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1.
Glutamine Synthetase in Rice: A COMPARATIVE STUDY OF THE ENZYMES FROM ROOTS AND LEAVES 总被引:1,自引:0,他引:1
Chromatographic, kinetic, and regulatory properties of glutamine synthetase in rice were investigated. By DEAE-Sephacel column chromatography, two forms (glutamine synthetase 1 and glutamine synthetase 2) were identified in leaves and one form (glutamine synthetase R) was identified in roots. Purification on hydroxyapatite and gel electrophoresis showed that glutamine synthetase R was distinct from the leaf enzymes. The three isoforms were purified to similar specific activities and their properties were studied. Heat lability, pH optimum about 8, K(m) for l-glutamate of 20 millimolar, and inhibition by glucosamine 6-phosphate were the main characteristics of glutamine synthetase 2. Heat stability, pH optimum about 7.5, K(m) for l-glutamate of 2 millimolar, and no effect of glucosamine 6-phosphate differentiated glutamine synthetase 1 from glutamine synthetase 2. Glutamine synthetase R was also a labile protein but its kinetic and regulatory properties were quite similar to those of glutamine synthetase 1. These results clearly demonstrate the existence of three isoforms of glutamine synthetase in rice, two of which are located in the leaves and the third in the roots. 相似文献
2.
Subcellular distribution and characteristics of trihydroxycoprostanoyl-CoA synthetase in rat liver. 总被引:2,自引:0,他引:2
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L Schepers M Casteels K Verheyden G Parmentier S Asselberghs H J Eyssen G P Mannaerts 《The Biochemical journal》1989,257(1):221-229
The subcellular distribution and characteristics of trihydroxycoprostanoyl-CoA synthetase were studied in rat liver and were compared with those of palmitoyl-CoA synthetase and choloyl-CoA synthetase. Trihydroxycoprostanoyl-CoA synthetase and choloyl-CoA synthetase were localized almost completely in the endoplasmic reticulum. A quantitatively insignificant part of trihydroxycoprostanoyl-CoA synthetase was perhaps present in mitochondria. Peroxisomes, which convert trihydroxycoprostanoyl-CoA into choloyl-CoA, were devoid of trihydroxycoprostanoyl-CoA synthetase. As already known, palmitoyl-CoA synthetase was distributed among mitochondria, peroxisomes and endoplasmic reticulum. Substrate- and cofactor- (ATP, CoASH) dependence of the three synthesis activities were also studied. Cholic acid and trihydroxycoprostanic acid did not inhibit palmitoyl-CoA synthetase; palmitate inhibited the other synthetases non-competitively. Likewise, cholic acid inhibited trihydroxycoprostanic acid activation non-competitively and vice versa. The pH curves of the synthetases did not coincide. Triton X-100 affected the activity of each of the synthetases differently. Trihydroxycoprostanoyl-CoA synthetase was less sensitive towards inhibition by pyrophosphate than choloyl-CoA synthetase. The synthetases could not be solubilized from microsomal membranes by treatment with 1 M-NaCl, but could be solubilized with Triton X-100 or Triton X-100 plus NaCl. The detergent-solubilized trihydroxycoprostanoyl-CoA synthetase could be separated from the solubilized choloyl-CoA synthetase and palmitoyl-CoA synthetase by affinity chromatograpy on Sepharose to which trihydroxycoprostanic acid was bound. Choloyl-CoA synthetase and trihydroxycoprostanoyl-CoA synthetase could not be detected in homogenates from kidney or intestinal mucosa. The results indicate that long-chain fatty acids, cholic acid and trihydroxycoprostanic acid are activated by three separate enzymes. 相似文献
3.
Distinct long chain and very long chain fatty acyl CoA synthetases in rat liver peroxisomes and microsomes 总被引:4,自引:0,他引:4
Mitochondria, peroxisomes, and microsomes were isolated from rat liver homogenates, and stearic acid and lignoceric acid beta-oxidation, as well as stearoyl CoA synthetase and lignoceroyl CoA synthetase activities in the three organelles, were compared. Stearic acid beta-oxidation in peroxisomes was sixfold greater compared to the oxidation in mitochondria. Lignoceric acid beta-oxidation, observed only in peroxisomes, was fivefold lower compared to stearic acid beta-oxidation. Stearoyl CoA synthetase was present whereas lignoceroyl CoA synthetase was absent in mitochondria. Stearoyl CoA synthetase and lignoceroyl CoA synthetase activities were present in microsomes and peroxisomes, but the activity of stearoyl CoA synthetase was several-fold greater compared to lignoceroyl CoA synthetase in both organelles. The differing responses to detergents and phospholipids of stearoyl CoA and lignoceroyl CoA synthetase activities in microsomes as well as peroxisomes indicated that each activity was catalyzed by a separate enzyme. Differences in detergent and phospholipid response were also noted when either stearoyl CoA or lignoceroyl CoA synthetase activity in one organelle was compared with the corresponding activity in the other organelle, suggesting that the same activity in different organelles may be catalyzed by separate enzyme proteins. 相似文献
4.
In vitro synthesis of alpha-carboxyl-linked folylpolyglutamates by an enzyme preparation from Escherichia coli 总被引:3,自引:0,他引:3
Extracts of Escherichia coli contained an enzymatic activity which catalyzed the addition of L-glutamate to the alpha-carboxyl of various polyglutamate substrates, including folylpolyglutamates. Much of the enzyme activity was separated by DE52 chromatography and gel filtration from the enzyme which adds the first three glutamates in the biosynthesis of folylpolyglutamates, dihydrofolate synthetase-folylpolyglutamate synthetase. The two enzyme activities differed in many properties. Whereas dihydrofolate synthetase-folylpolyglutamate synthetase preferred pteroate or pteroylmonoglutamate substrates, the folylpoly-alpha-glutamate synthetase preparations effectively utilized tetrahydropteroylpolyglutamates, pteroylpolyglutamates, p-aminobenzoylpolyglutamates (pAB(Glu)n), and even a polyglutamate tripeptide. Several di- and triglutamyl peptides were inhibitory to folylpoly-alpha-glutamate synthetase activity, but not to dihydrofolate synthetase-folylpolyglutamate synthetase. Conversely, dihydropteroate noncompetitively inhibits the folylpolyglutamate synthetase reaction of the dihydrofolate synthetase-folylpolyglutamate synthetase protein, but did not inhibit the folylpoly-alpha-glutamate synthetase reaction. Potassium chloride was inhibitory to folylpoly-alpha-glutamate synthetase activity (as were other salts and several polyanions), in contrast to the absolute requirement of dihydrofolate synthetase-folylpolyglutamate synthetase activity for a monovalent cation such as K+. Incubation of a folylpoly-alpha-glutamate synthetase preparation with (6S)-tetrahydropteroyltri(gamma)glutamate generated products which after chemical cleavage to pAB(Glu)n were identical to those from growing E. coli, in high performance liquid chromatography retention times and in pattern of digestion by alpha-COOH bond-specific carboxypeptidase Y. High performance liquid chromatography and mass spectral analysis of the products of the in vitro reactions of folylpoly-alpha-glutamate synthetase with several substrates also demonstrated the addition of glutamate residues via alpha-COOH linkages. Thus, there appear to be two folylpolyglutamate synthetase activities in E. coli, dihydrofolate synthetase-folylpolyglutamate synthetase which adds the first three glutamate residues by gamma-COOH linkages and the folylpoly-alpha-glutamate synthetase activity which extends the folylpolyglutamate chain via gamma-COOH peptide bonds. 相似文献
5.
Escherichia coli phenylalanyl-tRNA synthetase operon: characterization of mutations isolated on multicopy plasmids 总被引:3,自引:3,他引:0
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Plasmid pB1 carries the genes for threonyl-tRNA synthetase, phenylalanyl-tRNA synthetase, and translation initiation factor IF3. Strains carrying this plasmid overproduce phenylalanyl-tRNA synthetase about 100-fold. Spontaneous mutant plasmids were obtained which no longer caused the overproduction of the enzyme. Three classes of mutations were found. (i) Deletion mutations were found, some of which had the interesting property of fusing different genes together, e.g., putting phenylalanyl-tRNA synthetase under the control of the threonyl-tRNA synthetase promoter. (ii) Insertion mutations were found; one insertion in particular was studied. This insertion is located in front of the structural gene for phenylalanyl-tRNA synthetase and is shown to interrupt a cis-acting regulatory region. (iii) Mutations that showed no major change in DNA structure were found. One of these mutations is apparently purely structural, as it produces a small subunit of phenylalanyl-tRNA synthetase with a reduced molecular weight. This protein is less stable than the wild-type enzyme. These mutations represent useful tools to investigate how the phenylalanyl-tRNA synthetase operon is regulated. 相似文献
6.
Changes in certain aminoacyl transfer ribonucleic Acid synthetase activities in developing pea roots 总被引:2,自引:2,他引:0
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Tyrosyl-, arginyl-, leucyl-, and phenylalanyl-tRNA synthetase activities were measured in extracts from three root sections of 3-day-old pea seedlings. The sections 0 to 2, 3 to 7, and 8 to 22 millimeters from the root tip were chosen to represent the regions of cell division, elongation, and maturation, respectively. The specific activity for each aminoacyl-tRNA synthetase was highest in the 0- to 2-millimeter section and lowest in the 8 to 22 millimeter section. The changes in specific activity between the sections, however, varied with the particular synthetase. Tyrosyl-tRNA synthetase from each section was fractionated into two activity regions on a diethylaminoethyl cellulose column. Approximately 10, 22, and 44% of the total tyrosyl-tRNA synthetase activity in the 0 to 2, 3 to 7, and 8- to 22-millimeter sections, respectively, were associated with the first tyrosyl-tRNA synthetase region; the remaining activity was located in the second tyrosyl-tRNA synthetase region. Only one activity region for arginyl-tRNA synthetase was detected by diethylaminoethyl cellulose column fractionation. 相似文献
7.
Carbamoyl phosphate synthetase II encodes the first enzymic step of de novo pyrimidine biosynthesis. Carbamoyl phosphate synthetase II is essential for Toxoplasma gondii replication and virulence. In this study, we characterised the primary structure of a 28kb gene encoding Toxoplasma gondii carbamoyl phosphate synthetase II. The carbamoyl phosphate synthetase II gene was interrupted by 36 introns. The predicted protein encoded by the 37 carbamoyl phosphate synthetase II exons was a 1,687 amino acid polypeptide with an N-terminal glutamine amidotransferase domain fused with C-terminal carbamoyl phosphate synthetase domains. This bifunctional organisation of carbamoyl phosphate synthetase II is unique, so far, to protozoan parasites from the phylum Apicomplexa (Plasmodium, Babesia, Toxoplasma) or zoomastigina (Trypanosoma, Leishmania). Apicomplexan parasites possessed the largest carbamoyl phosphate synthetase II enzymes due to insertions in the glutamine amidotransferase and carbamoyl phosphate synthetase domains that were not present in the corresponding gene segments from bacteria, plants, fungi and mammals. The C-terminal allosteric regulatory domain, the carbamoyl phosphate synthetase linker domain and the oligomerisation domain were also distinct from the corresponding domains in other species. The novel C-terminal regulatory domain may explain the lack of activation of Toxoplasma gondii carbamoyl phosphate synthetase II by the allosteric effector 5-phosphoribosyl 1-pyrophosphate. Toxoplasma gondii growth in vitro was markedly inhibited by the glutamine antagonist acivicin, an inhibitor of glutamine amidotransferase activity typically associated with carbamoyl phosphate synthetase II, guanosine monophosphate synthetase, or CTP synthetase. 相似文献
8.
In extracts from the adductor muscle of the shell-fish, Pecten maximus, glycogen synthetase (EC.2.4.1.11) was found. The enzyme occurs predominantly as D form (glucose-6-P dependent for activity). An I form (G-6-P independent) was also present. Kinetics of glycogen synthetase showed that the Ka for G-6-P in the D form was 10 fold higher than in the I form. Both forms of glycogen synthetase were interconverted through reactions catalyzed by phosphatase and kinase enzymes respectively. Glucose-6-P and Mg+2 must be present to stabilize glycogen synthetase and to activate the synthetase D phosphatase, found in the 90,000 X g protein-glycogen complex. The conversion of synthetase D to I was inhibited by F-, glycogen, ATP and UTP. When F- was present the effect of G-6-P on synthetase and phosphatase suggested that conversion involved the existence of more than a single glycogen synthetase phosphatase enzyme. ATP and Mg+2 were necessary for the conversion of synthetase I to D, and the conversion was stimulated by cAMP. 相似文献
9.
Derepression of Synthesis of the Aminoacyl-Transfer Ribonucleic Acid Synthetases for the Branched-Chain Amino Acids of Escherichia coli 总被引:1,自引:4,他引:1
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The kinetics of derepression of valyl-, isoleucyl-, and leucyl-transfer ribonucleic acid (tRNA) synthetase formation was examined during valine-, isoleucine-, and leucine-limited growth. When valine was limiting growth, valyl-tRNA synthetase formation was maximally derepressed within 5 min, whereas the rates of synthesis of isoleucyl-, and leucyl-tRNA synthetases were unchanged. Isoleucine-restricted growth caused a maximal derepression of isoleucyl-tRNA synthetase formation in 5 min and derepression of valyl-tRNA synthetase formation in 15 min with no effect on leucyl-tRNA synthetase formation. When leucine was limiting growth, leucyl-tRNA synthetase formation was immediately derepressed, whereas valyl- and isoleucyl-tRNA synthetase formation was unaffected by manipulation of the leucine supply to the cells. These results support our previous findings that valyl-tRNA synthetase formation is subject to multivalent repression control by both isoleucine and valine. In contrast, repression control of iso-leucyl- and leucyl-tRNA synthetase formation is specifically mediated by the supply of the cognate amino acid. 相似文献
10.
J G Bode T Peters-Regehr R Kubitz D H?ussinger 《The journal of histochemistry and cytochemistry》2000,48(3):415-422
We studied the expression of glutamine synthetase in liver macrophages (Kupffer cells, KCs) in situ and in culture. Glutamine synthetase was detectable at the mRNA and protein level in freshly isolated and short-term-cultured rat liver macrophages. Enzyme activity and protein content were about 9% of that in liver parenchymal cells. In contrast, glutamine synthetase mRNA levels in liver macrophages apparently exceeded those in parenchymal liver cells (PCs). By use of confocal laser scanning microscopy and specific macrophage markers, immunoreactive glutamine synthetase was localized to macrophages in normal rat liver and normal human liver in situ. All liver macrophages stained positive for glutamine synthetase. In addition, macrophages in rat pancreas contained immunoreactive glutamine synthetase, whereas glutamine synthetase was not detectable at the mRNA and protein level in blood monocytes and RAW 264.7 mouse macrophages. No significant amounts of glutamine synthetase were found in isolated rat liver sinusoidal endothelial cells (SECs). The data suggest a constitutive expression of glutamine synthetase not only, as previously believed, in perivenous liver parenchymal cells but also in resident liver macrophages. 相似文献
11.
The seryl-tRNA synthetase from Saccharomyces cerevisiae interacts with the peroxisome biogenesis-related factor Pex21p. Several deletion mutants of seryl-tRNA synthetase were constructed and inspected for their ability to interact with Pex21p in a yeast two-hybrid assay, allowing mapping of the synthetase domain required for complex assembly. Deletion of the 13 C-terminal amino acids abolished Pex21p binding to seryl-tRNA synthetase. The catalytic parameters of purified truncated seryl-tRNA synthetase, determined in the serylation reaction, were found to be almost identical to those of the native enzyme. In vivo loss of interaction with Pex21p was confirmed in vitro by coaffinity purification. These data indicate that the C-terminally appended domain of yeast seryl-tRNA synthetase does not participate in substrate binding, but instead is required for association with Pex21p. We further determined that Pex21p does not directly bind tRNA, and nor does it possess a tRNA-binding motif, but it instead participates in the formation of a specific ternary complex with seryl-tRNA synthetase and tRNA(Ser), strengthening the interaction of seryl-tRNA synthetase with its cognate tRNA(Ser). 相似文献
12.
Glutamine accelerates the degradation of glutamine synthetase in hepatoma tissue culture cells. Compounds structurally related to glutamine were tested for their ability to mimic or antagonize this effect of glutamine. 6-Diazo-5-oxo-L-norleucine, like glutamine depressed the activity of glutamine synthetase in hepatoma tissue culture cells. L-Methionine sulfone, albizzine, L-methionine sulfoxide, L-gamma-glutamyl hydrazide and gamma-N-methyl-L-glutamine (listed in order of decreasing potency) were antagonists which prevented the effect of glutamine on glutamine synthetase activity. These antagonists had little effect on glutamine transport or protein synthesis of hepatoma tissue culture cells and their effects were reversible. The effects of compounds on gluatmine synthetase activity in cell-free extracts of the cells were examined. Diazo-oxonorleucine and albizzine inhibited neither the transferase nor the synthetase activity of glutamine synthetase. This observation is interpreted to mean that the glutamine-binding site involved in the regulation of glutamine synthetase activity of hepatoma tissue culture cells is not the active site of the enzyme. 相似文献
13.
A highly conserved protein motif characteristic of Class II aminoacyl tRNA synthetases was found to align with a region of Escherichia coli asparagine synthetase A. The alignment was most striking for aspartyl tRNA synthetase, an enzyme with catalytic similarities to asparagine synthetase. To test whether this sequence reflects a conserved function, site-directed mutagenesis was used to replace the codon for Arg298 of asparagine synthetase A, which aligns with an invariant arginine in the Class II aminoacyl tRNA synthetases. The resulting genes were expressed in E. coli, and the gene products were assayed for asparagine synthetase activity in vitro. Every substitution of Arg298, even to a lysine, resulted in a loss of asparagine synthetase activity. Directed random mutagenesis was then used to create a variety of codon changes which resulted in amino acid substitutions within the conserved motif surrounding Arg298. Of the 15 mutant enzymes with amino acid substitutions yielding soluble enzyme, 13 with changes within the conserved region were found to have lost activity. These results are consistent with the possibility that asparagine synthetase A, one of the two unrelated asparagine synthetases in E. coli, evolved from an ancestral aminoacyl tRNA synthetase. 相似文献
14.
Oscarsson J Gardmo C Edén S Mode A 《American journal of physiology. Endocrinology and metabolism》2001,280(2):E280-E286
S-adenosylmethionine synthetase (AdoMet synthetase) is responsible for the synthesis of the major methyl donor S-adenosylmethionine. The AdoMet synthetase gene was identified by subtractive suppressive hybridization as being expressed at higher levels in the liver of rats continuously exposed to growth hormone (GH) than in rats intermittently exposed to the hormone. Further studies on the regulation of AdoMet synthetase showed that the activity and mRNA levels were higher in female than in male rats. Hypophysectomy increased AdoMet synthetase mRNA in both male and female rats. Combined thyroxine and cortisol treatment of hypophysectomized rats had no effect on AdoMet synthetase mRNA levels. Two daily injections of GH for 7 days, mimicking the male secretory pattern of GH, decreased AdoMet synthetase activity and mRNA levels. A continuous infusion of GH, mimicking the female secretory pattern of GH, had small or no effects on AdoMet synthetase activity and decreased the mRNA levels to a lesser degree than two daily injections. It is concluded that the lower AdoMet synthetase activity in male rats is due to an inhibitory effect of the male characteristic pulsatile secretory pattern of GH on AdoMet synthetase mRNA expression. 相似文献
15.
Glycinamide ribonucleotide (GAR) transformylase from HeLa cells has been purified 200-fold to apparent homogeneity with a procedure using two affinity resins. The activities glycinamide ribonucleotide synthetase and aminoimidazole ribonucleotide synthetase were found to copurify with GAR transformylase. Glycinamide ribonucleotide synthetase and GAR transformylase were separable only after exposure to chymotrypsin. Antibodies raised to pure L1210 cell GAR transformylase were able to precipitate the glycinamide ribonucleotide transformylase and GAR synthetase activities from HeLa and L1210 cells both in their native and in their proteolytically shortened forms. The compound N-10-(bromoacetyl)-5,8-dideazafolate was found to inhibit formylation but to leave the ATP-requiring synthetase activities intact. 相似文献
16.
Structural role for a conserved region in the CTP synthetase glutamine amide transfer domain. 总被引:1,自引:0,他引:1
Site-directed mutations were introduced into a conserved region of the Escherichia coli CTP synthetase glutamine amide transfer domain. The amino acid replacements, valine 349 to serine, glycine 351 to alanine, glycine 352 to proline, and glycine 352 to cysteine, all increased the lability of CTP synthetase. The proline 352 replacement abolished the capacity to form the covalent glutaminyl-cysteine 379 catalytic intermediate, thus preventing glutamine amide transfer function; NH3-dependent CTP synthetase activity was retained. In CTP synthetase (serine 349), both glutamine and NH3-dependent activities were increased approximately 30% relative to that of the wild type. CTP synthetase mutants alanine 351 and cysteine 352 were not overproduced because of apparent instability and proteolytic degradation. We conclude that the conserved region between residues 346 and 355 in the CTP synthetase glutamine amide transfer domain has an important structural role. 相似文献
17.
Mutants of Escherichia coli B that contain essentially no detectable glutathione were isolated. These mutants had a very low activity of gamma-glutamylcysteine synthetase or glutathione synthetase. No significant differences in growth in minimal medium were observed between the mutants and the parental strain. The mutants lacking gamma-glutamylcysteine synthetase activity were more susceptible to toxic compounds than either the parental strain or a glutathione synthetase-deficient strain. The mutants lacking gamma-glutamylcysteine synthetase activity were also susceptible to oxygen. 相似文献
18.
Fatty acid synthetase messenger RNA was obtained from rat liver polysomal RNA and then injected into Xenopus laevis oocytes. The radioactive fatty acid synthetase protein synthesized in the oocytes was identified by immunoprecipitation with anti-fatty acid synthetase antibody and the immunoprecipitate was then characterized by electrophoresis on sodium dodecyl sulfate-polyacrylamide gel. Co-migration of authentic fatty acid synthetase and the labeled product synthesized in oocytes was observed. Based on sucrose density gradient analysis, the rat liver fatty acid synthetase mRNA has a sedimentation coefficient of approximately 33 S, which agrees with the predicted minimum size necessary to code for the fatty acid synthetase protein. In addition, this mRNA was partially purified with oligo(dT)-cellulose, which indicates that it has a polyadenylate region. The relative in vivo rate of synthesis of fatty acid synthetase and the level of fatty acid synthetase mRNA in liver were also determined during the course of dietary induction of this enzyme. The results indicate that the dietary-induced increase in the level of fatty acid synthetase is probably due to an increased level of the fatty acid synthetase mRNA. 相似文献
19.
In Bacillus subtilis, the activity of the nitrogen regulatory factor TnrA is regulated through a protein- protein interaction with glutamine synthetase. During growth with excess nitrogen, the feedback-inhibited form of glutamine synthetase binds to TnrA and blocks DNA binding by TnrA. Missense mutations in glutamine synthetase that constitutively express the TnrA-regulated amtB gene were characterized. Four mutant proteins were purified and shown to be defective in their ability to inhibit the in vitro DNA-binding activity of TnrA. Two of the mutant proteins exhibited enzymatic properties similar to those of wild-type glutamine synthetase. A model of B. subtilis glutamine synthetase was derived from a crystal structure of the Salmonella typhimurium enzyme. Using this model, all the mutated amino acid residues were found to be located close to the glutamate entrance of the active site. These results are consistent with the glutamine synthetase protein playing a direct role in regulating TnrA activity. 相似文献
20.
Abstract— C-6 glial cells in culture were utilized to define the role of glucocorticoid in the regulation of palmitic acid synthesis and the important lipogenic enzymes, fatty acid synthetase and acetyl-CoA carboxylase. Particular emphasis was given to fatty acid synthetase which exhibited more than a 50% reduction in specific activity when cells were exposed to hydrocortisone (10 μg/ml) for 1 week. Coordinate changes in acetyl-CoA carboxylase activity and in palmitic acid (and sterol) synthesis from acetate accompanied the alterations in fatty acid synthetase. Immunochemical techniques were utilized to show that the decrease in synthetase activity involved an alteration in enzyme content, not in catalytic efficiency. The changes in content of fatty acid synthetase were caused by alterations in enzyme synthesis. Glucocorticoids may regulate fatty acid synthetase in C-6 glial cells by a mechanism similar to that suggested for adipose tissue. The inhibition of palmitic acid synthesis may be relevant to other effects of glucocorticoids on developing brain. 相似文献