅读框1692 bp,编码563个氨基酸,预测蛋白质分子量为60.7 kDa,等电点7.9.实时荧光定量PCR分析表明:GR2基因在油菜根、茎、叶中均有表达,其中在叶中表达量最高.GR1和GR2基因的转录以及谷胱甘肽还原酶(GR)活性受到低温、高温、干旱、高盐胁迫的诱导,表明油菜谷胱甘肽还原酶在抵御低温、高温、干旱、高盐胁迫过程中发挥重要作用.脱落酸(ABA)预处理后再进行上述胁迫处理,与单独上述胁迫相比,GR1和GR2基因的转录以及GR活性水平明显上升,表明ABA可以诱导GR1和GR2基因表达和GR酶活性.MAPKK抑制剂U0126预处理后再进行上述胁迫处理,与单独上述胁迫相比,GR1和GR2基因的转录以及GR活性水平明显下降,表明U0126对GR1、GR2基因表达以及GR酶活性有抑制作用.     

UV cross-linking/immunoprecipitation assay: glucocorticoid receptor-adrenergic receptor gene sequence interaction

The rat beta 1-adrenergic receptor (beta 1-AR) gene contains glucocorticoid response element (GRE) half-sites at positions -2767 and -945. In electrophoretic mobility shift assay (EMSA) experiments, neither beta 1-AR GRE half-site recognized glucocorticoid receptors (GRs) obtained from baculovirus high-level expression systems or from mammalian cells. We have developed a sensitive UV cross-linking/immunoprecipitation assay, using a 524-bp fragment containing the prototypical GRE obtained from the rat tyrosine aminotransferase promoter sequence and using antibodies recognizing mammalian GR. Using this assay, we provide evidence that rat beta 1-AR gene sequences recognize mammalian GRs expressed in mouse 3T3 cells and that the site of GR interaction does not appear to specifically contain the beta 1-AR GRE half-sites. This represents one of the first reports demonstrating the utility of a UV cross-linking/immunoprecipitation assay in the detection of mammalian GR interaction with beta 1-AR sequences, is consistent with the lack of specific DNA-GR protein complexes observed in EMSA experiments using oligonucleotide probes containing the beta 1-AR GRE half-sites, and provides evidence that mammalian GR interaction occurs at complex rate beta 1-AR gene sequences.     

Disruption of glucocorticoid receptor exon 2 yields a ligand-responsive C-terminal fragment that regulates gene expression

Mice in which exon 2 of the glucocorticoid receptor (GR) has been disrupted [GR exon 2 knockout (GR2KO)] have been used as a model to study the requirement for this receptor in a number of biological systems. A recent report showed that these mice actually express a truncated ligand-binding GR fragment, prompting us to ask whether this mutation truly results in a glucocorticoid-insensitive phenotype. Based on cDNA microarray analysis of fetal thymocytes, we found that glucocorticoids were able to enhance or repress activation-induced gene expression in GR2KO and wild-type thymocytes to a similar degree. Moreover, although changes in gene expression induced by glucocorticoids alone were blunted, the expression of a substantial number of genes in GR2KO thymocytes was modulated by stimulation with glucocorticoids. Among these genes, as confirmed by quantitative real-time PCR, was the classic glucocorticoid-responsive gene glutamine synthetase as well as genes implicated in T cell development and function such as IL-7 receptor alpha-chain and glucocorticoid-induced leucine zipper (GIL2). Thus, the truncated C-terminal GR2KO product, which lacks the major transactivation domain, retains, to a large extent, the ability to regulate gene expression both positively and negatively in a ligand-responsive manner when expressed in vivo.     

Dehydroepiandrosterone and its 7-hydroxylated metabolites do not interfere with the transactivation and cellular trafficking of the glucocorticoid receptor

The human brain is a target tissue for glucocorticoids (GC). Dehydroepiandrosterone (DHEA) is a neurosteroid produced in the brain where it is transformed into 7alpha-hydroxy-DHEA and 7beta-hydroxy-DHEA. The antiglucocorticoid effects of both 7-hydroxylated metabolites have been investigated with evidence in mice that neither form of DHEA interfered with the binding of GC to its glucocorticoid receptor (GR), but contributed to a decreased nuclear uptake of the activated GR. Our objective was to use COS-7 cell culture to research DHEA, 7alpha-hydroxy-DHEA and 7beta-hydroxy-DHEA interferences with GR trafficking. These cells did not carry out the 7alpha-hydroxylation of DHEA and the oxidation of cortisol into cortisone. The cDNA of the human GR was inserted into pcDNA3 for a transient transfection of COS-7 cells. Human GR transactivation activity was measured from a luciferase-MMTV reporter gene. The transfected COS-7 cells were cultured using 10(-12) to 10(-5) M dexamethasone (DEX) or cortisol, which triggered the reporter expression. Treatment with 10(-12) to 10(-5) M DHEA, 7alpha-hydroxy-DHEA and 7beta-hydroxy-DHEA caused no change in the GC-induced GR transactivation. A reconstruction of the process associated EGFP to the human GR cDNA. Confocal microscopic examination of COS-7 cells transiently expressing the fusion protein EGFP-GR showed nuclear fluorescence 60 min after incubation with 10(-8) M DEX or cortisol. The addition of 10(-5) M DHEA, 7alpha-hydroxy-DHEA or 7beta-hydroxy-DHEA did not change its kinesis and intensity. These results contribute to the knowledge of DHEA, 7alpha-hydroxy-DHEA and 7beta-hydroxy-DHEA, in relation to antiglucocorticoid activity. We conclude that direct interference with GR trafficking can be discounted in the case of these hormones, therefore proposing new possibilities of investigation.     

Gene cloning and expression of cytosolic glutathione reductase in rice (Oryza sativa L.)

We have isolated a cDNA (RGRC2) encoding glutathione reductase (GR) from rice (Oryza sativa L.). The comparison of deduced amino acid sequences from RGRC2 and other plant GR cDNAs indicated that RGRC2 encodes a putative cytosolic isoform. The recombinant RGRC2 protein had enzymatic properties comparable to those of GR from rice embryo. Subcellular fractionation showed that the RGRC2 protein is localized primarily in cytosol. mRNA and protein of RGRC2 were observed mainly in roots and calli but little in leaf tissues. Southern blot analysis showed that the RGRC2 gene exists as a single copy gene. Here, we have also isolated a genomic clone completely corresponding to RGRC2. The RGRC2 gene is split into 16 exons spread about 7.4 kb of chromosomal DNA, with coding sequence beginning in the 2nd exon and ending in the 16th exon. From the presence of two ABA-responsive elements in the 5'-flanking region of RGRC2, we examined the expression in rice seedlings treated with ABA and the ABA-related environmental stresses, chilling, drought and salinity. The expression of RGRC2 was strongly induced by all these treatments. We suggest that the expression of the rice cytosolic GR gene is regulated via ABA-mediated signal transduction pathway under environmental stresses.     

Germination strategy of Striga hermonthica involves regulation of ethylene biosynthesis

Ethylene involvement in germination of Striga hermonthica (Del.) Benth., an important root parasitic weed on poaceous crops, was investigated at the physiological and molecular levels. Seeds, conditioned at 30°C for 14 days, were treated with ethylene, ethephon or 1-aminocyclopropane-1-carboxylic acid (ACC). Ethylene consistently induced low germination. Ethephon and ACC effectively stimulated germination at concentrations of 0.01 and 1 m M , respectively. In contrast to ethylene, both ethephon and ACC acted in a concentration-dependent manner. Germination induced by the synthetic strigolactone GR24 was inhibited by aminoethoxyvinylglycine (AVG) and 1-methylcyclopropene. ACC reversed the inhibition caused by AVG. When seeds were treated with GR24 in sealed vials, ethylene concentration in headspace gas increased prior to the onset of germination. Total RNA extracted from germinating seeds 12 h after GR24 treatment was used for PCR-based amplification of cDNA fragments encoding the ACC synthase- and oxidase-active site domains. Two distinct cDNA fragments encoding ACC synthase ( SHACS1 and SHACS2 ) and one encoding ACC oxidase ( SHACO1 ) were cloned and sequenced. Southern analysis suggested that each of the cloned genes was present as a single copy in the genome of S. hermonthica . Northern analyses showed that SHACS1 exhibited a temporal change in expression peaking at 10 h after GR24 treatment, which coincided with a steady increase in ethylene concentration. SHACS2 was expressed at a low level with a similar trend. SHACO1 exhibited a temporal change in expression peaking at 15 days during conditioning, when seed response to GR24 was maximal. In summary, expression of ACC synthase and ACC oxidase genes was found to be responsive to a germination stimulant and to conditioning, respectively. The implications of these findings with respect to germination of S. hermonthica under field conditions are discussed.     

Cloning of the glucocorticoid receptor (GR) in gilthead seabream (Sparus aurata). Differential expression of GR and immune genes in gilthead seabream after an immune challenge

In order to determine the cortisol response after an immune challenge in the gilthead seabream (Sparus aurata), a cortisol receptor (GR) was cloned, sequenced and its expression determined after lipopolysaccharide (LPS) treatment. To clone the gilthead seabream GR (sbGR), consecutive PCR amplifications and screening of a pituitary cDNA library were performed. We obtained a clone of 4586 bp encoding a 784aa protein. Northern blot analysis from head kidney, heart and intestine revealed that the full length sbGR mRNA was approximately 6.5 Kb. A LPS treatment, used as an acute stress model, was employed to characterise the expression of sbGR and some selected genes involved in the immune response (IL-1beta, TNF-alpha, Mx protein, cathepsin D and PPAR-gamma). All genes were expressed in all tissues examined and responses were tissue and time dependent revealing differential gene expression profiles after LPS administration. Furthermore, analysis of plasma cortisol levels after LPS injection, showed an acute response to inflammatory stress with a significant increase two and six h after injection, recovering to basal levels 12 h post-stress in all LPS concentrations tested.