Adrenocorticotropic hormone-mediated changes in rat adrenal mitochondrial phospholipids

We examined the subcellular localization of ACTH (adrenocorticotropic hormone)-induced changes in adrenal phospholipids using dexamethasone-treated rats. In adrenal mitochondrial fraction, ACTH significantly enhanced both concentrations and contents of phosphatidylinositol (37%), phosphatidylcholine (22%), and phosphatidylethanolamine (20%). Other mitochondrial phospholipids including cardiolipin did not change upon administration of ACTH. In adrenal plasma membrane, endoplasmic reticulum, and peroxisomes, no increase in phospholipids was observed. The ACTH-induced increases in mitochondrial phosphatidylinositol, phosphatidylcholine, and phosphatidylethanolamine were specific to adrenal among tissues tested. These changes were observed specifically in cortical cells rather than medulla. Nonsteroidogenic ACTH fragments and related peptides were unable to induce the change in adrenal mitochondrial phospholipids. From the dose-response profile with ACTH, the changes in mitochondrial phospholipids were closely related to ACTH-dependent stimulation of steroidogenesis. Furthermore, in vitro treatment with cyclic AMP enhanced both concentrations and contents of mitochondrial phosphatidylinositol, phosphatidylcholine, and phosphatidylethanolamine similar to those by the in vivo administration of ACTH. Both in vivo and in vitro experiments revealed that the hormone-induced changes in mitochondrial phospholipids were sensitive to a protein-synthesis inhibitor, cycloheximide. However, aminoglutethimide and cytochalasin B, which strongly inhibited the hormone-induced formation of corticosterone, did not affect the increases in mitochondrial phospholipids. These results suggest that the hormone-induced increases in these phospholipids occur between ACTH-mediated ribosomal protein synthesis and corticosterone formation.     

Structural analysis of a maize gene coding for glutathione-S-transferase involved in herbicide detoxification

Summary We have used the cDNA clone encoding maize glutathione-S-transferase (GST I) to isolate a genomic DNA clone containing the complete GST I gene. Nucleotide sequence analysis of the cDNA and genomic clones has yielded a complete amino acid sequence for maize GST I and provided the exon-intron map of its gene. The mRNA homologous sequences in the maize GST I gene consist of a 107 bp 5 untranslated region, a 642 bp coding region and 340 bp of the 3 untranslated region. They are divided into three exons by two introns which interrupt the coding region. The 5 untranslated spacer contains an unusual sequence of pentamer AGAGG repeated seven times. The inbred maize line (Missouri 17) contains a single gene for GST I, whereas the hybrid line (3780A) contains two genes. Nucleotide sequence analysis of the primer extended cDNA products reveals that the 5 untranslated regions of the two genes in the hybrid 3780A are identical except for a 6 bp internal deletion (or insertion). The amino acid sequence of maize GST I shares no apparent sequence homology with the published sequences of animal GST's and represents the first published sequence of a plant GST. re]19850813 ac]19851126     

Effect of polyamines on synthesis and degradation of guanosine 5'-diphosphate 3'-diphosphate

The effect of polyamines on the in vitro and in vivo synthesis and degradation on guanosine 5'-diphosphate 3'-diphosphate (ppGpp) has been studied in Escherichia coli. The presence of 2 mM spermidine lowered the optimal Mg2+ concentration for ppGpp formation from 17 mM to 11 mM. The formation of ppGpp in the presence of 2 mM spermidine and 11 mM Mg2+ was about 15% greater than that in the presence of 17 mM Mg2+. At a concentration of less than 11 mM Mg2+, spermidine was found to stimulate ppGpp formation greatly. Putrescine did not cause any effect. When a polyamine-requiring mutant of E. coli (EWH319) was starved for an amino acid by the addition of valine, spermidine stimulated ppGpp formation. The degradation of ppGpp was not influenced significantly by polyamines.     

Expression of human immunoglobulin E epsilon chain cDNA in E. coli.

Using the cDNA of human epsilon chain, three expression plasmids that code directly the constant portion of the epsilon chain (C epsilon 1-C epsilon 4, C epsilon 2-C epsilon 4 and C epsilon 3-C epsilon 4 domains) were constructed. These epsilon chain peptides were synthesized in E. coli under the control of the trp promoter-operator. The bacterially produced peptides have the antigenicity of human epsilon chain and gave the molecular weights equal to the values calculated from the amino acid sequence of the constructed plasmids.     

Molecular cloning and nucleotide sequencing of human immunoglobulin epsilon chain cDNA.

DNA complementary to mRNA of human immunoglobulin E heavy chain (epsilon chain) isolated and purified from U266 cells has been synthesized and inserted into the PstI site of pBR322 by G-C tailing. This recombinant plasmid was used to transform E. coli chi 1776 to screen 1445 tetracycline resistant colonies. Nine clones (pGETI - 9) containing cDNA coding for the human epsilon chain were recognized by colony hybridization and Southern blotting analysis with a nick-translated human IgE genome fragment. The nucleotide sequence of the longest cDNA contained in pGET2 was determined. The results indicate that the sequence of 1657 nucleotides codes for 494 amino acids covering a part of the variable region and all of the constant region of the human epsilon chain. Most of the amino acid sequence deduced from the nucleotide sequence is in substantial agreement with that reported. Furthermore a termination codon after the -COOH terminal amino acid marks the beginning of a 3' untranslated region of 125 nucleotides with a poly A tail. Taking this into account, the structure of the human epsilon chain mRNA, except a part of the 5' end, is conserved fairly well in the cDNA insert in pGET2.