Allogibberic acid: An inhibitor of flowering in Lemna perpusilla

Allogibberic acid (I) has been identified as the compound responsible for the inhibition of flowering, increase in frond multiplication rate and decrease in frond size produced in Lemna perpusilla 6746 by autoclaved, unbuffered aqueous solutions of gibberellic acid (VII). 13-Deoxyallogibberic acid (IV), a product of autoclaving aq. GA₇ (VIII) solutions, also inhibits flowering in L. perpusilla and is about 10 times more active than allogibberic acid.     

Effect of growth phase on phospholipid biosynthesis in Saccharomyces cerevisiae.

The effect of growth phase on the membrane-associated phospholipid biosynthetic enzymes CDP-diacylglycerol synthase, phosphatidylserine synthase, phosphatidylinositol synthase, and the phospholipid N-methyltransferases in wild-type Saccharomyces cerevisiae was examined. Maximum activities were found in the exponential phase of cells grown in complete synthetic medium. As cells entered the stationary phase of growth, the activities of the CDP-diacylglycerol synthase, phosphatidylserine synthase, and the phospholipid N-methyltransferases decreased 2.5- to 5-fold. The subunit levels of phosphatidylserine synthase and the cytoplasmic-associated enzyme inositol-1-phosphate synthase were not significantly affected by the growth phase. When grown in medium supplemented with inositol-choline, cells in the exponential phase of growth had reduced CDP-diacylglycerol synthase, phosphatidylserine synthase, and phospholipid N-methyltransferase activities, with repressed subunit levels of phosphatidylserine synthase and inositol-1-phosphate synthase compared with cells grown without inositol-choline. Enzyme activity levels remained reduced in the stationary phase of growth of cells supplemented with inositol-choline. The phosphatidylserine synthase and inositol-1-phosphate synthase subunit levels, however, were depressed. Phosphatidylinositol synthase (activity and subunit) was not affected by growth in medium supplemented with or without inositol-choline or the growth phase of the culture. The phospholipid composition of cells in the exponential and stationary phase of growth was also examined. The phosphatidylinositol to phosphatidylserine ratio doubled in stationary-phase cells. The phosphatidylcholine to phosphatidylethanolamine ratio was not significantly affected by the growth phase of cells.     

Inhibition of apolipoprotein B net synthesis and secretion from cultured rat hepatocytes by the calcium-channel blocker diltiazem.

1. The effect of the Ca2+-channel blocker diltiazem on hepatic apolipoprotein B (apo B) synthesis and secretion was studied in 12-18 h cultures of collagenase-dispersed rat hepatocytes. 2. The presence of diltiazem in the medium decreased apo B secretion by hepatocytes in a concentration-dependent manner. At 25 microM, diltiazem inhibited apo B secretion by approx. 36%, but there was no evidence of intracellular accumulation of apo B. 3. The inhibition of apo B secretion by hepatocytes was significantly correlated with cell-associated diltiazem (r = 0.72, P less than 0.01). 4. The rate of apo B secretion remained linear over 16 h even in the presence of 50 microM-diltiazem. 5. At diltiazem concentrations in the medium which were inhibitory for apo B secretion, [14C]acetate incorporation into cellular lipids and [35S]methionine incorporation into protein were enhanced. 6. Diltiazem inhibited the secretion of the apo B variants with a preferential inhibition of the higher-molecular-mass form of apo B (apo BH) over the lower-molecular-mass form (apo BL) at diltiazem concentrations in the medium greater than 25 microM. 7. Together, these results suggest that Ca2+ may play an important role in the synthesis and secretion of apo B-containing lipoproteins.     

Characterization and chromosomal mapping of the gene encoding the cellular DNA binding protein ILF.

Recently we isolated a cellular DNA binding protein, designated interleukin enhancer binding factor (ILF), that binds to purine-rich regulatory motifs in both the HIV-1 LTR and the IL2 promoter. Further analysis of the ILF gene reveals the existence of two mRNA species, both of which encode proteins containing the recently described fork head DNA binding domain. Gel retardation analysis demonstrates that the portion of the ILF protein with homology to the fork head domain is sufficient to mediate DNA binding to a number of related purine-rich sequences. ILF mRNA is expressed constitutively in both lymphoid and nonlymphoid tissues. Chromosomal mapping localizes the ILF gene to human chromosome 17q25, which is a site of chromosomal translocations in some cases of human acute myelogous leukemias. These studies further characterize the structure of the cellular DNA binding protein ILF and may prove valuable in the molecular analysis of possible translocations affecting this gene.     

Subcellular localization of rice leaf aryl acylamidase activity

The intracellular localization of aryl acylamidase (aryl-acylamide amidohydrolase, EC 3.5.1.13) in rice (Oryza sativa L. var Starbonnet) leaves was investigated. The enzyme hydrolyzes and detoxifies the herbicide propanil (3,4-dichloropropionanilide) thereby accounting for immunity of the rice plant to herbicidal action. Fractionation of mesophyll protoplasts by differential centrifugation yielded the highest specific activity of amidase in the crude mitochondrial fraction. Further separation of density gradients of the silica sol Percoll also indicated that this enzyme was mitochondrial. By the use of biochemical markers, the purified mitochondrial fraction was shown to be substantially free of contamination from nuclei, chloroplasts, golgi, and plasma membranes. Subfractionation of the purified mitochondria suggests that this enzyme is located on the outer membrane.     

1,10-Phenanthroline-copper, a footprinting reagent for single-stranded regions of RNAs.

The 1,10-phenanthroline-cuprous complex (OP-Cu) with hydrogen peroxide as a coreactant nicks the single-stranded loops and bulges of RNA stem-loop structures more rapidly than the double-stranded stems. This chemical nuclease is therefore a useful footprinting reagent for these regions and can be used to monitor both intramolecular and intermolecular hybridization of single-stranded domains. The formation of A-form structures characteristic of either RNA-RNA or RNA-DNA duplexes inhibits scission because it blocks the binding site of the coordination complex in single-stranded loops and not because the oxidatively sensitive hydrogens of the ribose moiety are blocked. The C-4' and C-1' hydrogens are accessible to solvent in A-structures.     

TAR independent activation of the human immunodeficiency virus in phorbol ester stimulated T lymphocytes.

Multiple regulatory elements in the human immunodeficiency virus long terminal repeat (HIV LTR) are required for activation of HIV gene expression. Previous transfection studies of HIV LTR constructs linked to the chloramphenicol acetyltransferase gene indicated that multiple regulatory regions including the enhancer, SP1, TATA and TAR regions were important for HIV gene expression. To characterize these regulatory elements further, mutations in these regions were inserted into both the 5' and 3' HIV LTRs and infectious proviral constructs were assembled. These constructs were transfected into either HeLa cells, Jurkat cells or U937 cells in both the presence and absence of phorbol esters which have previously been demonstrated to activate HIV gene expression. Viral gene expression was assayed by the level of p24 gag protein released from cultures transfected with the proviral constructs. Results in all cell lines indicated that mutations of the SP1, TATA and the TAR loop and stem secondary structure resulted in marked decreases in gene expression while mutations of the enhancer motif or TAR primary sequence resulted in only slight decreases. However, viruses containing mutations in either the TAR loop sequences or stem secondary structure which were very defective for gene expression in untreated Jurkat cells, gave nearly wild-type levels of gene expression in phorbol ester-treated Jurkat cells but not in phorbol ester-treated HeLa or U937 cells. High level gene expression of these TAR mutant constructs in phorbol ester-treated Jurkat cells was eliminated by second site mutations in the enhancer region or by disruption of the tat gene.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential growth kinetics are exhibited by human immunodeficiency virus type 1 TAR mutants.

The human immunodeficiency virus type 1 (HIV-1) TAR element is critical for the activation of gene expression by the transactivator protein, Tat. Mutagenesis has demonstrated that a stable stem-loop RNA structure containing both loop and bulge structures transcribed from TAR is the major target for tat activation. Though transient assays have defined elements critical for TAR function, no studies have yet determined the role of TAR in viral replication because of the inability to generate viral stocks containing mutations in TAR. In the current study, we developed a strategy which enabled us to generate stable 293 cell lines which were capable of producing high titers of different viruses containing TAR mutations. Viruses generated from these cell lines were used to infect both T-lymphocyte cell lines and peripheral blood mononuclear cells. Viruses containing TAR mutations in either the upper stem, the bulge, or the loop exhibited dramatically decreased HIV-1 gene expression and replication in all cell lines tested. However, we were able to isolate lymphoid cell lines which stably expressed gene products from each of these TAR mutant viruses. Though the amounts of virus in these cell lines were roughly equivalent, cells containing TAR mutant viruses were extremely defective for gene expression compared with cell lines containing wild-type virus. The magnitude of this decrease in viral gene expression was much greater than previously seen in transient expression assays using HIV-1 long terminal repeat chloramphenicol acetyltransferase gene constructs. In contrast to the defects in viral growth found in T-lymphocyte cell lines, several of the viruses containing TAR mutations were much less defective for gene expression and replication in activated peripheral blood mononuclear cells. These results indicate that maintenance of the TAR element is critical for viral gene expression and replication in all cell lines tested, though the cell type which is infected is also a major determinant of the replication properties of TAR mutant viruses.