Codon recognition by enzymatically mischarged valine transfer ribonucleic acid.

Direct evidence for the adaptor hypothesis has been obtained by examining the codon recognition of a purified Escherichia coli valine transfer ribonucleic acid which was enzymatically mischarged with phenylalanine labeled with carbn-14 by reaction with purified phenylalanyl-transfer ribonucleic acid synthetase from Neurospora crassa. The mischarged transfer ribonucleic acid recognized the valine codons but failed to recognize the phenylalanine codon when tested in trinucleotide-directed ribosomal binding assay.

Novel Pseudomonas product stimulates interleukin-8 production in airway epithelial cells in vitro.

Because high concentrations of IL-8 are found in the sputum of cystic fibrosis patients, we hypothesized that Pseudomonas aeruginosa (PA) induces the production of IL-8 in airway epithelial cells and in monocytes. Therefore, we incubated the supernatant from PA culture with human transformed bronchial epithelial cells (16-HBE) or with monocytes. The culture medium of 16-HBE cells that had been incubated with PA supernatant for 6 h had chemotactic activity that was inhibited by an antibody to human IL-8. The PA supernatant induced IL-8 production by primary bronchial epithelial cells, by 16-HBE cells, and by monocytes. After incubation with PA supernatant, 16-HBE cells showed a marked increase in the levels of IL-8 gene expression. The PA product responsible for IL-8 production resisted freezing, boiling, and proteolysis. This product was not lipid extractable and was present in a 1-kD filtrate. We conclude that a small molecular mass product of PA stimulates IL-8 production by 16-HBE cells and by monocytes, and that the chemotactic activity produced by 16-HBE cells after exposure to PA is due principally to IL-8.

Splicing mutants and their second-site suppressors at the dihydrofolate reductase locus in Chinese hamster ovary cells.

Point mutants induced with a variety of mutagens at the dihydrofolate reductase (dhfr) locus in Chinese hamster ovary (CHO) cells were screened for aberrantly spliced dhfr mRNA by RNase protection and/or reverse transcriptase coupled with cDNA amplification by the polymerase chain reaction (PCR). Of 115 mutants screened, 28 were found to be affected in splicing. All exhibited less than 1% correct splicing, probably because the selection procedure was stringent. All 26 unique mutations were located within the consensus splice sequences; changes were found at 9 of 10 possible sites in this 25-kb six-exon gene. Mutations at the sites flanking the first and last exons resulted in the efficient recruitment of a cryptic site within each exon. In contrast, mutations bordering internal exons caused predominantly exon skipping. In many cases, multiple exons were skipped, suggesting the clustering of adjacent exons prior to actual splicing. Six mutations fell outside the well-conserved GU and AG dinucleotides. All but one were donor site single-base substitutions that decreased the agreement with the consensus and resulted in little or no correct splicing. Starting with five of these donor site mutants, we isolated 31 DHFR+ revertants. Most revertants carried a single-base substitution at a site other than that of the original mutation, and most had only partially regained the ability to splice correctly. The second-site suppression occurred through a variety of mechanisms: (i) a second change within the consensus sequence that produced a better agreement with the consensus; (ii) a change close to but beyond the consensus boundaries, as far as 8 bases upstream in the exon or 28 bases downstream in the intron; (iii) mutations in an apparent pseudo 5' site in the intron, 84 and 88 bases downstream of a donor site; and (iv) mutations that improved the upstream acceptor site of the affected exon. Taken together, these second-site suppressor mutations extend the definition of a splice site beyond the consensus sequence.

Multiple sites of action of N-hydroxy-2-acetylaminofluorene rat hepatic nuclear transcription.

This study attempts to identify the site(s) of action of N-hydroxy-2-acetylaminofluorene (N-OH-AAF) in relation to its inhibition of rat hepatic nuclear RNA synthesis. Two hr after N-OH-AAF injection (3 mg/100 g body weight), rat hepatic nuclear synthesis and nucleolar RNA synthesis in vitro were inhibited by 60 and 80%, respectively. When total nuclear RNA polymerases were solubilized and assayed in the presence of alpha-amanitin (3.2 mug/ml), only alpha-amanitin-sensitive activity was reduced (50%) by N-OH-AAF. Diethylamino-ethyl-Sephadex column chromatography confirmed this finding and further demonstrated that RNA polymerase II was the activity selectively inhibited. Since N-OH-AAF dramatically inhibited nucleolar RNA synthesis but had little effect on RNA polymerase I activity, per se, we therefore concluded that, in addition to its direct inhibitory effect on the enzymic function of RNA polymerase II, N-OH-AAF must also cause impairment of the nucleolar DNA template function.

Inhibition of oxidative stress in HeLa cells by chemopreventive agents.

12-O-Tetradecanoylphorbol-13-acetate (TPA)-mediated oxidative stress in HeLa cells and its inhibition were studied by fluorometric measurement of H2O2 and by 3H-postlabeling of the oxidized bases 8-hydroxyl-2'-deoxyguanosine (8-OHdG) and 5-hydroxymethyl-2'-deoxyuridine (HMdU). TPA treatment (10 fmol/cell) caused approximately 7-fold increase in H2O2 levels (0.1 nmol TPA/ml), and 5-10-fold increase in 8-OHdG and HMdU (10 nmol TPA/ml). Naturally occurring compounds [caffeic acid phenethyl ester (CAPE), (-).epigallocatechin gallate (EGCG), penta-O-galloyl-beta-D-glucose (PGG) and sarcophytol A (Sarp A)] and the anticancer drug tamoxifen (TAM) were tested as potential chemopreventive agents. These agents dose-dependently inhibited TPA-induced H2O2, 8-OHdG and HMdU. The doses required for a 50% decrease in H2O2 were approximately 2.5 microM for TAM; 5 microM for CAPE, EGCG and PGG; and 75 microM for Sarp A. TAM and PGG (10 microM), EGCG (25 microM), and CAPE (50 microM) abolished TPA-mediated H2O2 production, even below the normal cellular levels. TAM (2.5-20 microM) decreased TPA-mediated HMdU and 8-OHdG formation 2-29 times. Maximum inhibition occurred at 20 microM TAM, which caused an approximately 95% decline in HMdU and 8-OHdG. CAPE was effective at 0.5-50 microM. CAPE (25 microM) decreased 8-OHdG 95%, and HMdU 58%, while Sarp A (250 microM) reduced 8-OHdG by 93% and HMdU by 78%. EGCG (1-25 microM) and PGG (1-10 microM) inhibited of 8-OHdG and HMdU dose-dependently. However, higher doses (50 and 100 microM) decreased the efficacy of that inhibition. Of those agents tested, TAM appears to be the most and Sarp A the least effective. Our results point to these 5 compounds as being potential chemopreventive agents, which at very low doses decrease the tumor promoter-mediated oxidative processes.

Aberrant de novo methylation of DNA after treatment of murine cells with N-acetoxy-N-2-acetylaminofluorene.

The ultimate chemical carcinogen N-acetoxy-N-2-acetylaminofluorene inhibits the enzymatic methylation of newly replicated DNA in cultured mouse P815 cells in a dose-dependent manner. After removal of the carcinogen, a significant de novo methylation of newly replicated DNA takes place, the level of methylation being higher than in control cultures. This aberrant methylation persists in the absence of N-acetoxy-N-2-acetylaminofluorene in subsequent cell cycles. Cellular cloning experiments suggest that N-acetoxy-N-2-acetylaminofluorene treatment leads to two distinct sets of cells, one with a higher and another with a lower extent of enzymatic methylation of DNA, contrasting to the apparent uniform methylation pattern in control clones.

Differential excision from DNA of the C-8 and N2 guanosine adducts of N-acetyl-2-aminofluorene by single strand-specific endonucleases.

Purified duck reticulocyte DNA was reacted in vitro with [9-14C]-N-acetoxy-N-acetyl-2-aminofluorene. Hydrolysis of the [14C]-N-acetyl-2-aminofluorene-modified DNA followed by Sephadex LH-20 column chromatography showed that 85% of the DNA-bound [14C]-N-acetyl-2-aminofluorene was N-(deoxyguanosin-8-yl)-N-acetyl-2-aminofluorene and 15% was 3-(deoxyguanosin-N2-yl)-N-acetyl-2-aminofluorene. When this modified DNA was incubated with the single strand-specific nuclease, S1, and the undigested fraction of the DNA was analyzed, there was preferential loss of the quanosine C-8 adduct from the DNA. Moreover, analysis of the nucleosides released by exposure of N-acetyl-2-aminofluorene-modified DNA to a single strand-specific nuclease from Neurospora crassa showed only the guanosine C-8 adduct in the supernatant fraction. These results suggest that, whereas the N-(deoxyguanosin-8-yl)-N-acetyl-2-aminofluorene adduct in DNA causes major conformational changes in the double-stranded helix and localized regions of denaturation, the 3-(deoxyguanosin-N2-yl)-N-acetyl-2-aminofluorene adduct does not cause major distortions of the native DNA structure.

Caffeic acid phenethyl ester stimulates human antioxidant response element-mediated expression of the NAD(P)H:quinone oxidoreductase (NQO1) gene.

Caffeic acid phenethyl ester (CAPE) is a phenolic antioxidant derived from the propolis of honeybee hives. CAPE was shown to inhibit the formation of intracellular hydrogen peroxide and oxidized bases in DNA of 12-O-tetradecanoylphorbol-13-acetate (TPA)-treated HeLa cells and was also found to induce a redox change that correlated with differential growth effects in transformed cells but not the nontumorigenic parental ones. Mediated via the electrophile or human antioxidant response element (hARE), induction of the expression of NAD(P)H quinone oxidoreductase (NQO1) and glutathione S-transferase Ya subunit genes by certain phenolic antioxidants has been correlated with the chemopreventive properties of these agents. Here, we determined by Northern analysis that CAPE treatment of hepatoma cells stimulates NQO1 gene expression in cultured human hepatoma cells (HepG2), and we characterized the effects of CAPE treatment on the expression of a reporter gene either containing or lacking the hARE or carrying a mutant version of this element in rodent hepatoma (Hepa-1) transfectants. A dose-dependent transactivation of human hARE-mediated chloramphenicol acetyltransferase (cat) gene expression was observed upon treatments of the Hepa-1 transfectants with TPA, a known inducer, as well as with CAPE. The combined treatments resulted in an apparent additive stimulation of the reporter expression. To learn whether this activation of cat gene expression was effected by protein kinase C in CAPE-treated cells, a comparison was made of cat gene activity after addition of calphostin, a protein kinase C inhibitor. Calphostin reduced the cat gene induction by TPA but not by CAPE, suggesting that stimulation of gene expression in this system by these agents proceeds via distinct mechanisms. Band-shift experiments to examine binding of transactivator proteins from nuclear extracts of treated and untreated cells to a hARE DNA probe showed that TPA exposure increased the binding level. In contrast, binding of factors to this probe was inhibited after either in vivo treatment of cells with CAPE or in vitro addition of this compound to the nuclear extract. In view of the clear stimulation by CAPE of gene expression mediated by hARE, possible explanations of this result are discussed.

Growth suppression and toxicity induced by caffeic acid phenethyl ester (CAPE) in type 5 adenovirus-transformed rat embryo cells correlate directly with transformation progression.

The active component of the honeybee hive product propolis, caffeic acid phenethyl ester (CAPE), induces a selective growth suppressive and toxic effect toward cloned rat embryo fibroblast cells transformed by adenovirus type 5 (Ad5) or the Ad5 E1A transforming gene versus untransformed cloned rat embryo fibroblast cells (Z-z. Su et al., Mol. Carcinog., 4: 231-242, 1991). The present study was conducted to determine whether CAPE-induced growth suppression/toxicity was a direct result of expression of the Ad5 E1A and E1B transforming genes or a consequence of the action of these genes resulting in the transformed state. For this investigation we used somatic cell hybrids and 5-azacytidine-treated Ad5-transformed rat embryo cells that display different stages of expression of the transformed phenotype. This series of cell lines has permitted us to determine whether expression of the transformed state and the stage of transformation progression regulates CAPE sensitivity. Evidence is presented indicating that sensitivity to CAPE is directly determined by the state of expression of the transformed progression phenotype, as opposed to simply the expression of the Ad5 E1A and E1B transforming genes. These results provide further evidence that CAPE may represent a unique compound that can specifically target progressed transformed cells for growth suppression and toxicity. An understanding of the mechanism underlying this selective effect of CAPE could result in the identification of important biochemical pathways mediating cellular transformation and progression of the transformed state.

Apoptosis and altered redox state induced by caffeic acid phenethyl ester (CAPE) in transformed rat fibroblast cells.

Caffeic acid phenethyl ester (CAPE), which is derived from the propolis of bee hives, was shown previously to block tumor promoter- and carcinogen-generated oxidative processes in several assays and to engender differential toxicity to some transformed cells. To study the mechanisms of CAPE-induced differential cytotoxicity, nontumorigenic rat embryo fibroblasts (CREF) and adenovirus (type 5)-transformed CREF cells (Wt3A) were used. As shown by nucleosomal-length DNA degradation, morphological alterations by electron microscopy, in situ labeling of 3'-OH ends, and the appearance of a hypodiploid cell population by bivariant flow cytometry, cell death induced by CAPE in the transformed Wt3A cells was apoptosis. Under the same CAPE treatment conditions, CREF cells transiently growth arrested. Both CREF and Wt3A cells were radioresistant, suggesting deficiencies in the proteins controlling the G1 checkpoint. To explore possible mechanisms of CAPE-induced apoptosis, it was determined whether CAPE-induced toxicity was influenced by the redox state of the cells. Depletion of cellular glutathione (GSH) with buthionine sulfoximine before CAPE treatment caused CREF sensitive to CAPE-induced cell death. GSH levels were also determined in CAPE-treated CREF and Wt3A cells. The GSH level in the CREF cells was unaffected by CAPE, whereas the Wt3A cells showed a significant reduction. When the GSH levels were increased in Wt3A cells by treatment with the reducing agent, N-acetyl-cysteine before CAPE treatment, the Wt3A cells were partially rescued. Furthermore, Bcl2, which protects cells from oxidative stress, had a protective effect against CAPE-induced apoptosis in Wt3A cells. Finally, the sensitivity of Wt3A cells to a known oxidant, hydrogen peroxide (H2O2), was examined. Wt3A cells were killed by H2O2-induced apoptosis, whereas CREF cells remained resistant. When Wt3A cells were treated with catalase, a cellular enzyme that inactivates H2O2, CAPE-induced apoptosis in Wt3A cells was reduced, further proving that Wt3A cells were more sensitive than CREF cells to oxidative stress. These results suggest that CAPE can modulate the redox state of cells. Sensitivity of cells to CAPE-induced cell death may be determined by the loss of normal redox state regulation in transformed cells.

Quantitation of benzo(a)pyrene-deoxyguanosine adducts by radioimmunoassay.

Calf thymus DNA was modified with the benzo(a)pyrene (BP) derivative, (+/-)7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene [(+/-) BPDE I], under conditions which yielded greater than 99% of the binding product in the form of trans-(7R)-N2-(10[7 beta,8 alpha,9 alpha-trihydroxy-7,8,9,10-tetrahydrobenzo(a)pyrene]yl) deoxyguanosine. Rabbits were immunized with modified DNA coupled to methylated bovine serum albumin, and the resulting antiserum was utilized in a competition radioimmunoassay for the quantitation of products of BP covalently bound to DNA. The antiserum was specific for both native and denatured immunogen DNA's as well as for the major isolated BP binding product, but it did not recognize BP, the tetrol of (+/-)BPDE I, or unmodified deoxyguanosine. The modified DNA was assayed in quantities as low as 2 pmol of adduct, a sensitivity sufficient to quantitate the extent of modification of cellular DNA when epidermal cell cultures were exposed either to BP or to (+/-)BPDE I. High-pressure liquid chromatographic analysis of DNA hydrolsates, obtained from epidermal cells exposed to BP or to (+/-)BPDE I, indicated that the major adduct was the same as than on the immunogen DNA. This approach should prove valuable for further studies on the mechanism of carcinogenesis and for monitoring human exposure to this ubiquitous carcinogen.

Relationships between benzo(a)pyrene-DNA adduct levels and genotoxic effects in mammalian cells.

The effectiveness of benzo(a)pyrene [B(a)P]-DNA binding as an internal dosimeter was evaluated. Data were obtained from concurrent studies, measuring B(a)P induced genotoxic effects and DNA adducts in several short-term bioassay systems: cytotoxicity, gene mutation, and sister chromatid exchange in Chinese hamster V79 cells; cytotoxicity, gene mutation, and chromosome aberrations in mouse lymphoma L5178Y TK+/-; cytotoxicity and enhanced virus transformation in Syrian hamster embryo cells; and cytotoxicity and morphological transformation in C3H10T1/2CL8 mouse embryo fibroblasts. Both total B(a)P-DNA binding and specific B(a)P-DNA adducts were measured. N2-(10 beta-[7 beta,8 alpha,9 alpha-trihydroxy-7,8,9,10-tetrahydrobenzo(a)pyrene]yl)deoxyguanosine [BPDE I-dGuo] was one of the major adducts identified in all bioassay systems. DNA binding and genotoxic responses varied significantly between bioassays. Each genetic end point was induced with a differing efficiency on a per adduct basis. However, the relationships between frequency of genetic effect or morphological transformation and B(a)P-DNA binding or BPDE I-dGuo were linear within a given assay. In order to compare biological end points of diverse frequencies in diverse biological systems, a doubling adduct level, expressed as the number of BPDE I-dGuo adducts per unit of DNA required to double the induced frequency of biological response, was applied to the data.

Inhibition of tumor promoter-mediated processes in mouse skin and bovine lens by caffeic acid phenethyl ester.

Caffeic acid phenethyl ester (CAPE) was isolated from propolis (a product of honeybee hives) that has been used in folk medicine as a potent antiinflammatory agent. CAPE is cytotoxic to tumor and virally transformed but not to normal cells. Our main goal was to establish whether CAPE inhibits the tumor promoter (12-O-tetradecanoylphorbol-13-acetate)-induced processes associated with carcinogenesis. Topical treatment of SENCAR mice with very low doses (0.1-6.5 nmol/topical treatment) of CAPE strongly inhibits the following 12-O-tetradecanoylphorbol-13-acetate-mediated oxidative processes that are considered essential for tumor promotion: (a) polymorphonuclear leukocyte infiltration into mouse skin and ears, as quantified by myeloperoxidase activity; (b) hydrogen peroxide (H2O2) production; and (c) formation of oxidized bases in epidermal DNA, as measured by 5-hydroxymethyluracil and 8-hydroxylguanine. A 0.5-nmol dose of CAPE suppresses the oxidative burst of human polymorphonuclear leukocytes by 50%. At higher doses (1-10 mumol), CAPE inhibits edema and ornithine decarboxylase induction in CD-1 and SENCAR mice. Interestingly, we discovered that 12-O-tetradecanoylphorbol-13-acetate-induced H2O2 production in bovine lenses also is inhibited by CAPE. Cumulatively, these findings point to CAPE as being a potent chemopreventive agent, which may be useful in combating diseases with strong inflammatory and/or oxidative stress components, i.e., various types of cancer and possibly cataract development.

Measurement of benzo(a)pyrene-DNA adducts by enzyme immunoassays and radioimmunoassay.

Ultrasensitive enzymatic radioimmunoassay (USERIA) was compared to radioimmunoassay and enzyme-linked immunosorbent assay in determining the amount of benzo(a)pyrene [B(a)P] metabolite covalently bound to guanine in DNA. In the USERIA approach, DNA either with or without B(a)P metabolite modification was adsorbed on the wells of microtiter plates, and rabbit antiserum to B(a)P metabolite-modified DNA [B(a)P-DNA] was then added. Antibodies reacted specifically with the B(a)P-DNA attached to the surface of the plate. After reaction between goat anti-rabbit IgG conjugated to alkaline phosphatase and rabbit IgG bound to the solid phase, this specific antigen-antibody reaction was enzymatically amplified by alkaline phosphatase conversion of [3H]adenosine 5'-monophosphate to [3H]adenosine. Following chromatographic separation from [3H]adenosine5'-monophosphate, [3H]adenosine was measured by liquid scintillation counting. The amount of [3H]-adenosine formed was linearly related to the amount of B(a)P-DNA in 10 ng DNA attached to the solid phase. As little as 3 fmol of bound B(a)P metabolite can be detected by noncompetitive USERIA, while 10 fmol of the adducts in 25 microgram DNA [1 B(a)P-DNA adduct per 7 X 10(6) nucleotides] can be measured by the competitive USERIA approach. Under our standard competitive procedure with 1 microgram DNA in the antigen-antibody reaction mixture, USERIA is approximately 500-fold more sensitive than radioimmunoassay and 5-fold more sensitive than enzyme-linked immunosorbent assay for the detection of B(a)P-DNA adducts. These highly sensitive assays should be extremely useful for studies in DNA damage and repair by B(a)P metabolites as well as in studies on environmental exposure to this ubiquitous carcinogen.

Inhibitory effects of caffeic acid phenethyl ester on the activity and expression of cyclooxygenase-2 in human oral epithelial cells and in a rat model of inflammation.

We investigated the mechanisms by which caffeic acid phenethyl ester (CAPE), a phenolic antioxidant, inhibited the stimulation of prostaglandin (PG) synthesis in cultured human oral epithelial cells and in an animal model of acute inflammation. Treatment of cells with CAPE (2.5 microg/ml) suppressed phorbol ester (12-O-tetradecanoylphorbol-13-acetate; TPA) and calcium ionophore (A23187)-mediated induction of PGE2 synthesis. This relatively low concentration of CAPE did not affect amounts of cyclooxygenase (COX) enzymes. CAPE nonselectively inhibited the activities of baculovirus-expressed hCOX-1 and hCOX-2 enzymes. TPA- and A23187-stimulated release of arachidonic acid from membrane phospholipids was also suppressed by CAPE (4-8 microg/ml). Higher concentrations of CAPE (10-20 microg/ml) suppressed the induction of COX-2 mRNA and protein mediated by TPA. Transient transfections using human COX-2 promoter deletion constructs were performed; the effects of TPA and CAPE were localized to a 124-bp region of the COX-2 promoter. In the rat carrageenan air pouch model of inflammation, CAPE (10-100 mg/kg) caused dose-dependent suppression of PG synthesis. Amounts of COX-2 in the pouch were markedly suppressed by 100 mg/kg CAPE but were unaffected by indomethacin. These data are important for understanding the anticancer and anti-inflammatory properties of CAPE.

Resolution of plasma membrane lipid fluidity in intact cells labelled with diphenylhexatriene.

The partitioning of fluorescence probes into intracellular organelles poses a major problem when fluorescence methods are applied to evaluate the fluidity properties of cell plasma membranes with intact cells. This work describes a method for resolution of fluidity parameters of the plasma membrane in intact cells labelled with the fluorescence polarization probe 1,6-diphenyl-1,3,5-hexatriene (DPH). The method is based on selective quenching, by nonradiative energy transfer, of the fluorescence emitted from the plasma membrane after tagging the cell with a suitable membrane impermeable electron acceptor. Such selective quenching is obtained by chemical binding of 2,4,6-trinitrobenzene sulfonate (TNBS), or by incorporation of N-bixinoyl glucosamine (BGA) to DPH-labelled cells. The procedures for determination of lipid fluidity in plasma membranes of intact cells by this method are simple and straightforward.

Phospholipid fatty acid composition of pulmonary airway epithelial cells: potential substrates for oxygenation.

To determine possible substrates for airway epithelial lipoxygenase and cyclooxygenase activities, we examined the amounts and distributions of fatty acids in phospholipids of human, dog and sheep airway epithelial cells. We found that the cells contained significant levels of n-6 and n-3 fatty acids in species-specific amounts: dog cells were relatively enriched in the n-6 series and poor in n-3, while sheep cells were enriched in the n-3 series and poor in n-6. Despite differences in fatty acid content, cells from each species expressed a constant phospholipid composition and distributed their n-6 and n-3 fatty acids in a stereotyped fashion among phospholipid classes. The analysis shows that the species differences in oxygenation activities reported previously are paralleled by heterogeneity in potential substrates.

Overexpression, purification and characterization of human recombinant 15-lipoxygenase.

Human 15-lipoxygenase was expressed to high levels (approx. 20% of cellular protein) in a baculovirus/insect cell expression system. Catalytically active enzyme was readily purified (90-95% pure) from cytosolic fractions by anion-exchange chromatography on a Mono Q column with approx. 95% recovery of enzymatic activity. Routinely, a yield of 25-50 mg of pure enzyme per L of culture and a specific activity of 7.1-21 mumol 13-hydroxyoctadecadienoic acid (13-HODE)/mg.min (turnover rate of 8.4-25 s-1) were obtained. Both the specific activity and the enzyme's iron content was significantly increased by the addition of ferrous ions to either the purified enzyme or to the insect cell culture medium during production. An isoelectric point of 5.85 was determined and the N-terminal amino acid sequence was found to be identical to that predicted from the cDNA. The purified recombinant enzyme exhibits a dual positional specificity with arachidonic acid (formation of 15S- and 12S-hydroxyeicosatetraenoic acid (12S-HETE) in a ratio of 12:1). Double oxygenation products 14R,15S- and various 8,15-DiHETE isomers were also identified. With linoleic acid as substrate, a pH-optimum of 7.0 and a KM of 3 microM were determined. The enzyme undergoes suicidal inactivation during fatty acid oxygenation, is sensitive to standard lipoxygenase inhibitors, and oxygenates phospholipids, cholesterol esters, biomembranes and human low-density lipoprotein. Contrary to prior studies on the rabbit enzyme, no glycosylation was detected.

DNA base changes and RNA levels in N-acetoxy-2-acetylaminofluorene-induced dihydrofolate reductase mutants of Chinese hamster ovary cells.

Formerly, we isolated a series of dihydrofolate reductase-deficient Chinese hamster ovary cell mutants that were induced by N-acetoxy-2-acetylaminofluorene. Deletions and complex gene rearrangements were detected in 28% of these mutants; 72% contained putative point mutations. In the present study, we have localized the putative point mutations in the 25,000 base dhfr gene by RNase heteroduplex mapping. Assignment of a position for each mutation was successful in 16 of 19 mutants studied. We cloned DNA fragments containing the mapped mutations from nine mutants into a bacteriophage lambda vector. In the case of 11 other mutants, DNA was amplified by the polymerase chain reaction procedure. Sequence analysis of cloned and amplified DNA confirmed the presence of point mutations. Most mutants (90%) carried base substitutions; the rest contained frameshift mutations. Of the point mutations, 75% were G.C to T.A transversions in either the dhfr coding sequence or at splice sites; transition G.C to A.T mutations were found in two mutants (10%). In one of these transition mutants, the base substitution occurred at the fifth base of the third intron. Of the frameshift mutations, one was a deletion of G.C pair and the other was an insertion of an A.T pair. Of the mapped mutants, 38% exhibited greatly reduced (approximately 10-fold) steady-state levels of dhfr mRNA. All eight sequenced mutants displaying this phenotype contained premature chain termination codons. Normal levels of dhfr mRNA were observed in five missense mutants and in five mutants carrying nonsense codons in the translated portion of exon VI. Taken together with the results of other mutagens at this locus, we conclude that the low dhfr mRNA phenotype is correlated with the presence of nonsense codons in exons II to V but not in the last exon of the dhfr gene.

Hydroxylated aromatic inhibitors of HIV-1 integrase.

Efficient replication of HIV-1 requires integration of a DNA copy of the viral genome into a chromosome of the host cell. Integration is catalyzed by the viral integrase, and we have previously reported that phenolic moieties in compounds such as flavones, caffeic acid phenethyl ester (CAPE, 2), and curcumin confer inhibitory activity against HIV-1 integrase. We now extend these findings by performing a comprehensive structure-activity relationship using CAPE analogues. Approximately 30 compounds have been prepared as HIV integrase inhibitors based on the structural lead provided by CAPE, which has previously been shown to exhibit an IC50 value of 7 microM in our integration assay. These analogues were designed to examine specific features of the parent CAPE structure which may be important for activity. Among the features examined for their effects on inhibitory potency were ring substitution, side chain length and composition, and phenyl ring conformational orientation. In an assay which measured the combined effect of two sequential steps, dinucleotide cleavage and strand transfer, several analogues have IC50 values for 3'-processing and strand transfer lower than those of CAPE. Inhibition of strand transfer was assayed using both blunt-ended and "precleaved" DNA substrates. Disintegration using an integrase mutant lacking the N-terminal zinc finger and C-terminal DNA-binding domains was also inhibited by these analogues, suggesting that the binding site for these compounds resides in the central catalytic core. Several CAPE analogues were also tested for selective activity against transformed cells. Taken together, these results suggest that the development of novel antiviral agents for the treatment of acquired immune deficiency syndrome can be based upon inhibition of HIV-1 integrase.