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Biomedical subjects

D Grunberger

Publications and source records attributed to D Grunberger.

At least 55 records · Page 3Linked to original sources
PubMed1988

Arachidonate 15-lipoxygenase (omega-6 lipoxygenase) from human leukocytes. Purification and structural homology to other mammalian lipoxygenases.

The enzyme responsible for 15-lipoxygenation of arachidonic acid was purified to homogeneity from human eosinophil-enriched leukocytes using a combination of ammonium sulfate precipitation, hydrophobic interaction chromatography, and high pressure liquid chromatography on hydroxyapatite and cation-exchange columns. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified protein revealed a single major band (apparent Mr 70,000). Amino acid sequence analysis yielded a single N-terminal sequence. Comparison of the N-terminal 15 residues reveals 71% sequence identity to the rabbit reticulocyte lipoxygenase and 36% sequence identity to the rat basophilic leukemia 5-lipoxygenase. In contrast, sequence identity to the soybean lipoxygenase-1 is not observed. These results demonstrate that human 15-lipoxygenase can be isolated from eosinophil-enriched leukocytes and is accessible for direct sequence analysis. Furthermore, we present initial evidence that the mammalian lipoxygenases constitute an homologous family of enzymes. The availability of homogeneous human 15-lipoxygenase will play a key role in elucidating other relationships in this family of enzymes.

Amino Acid Sequence↗
PubMed1988

Preferential cytotoxicity on tumor cells by caffeic acid phenethyl ester isolated from propolis.

The honeybee hive product, propolis, is a folk medicine employed for treating various ailments. Many important pharmaceutical properties have been ascribed to propolis, including anti-inflammatory, antiviral, immunostimulatory and carcinostatic activities. Propolis extracts have provided an active component identified as caffeic acid phenethyl ester (CAPE), which was readily prepared in one step. Differential cytotoxicity has been observed in normal rat/human versus transformed rat/human melanoma and breast carcinoma cell lines in the presence of CAPE.

Animals↗
PubMed1988

Arachidonate 15-lipoxygenase from human eosinophil-enriched leukocytes: partial purification and properties.

Arachidonate 15-lipoxygenase was purified from human eosinophil-enriched leukocytes after showing that 15-lipoxygenase activity was 100-fold greater in eosinophils than in neutrophils. Partial purification was achieved using ammonium sulfate precipitation, cation-exchange and hydrophobic-interaction chromatography. New evidence is presented suggesting that 15-lipoxygenase has electrostatic and hydrophobic properties distinct from 5-lipoxygenase. In addition, ATP is shown to inhibit, and phosphatidylcholine is shown to stimulate, 15-lipoxygenase, suggesting a regulatory role for these compounds in the lipoxygenation of arachidonic acid.

Adenosine Triphosphate↗
PubMed1988

Mapping and characterization of mutations induced by benzo[a]pyrene diol epoxide at dihydrofolate reductase locus in CHO cells.

Chinese hamster ovary cells were mutagenized with benzo[a]pyrene diol epoxide (BPDE), an aromatic hydrocarbon carcinogen, and mutants at the dihydrofolate reductase (dhfr) locus were isolated. Of 15 mutants analyzed by Southern blotting, one contained a large deletion that spanned all six exons of the 25-kb dhfr gene; the remaining mutants exhibited no detectable changes. Three of these putative point mutations were localized by the loss of a restriction site: a SacI site in exon III, an MspI site in exon III, and a KpnI site in exon VI. The affected regions in two of these mutants were cloned and sequenced. The SacI- mutant was caused by a G:C----T:A transversion resulting in an amber termination codon. In the MspI- mutant, the deletion of a single C:G resulted in a frameshift and a downstream ochre termination codon. On the basis of overlapping restriction site sequences, the KpnI- mutant was deduced to be a splicing mutant involving the most 3' G in intron V. The location of these and the remaining 11 putative point mutations was sought using RNA heteroduplex mapping. Mismatched bases between riboprobes complementary to wild-type dhfr mRNA and mutant mRNA molecules were detected in 10 of the 14 mutants analyzed. These mutations mapped to four of the six exons or exon splice sites. Surprisingly, over half of these mutants exhibited greatly reduced (approximately 10-fold) steady-state levels of dhfr mRNA.

Animals↗
PubMed1987

Circular dichroism of poly(dG-dC) modified by the carcinogens N-methyl-4-aminoazobenzene or 4-aminobiphenyl.

Poly(dG-dC) was modified to different extents by the carcinogens 4-aminobiphenyl (ABP) or N-methyl-4-aminoazobenzene (MAB). HPLC analysis of the enzymatically hydrolyzed modified polymers indicates that more than 90% of the ABP and 81% of the MAB modification occurs at the C8 position of guanine. The conformational changes of the unmodified and modified polymers were studied as a function of ethanol and magnesium ion concentrations by the use of circular dichroism (CD). The modified polymers show a CD inversion pattern similar to that of the salt-induced B to Z transition of poly(dG-dC). Both of the modified polymers require less salt or ethanol than the unmodified polymer for the inversion of the spectra. The amount of ethanol or magnesium needed to induce the inverted CD spectrum is inversely proportional to the percentage of bound ABP or MAB. These data indicate that ABP and MAB can enhance conversion from B to Z conformation in alternating purine-pyrimidine sequences.

Aminobiphenyl Compounds↗
PubMed1987

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.

Animals↗
PubMed1987

Properties of covalent benzo[a]pyrene diol epoxide-DNA adducts investigated by fluorescence techniques.

The spectroscopic absorption and fluorescence properties of adducts derived from the covalent binding of (+/-)trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyre ne (BPDE) to DNA are re-examined in view of conflicting interpretations regarding the conformations of these adducts which currently exist in the literature. The fluorescence decay profiles were accurately determined utilizing synchrotron-pulsed light source excitation and the time-correlated single photon counting technique. The conformational properties of the adducts were probed by determining their accessibilities to acrylamide, a known fluorescence quencher, and by comparing the accessibilities of the BPDE-DNA adducts with those of known model systems with intercalative, partially intercalative and minor groove binding conformations. In contrast to any of these model systems, the fluorescence of the aromatic pyrenyl residues in the covalent BPDE-DNA adducts exhibit significant sensitivity to acrylamide, suggesting that these residues are located at binding sites with significant solvent exposure. A quantitative analysis of the acrylamide fluorescence quenching according to a dynamic Stern-Volmer quenching model suggests the following characteristics: the major (65%) component (1.4 ns lifetime) is characterized by significant exposure to the solvent environment; the second component (6-7 ns lifetime) can be subdivided into a solvent-accessible and a solvent-inaccessible component, the inaccessible fraction being attributed to minor adducts, possibly with a quasi-intercalative conformation. The amplitude of the third, long-lived (200-ns) component is variable; it arises from the photochemical decomposition of the adducts which gives rise to tetraols (7,8,9,10-tetrahydro-tetrahydroxybenzo[a]pyrene). The variable content of these degradation products accounts for most discrepancies in the fluorescence properties of the covalent BPDE-DNA adducts previously reported.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1986

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.

Animals↗
PubMed1986

Enhanced expression of the bacterial chloramphenicol acetyltransferase gene in mouse cells cotransfected with synthetic polynucleotides able to form Z-DNA.

Recent studies have demonstrated that the left-handed, Z-DNA conformation is favored in polymers containing alternating purine/pyrimidine sequences that can exist in vivo and may play a role in gene expression. On the basis of this assumption, we have studied the effect of various cotransfected polynucleotides on the transient expression of the chloramphenicol acetyltransferase (CAT) gene in thymidine kinase-deficient murine L cells. Cotransfections were performed by calcium phosphate coprecipitation of CAT gene plasmids with various polymers, and the CAT enzymatic activity was measured in cell lysates after 48 hr. About 2- to 10-fold stimulation of CAT gene expression was observed when the cells were cotransfected with 10 micrograms (per 10-cm culture dish) of plasmid pSV2cat, which contains simian virus 40 (SV40) promoter and enhancer sequences, and 2-10 micrograms of polymers that can form Z-DNA, such as poly(dG-m5dC) X poly(dG-m5dC) or poly(dG-dC) X poly(dG-dC), as compared to transfection with pSV2cat alone. Further, enhanced CAT gene expression was also observed when cotransfections were performed with these polymers and two other plasmid vectors, one containing the SV40 promoter but no enhancer and the other lacking any SV40 regulatory sequences. However, poly(dA-dC) X poly(dG-dT), which can form Z-DNA, did not induce any stimulation. Similarly, no or very little stimulation was observed after cotransfection of pSV2cat with either poly(dG) X poly(dC) or poly(dA-dT) X poly(dA-dT), which do not adopt the Z conformation. These results suggest that certain polynucleotides may enhance transcription of the CAT gene.

Acetyltransferases↗
PubMed1986

Characterization of mutations induced by 2-(N-acetoxy-N-acetyl)aminofluorene in the dihydrofolate reductase gene of cultured hamster cells.

To determine the types of alterations in gene structure that are induced by the carcinogen 2-(N-acetoxy-N-acetyl)aminofluorene, we used this compound to generate mutations at the dihydrofolate reductase (DHFR) locus (DHFR) in Chinese hamster ovary cells. Twenty-nine independent enzyme-deficient mutants were isolated. A profile of the 26-kilobase (kb)-long gene was obtained by Southern blot analysis of the mutant and parental DNAs digested with BstEII/Kpn I. Hybridization to a mixed probe of 10 DHFR genomic and cDNA fragments revealed 12 bands that scan 34 kb. Twenty-one DHFR- clones (72%) contained small mutations (changes less than 100 base pairs in size). Large or small deletions involving various parts of the gene occurred in eight of the mutants (28%). A large deletion (greater than 35 kb) with 5' and 3' breakpoints mapping to approximately the same location was noted in four mutants. One mutant has undergone a deletion of 550-900 bp that eliminated the first coding exon. Concomitantly, a chromosomal event (either translocation, insertion, or inversion) has separated the 5' flank from the body of the gene. In another mutant, four deletions have occurred at the DHFR 5' end and internally. Restriction fragment length polymorphism analysis of the mutant DNAs with exon-specific probes localized three mutations. One mutant has lost a Taq I (TCGA) site, and another has lost a Sac I (GAGCTC) site. In a third, a GC----TA transversion has created a BstEII (GGTNACC) site. Finally, we used HPLC to determine the ratio of acetylated (12%) to deacetylated (88%) 2-aminofluorene adducts formed in the parental cells. A correlation between the mutational specificities and the conformational changes induced by the two types of DNA adducts is discussed.

2-Acetylaminofluorene↗
PubMed1985

Inhibition of the herpes simplex virus thymidine kinase gene transfection in Ltk- cells by potential Z-DNA forming polymers.

It has been demonstrated that certain alternating purine and pyrimidine sequences may assume a left-handed Z-DNA conformation. In order to evaluate the possibility that Z-DNA is involved in the modulation of gene expression, we examined the ability of various synthetic DNA polymers to affect the transfection of herpes simplex virus thymidine kinase (HSVtk) gene in Ltk- cells using the DNA-calcium phosphate cotransfection technique. We found that potential Z-DNA forming polymers such as, poly(dG-m5dC) X poly(dG-m5dC) and poly(dG-dC) X poly(dG-dC), cotransfected with the tk gene decreased the level of Tk+ transformed colonies. In contrast, cotransfection of the tk gene with polymers which do not assume Z-conformation such as, poly(dG) X poly(dC) or poly(dA-dT) X poly(dA-dT) showed no effect on the number of colonies formed. About 50% inhibition of the Tk+ colony formation was obtained by 0.4 micrograms of poly(dG-m5dC) X poly(dG-m5dC), or by 2 micrograms of poly(dG-dC) X poly(dG-dC). DNA uptake into Ltk- cells was not significantly affected by any of these polymers. Approximately 20-42 base pairs (bp) long alternating dG-dC sequence linked at either the 5'-end or 3'-end of tk gene were cloned into plasmids. These recombinant plasmids, however, showed no remarkable effect upon the transfection of Ltk- cells. The DNAs of Tk+ colonies obtained by transfecting these recombinant plasmids were digested with BssH II and analyzed by Southern blotting. We demonstrated that the dG-dC sequences proximal to the tk gene were integrated into cellular DNA. All the presented results indicate that only larger polymers with the potential to assume a Z-DNA conformation may affect tk gene transfection either by inhibiting transcription or more probably by affecting the stable integration of the tk gene into the host chromosome.

Animals↗
PubMed1984

An immunochemical examination of acetylaminofluorene-modified poly(dG-dC) X poly(dG-dC) in the Z-conformation.

Immunization of rabbits with a complex of methylated bovine serum albumin and N-2-acetylaminofluorene (AAF)-modified poly(dG-dC) X poly(dG-dC), a polynucleotide that can assume the Z-DNA conformation, yielded several populations of antibodies specific for Z-DNA determinants. The Z-DNA determinants were analyzed by examination of the antisera and of antibody preparations purified on immunoadsorbents. The following was found: AAF-poly(dG-dC) X poly(dG-dC) shared Z-DNA determinants in common with poly(dG-dC) X poly(dG-dC) in 3.0 M NaCl, poly(dG-m5dC) X poly(dG-m5dC) in 1.5 M NaCl, and brominated poly(dG-dC) X poly(dG-dC) in 0.2, 1.5, and 3.0 M NaCl. Included among the antibodies induced by these determinants was a subpopulation whose reaction with brominated poly(dG-dC) X poly(dG-dC) was sensitive to increased ionic strength. Another distinct population of antibodies recognized determinants present on AAF-poly(dG-dC) X poly(dG-dC) but not on the other Z-DNAs. Only a small portion of this population was specific for the AAF moiety; the greater part appeared to recognize Z-DNA-associated conformational characteristics that were unique to AAF-poly(dG-dC) X poly(dG-dC). These findings are consistent with the existence of a continuum of Z-DNA determinants, which might be capable of functioning as recognition signals for regulatory DNA-binding proteins.

2-Acetylaminofluorene↗
PubMed1984

Impaired enzymatic methylation of BPDE-modified DNA.

Highly purified DNA methyltransferase from human placenta methylates hemimethylated and 5-methylcytosine-free DNA substrates suggesting that one enzyme molecule may exercise both, the maintenance and de novo activities. Modification of these methyl accepting polymers with (+/-)-r-7,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene, anti (BPDE) interferes with the methylation reaction, and this inhibition is proportional to the degree of BPDE-modification. This indicates that BPDE - DNA adducts affect both the maintenance and the de novo DNA methyltransferase activities. The mechanism responsible for such inhibition is related neither to interference of BPDE - DNA adducts with the initial binding of the enzyme to DNA nor with the processive mode of action of the enzyme on the modified DNA template. More likely, the BPDE - DNA adducts inhibit the transmethylation reaction directly at the sites of modification.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1983

Molecular cloning, sequence analysis and in vitro expression of a rat tRNA gene cluster.

A rat genomic DNA fragment containing a tRNA gene cluster was isolated from a lambda phage library. Hybridization and nucleotide sequence analysis revealed the presence of a 83 bp tRNALeuCUG gene and a 72 bp tRNAAspGUG gene. Both genes possessed intact coding regions and putative transcription termination signals at their respective 3' ends. In vitro transcription analysis of the two subcloned genes in a HeLa cell S-100 system demonstrated the specific synthesis of a number of RNAs by RNA polymerase III. Studies carried out in the presence of alpha-amanitin showed that the larger RNAs are precursors for the final processed transcripts of the tRNALeu and tRNAAsp genes, respectively. Further nucleotide sequence analysis of the cluster revealed the presence of tRNAGly and a tRNAGlu pseudogenes with missing areas within their coding regions which are essential for transcription by RNA polymerase III. Within the region of DNA between the tRNALeu and tRNAAsp genes is a sequence which is 65% homologous to a region of the rat B1 element. The significance of this latter structure within the gene cluster is unknown.

Animals↗
PubMed1983

Alterations in post-transcriptional modification of the Y base in phenylalanine tRNA from tumor cells.

Various tumor cells contain chromatographically distinct isoacceptor tRNA species. To decide whether the tumor-specific species represent an expression of a separate tRNA gene or only an undermodified form of normal tRNAPhe, nucleotide sequences of tRNAPhe isolated from neuroblastoma and normal mouse liver were determined by postlabeling techniques. The results showed identical sequences except for the changes of post-transcriptional modifications in the anticodon loop. Normal mouse liver tRNAPhe contained Cm32, Gm34, and the hypermodified YOH next to the 3' end of the anticodon. On the contrary, tRNAPhe from neuroblastoma contained C32, G34, and, instead of YOH base m1G. A small proportion of tRNAPhe species contained an undermodified YOH base. For the examination of the conditions leading to the undermodified tRNAPhe, Vero cells derived from the kidney of African green monkey in culture were used. In these cells, deprivation of methionine or lysine resulted in changes in tRNAPhe modification similar to those in tumor cells. Ehrlich ascites tumor cells were examined to determine whether the presence of altered tRNAPhe species in various tumors is also the result of starvation of some nutritional factors. Results obtained with these cells showed that tRNAPhe species lacking the Y base disappeared in tumor-bearing mice after intraperitoneal injection with a mixture of amino acids and vitamins. Thus it is concluded that tumor-specific tRNAPhe species are the products of aberrant post-transcriptional modification, not the transcripts of different, normally repressed genes.

Animals↗
PubMed1983

The correlation between benzo[a]pyrene-induced mutagenicity and DNA adduct formation in Salmonella typhimurium TA100.

In an attempt to stabilize the dose response in the Salmonella typhimurium test (STT), the use of DNA-bound products from BP was evaluated as a measure of the biologically effective dose. In addition to the previously documented interlaboratory variation, we observed a 3-fold difference in the dose response of TA100 to BP even when the assay was repeated with the same experimental conditions. When overall BP-DNA adduct formation was related to the level of His+ revertants, a series of responses emerged with two predominating. In the first type of response around 70 revertants per plate were generated for every BP molecule bound per 10(6) nucleotides of cellular DNA. The second response gave about 1400 revertants per plate for one BP bound in every 10(6) nucleotides. Several intermediates curves were also detected. The variation in the mutational response to binding levels occurred regardless of the source of S9 or the growth stage of the cells. These experiments indicate that there was no constant level of DNA damage that would lead to a specified number of revertants of TA100 by BP and that DNA modification was not solely responsible for mutagenic potency. It is possible that an induction of an error-prone repair function of the muc gene carried by the plasmid pKM101 in TA100 may be affecting the relationship between the measured adduct level and reversion frequency.

Benzopyrenes↗
PubMed1983

Cell specific activation of benzo[a]pyrene by fibroblasts and hepatocytes.

The cell specific activation of benzo[a]pyrene (BP) by embryonic fibroblasts and by mature hepatocytes to intermediates that can interact with DNA, or cause mutations in Chinese hamster V79 cells has been investigated. At BP concentrations of up to 15 muM, BP was activated to mutagenic intermediates for the V79 cells by embryonic fibroblasts but not by hepatocytes. However, hepatocytes from rats that had been pretreated with an inducer of the mixed function oxidases, 3-methylcholanthrene, did metabolize higher doses of BP (greater than 15 muM) to mutagenic intermediates. BP was extensively metabolized by both cell types, but the hepatocytes and fibroblasts showed differences both in the profiles of BP metabolites and the nature of the BP-DNA adducts formed. Hepatocytes metabolized BP principally to 4,5-dihydro-4,5-dihydroxybenzo[a]pyrene, phenols, and quinones, which underwent further metabolism to water-soluble metabolites. Metabolism of BP to 7,8-dihydro-7,8-dihydroxybenzo[a]-pyrene (BP-7,8-diol) occurred but proceeded rapidly to the formation of triols and tetraols. Fibroblasts metabolized BP predominantly toward the formation of BP-7,8-diol. The proportion of primary metabolites undergoing further metabolism to conjugates was less extensive than in the hepatocytes. Hepatocytes bound more BP to their DNA than the fibroblasts. In the hepatocytes the major DNA adducts formed were hydrophilic derivatives, and no [+/-]7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) adducts were detected even after treatment with BP-7,8-diol. In the fibroblasts, the major BP--DNA adduct was derived from the reaction of BPDE with deoxyguanosine. These results suggest that the differences in the response of embryonic fibroblasts and mature hepatocytes in the activation of BP to a mutagen for mammalian cells is determined at least in part by the overall balance of oxidation and detoxification processes in the cells and, hence, by the levels of critical oxidative intermediates that interact with DNA.

Animals↗