PubMed Health⌕ Search

Biomedical subjects

F L Yu

Publications and source records attributed to F L Yu.

At least 19 recordsLinked to original sources
PubMed1999

The effect of 17beta-estradiol-DNA adducts on the replication of exon # 5 of the human suppressor gene p53.

Using a PCR technique, exon # 5 of the human tumor suppressor gene p53 was amplified and ligated into the pCRII vector and transformed into Escherichia coli INV alphaF' competent cells. The cloned exon # 5 was 184 bp long. Evidence is presented to show that after dimethyldioxirane epoxidation, 17beta-estradiol was able to form 17beta-estradiol-DNA adducts and to strongly inhibit the replication of the cloned exon # 5 producing smaller sizes of DNA fragments and introducing errors of incorporation at the 3'-end of the terminating DNAs. The errors occurred mainly at the clusters of the complementary 'G' and 'A' bases on the template strand DNA, presumably, the major sites where the 17beta-estradiol-DNA adducts were formed.

Base Sequence↗
PubMed1998

Evidence for the DNA binding and adduct formation of estrone and 17beta-estradiol after dimethyldioxirane activation.

Estrogens, used widely from hormone replacement therapy to cancer treatment, are themselves carcinogenic, causing uterine and breast cancers. However, the mechanism of their carcinogenic action is still not known. Recently, we found that estrone (E1) and 17beta-estradiol (E2) could be activated by the versatile epoxide-forming oxidant dimethyldioxirane (DMDO), resulting in the inhibition of rat liver nuclear and nucleolar RNA synthesis in a dose-dependent manner in vitro. Since epoxidation is often required for the activation of chemical carcinogens, we proposed that estrogen epoxidation is the underlying mechanism for the initiation of estrogen carcinogenesis (Carcinogenesis 17 (1996) 1957-1961). It is known that initiation requires the binding of a carcinogen to DNA with the formation of DNA adducts. One of the critical tests of our hypothesis is therefore to determine whether E1 and E2 after activation are able to bind DNA. This paper reports that after DMDO activation, [3H]E1 and [3H]E2 were able to bind to both A-T and G-C containing DNAs. Furthermore. the formation of E1-DNA and E2-DNA adducts was detected by 32P-postlabeling analysis.

Animals↗
PubMed1998

The transcriptional effects and DNA-binding specificities of 17beta-estradiol after dimethyldioxirane activation.

It was found recently that 17beta-estradiol (E2) could be activated by the epoxide-forming oxidant dimethyldioxirane (DMDO) resulting in the inhibition of rat liver nuclear and nucleolar RNA synthesis in vitro (Carcinogenesis, 17, 1957-1961, 1996). To further study the mechanism of this inhibition, several synthetic DNAs with different base content and sequence were used to study the transcriptional effects and binding specificities of E2 after DMDO activation in vitro. The results show: (1) E2 after activation had a strong inhibitory effect on the template function of both A-T and G-C containing double-stranded DNAs, e.g. poly[d(A-T)], polydG x polydC and poly[d(I-C)], and only a weak inhibition on the single-stranded DNA template, polydC. The inhibition was dose-dependent, and only after DMDO activation. (2) 3H-labeled E2, after DMDO activation, was able to bind DNAs containing both A-T and G-C bases. The order of the binding preference was: calf thymus DNA > poly[d(A-T)] > poly[d(G-C)]. (3) The covalent binding nature of E2 to DNA after activation was further confirmed by 32P-post-labeling analysis using calf thymus DNA. (4) The absorption spectrum of E2 changed, after DMDO treatment, from a peak around 280-290 nm to 260-270 nm with a shoulder appearing around 300-320 nm. These studies have not only confirmed our earlier observation that E2, after DMDO activation, can inhibit DNA-dependent RNA synthesis, but also provided new insights into the DNA-binding properties after activation. Additionally, since epoxidation is often required for the activation of chemical carcinogens to bind DNA, these studies lend further support to our proposed hypothesis that E2 epoxidation may play an initiation role in estrogen carcinogenesis.

Animals↗
PubMed1996

Effect of aflatoxin B1-8,9-epoxide-DNA adducts on transcription of a supF gene fragment.

A linearized template, obtained from the vector pGEM-3Zf(+) containing a supF gene fragment, was treated with aflatoxin B1-8,9-epoxide (AFB1 epoxide) and transcription in vitro was then studied. The template functions of both strands of the supF gene were similarly inhibited as shown by transcription with both T7 and SP6 RNA polymerases. This inhibition was dose-dependent and affected the elongation step more extensively than the initiation step. Gel electrophoretic analysis of RNA formed by T7 RNA polymerase indicated that template treated with different AFB1 epoxide doses yielded the same three major truncated RNA fragments. Sequence analysis showed that these major sites of RNA truncation occurred in the vicinity of adjacent guanine residues in the template.

Aflatoxin B1↗
PubMed1996

Activation of 17beta-estradiol and estrone by dimethyldioxirane and inhibition of rat liver nuclear and nucleolar RNA synthesis in vitro.

17beta-estradiol (E2), estrone and diethylstilbestrol (DES) had no effect on nuclear and nucleolar RNA synthesis in vitro. However, after reacting with dimethyldioxirane (DMDO), a versatile epoxide-forming oxidant, these estrogens were able to inhibit and in a dose-dependent manner nuclear and nucleolar RNA synthesis in vitro. It was also found that the time required for the maximal activation of these chemicals by DMDO varied: estrone, 10 min; E2, 30 min; DES, 60 min. Tamoxifen (TAM) was also able to inhibit nuclear and nucleolar RNA synthesis in a dose-dependent manner, but the mechanism of this inhibition was more complex. Control experiments clearly indicated, unlike E2, estrone and DES, TAM per se was able to directly inhibit RNA synthesis in vitro. TAM after activation by DMDO was able to further inhibit RNA synthesis contributing part of the total observed inhibition. These data show for the first time that E2, estrone, DES and TAM can be activated by DMDO and possibly to epoxides. We propose that epoxidation of E2 and estrone may be the underlying mechanism of carcinogenesis for these estrogens in vivo.

Animals↗
PubMed1996

[Effects of AF64A on neurons containing both nitric oxide synthase and choline acetyltransferase in the rat septal complex].

Ethylcholine mustard aziridinium ion (AF64A), a neurotoxic choline analog, was injected (ICV) bilaterally (1.5 nmol/ventricle, n = 10) into male adult rats to induce a model of Alzheimer's disease (AD). One month later, using NADPH-diaphorase (NADPH-d) histochemistry followed by choline acetyltransferase (ChAT) immunocytochemistry (PAP) on the coronal sections of the septal complex, double-staining experiments were performed to assay the alterations of septal cholinergic neurons coexisted with nitric oxide synthase (NOS). Compared to controls, AF64A can significantly reduce the numbers of ChAT single labelled neurons and NADPH-d + ChAT double labelled neurons in the dorsal subgroup (29.5% and 26.7%, respectively, P < 0.01). Moreover, the dendrites of these neurons were damaged. While administration of AF64A resulted in a significant decrease in the number of ChAT single labelled neurons (35.2%, P < 0.01) in the intermediate subgroup (rostral extension of the nucleus/substantia innominata) NADPH-d + ChAT double labelled neurons were unchanged (P > 0.05). In the midline and the ventral subgroups, both of these two kinds of cholinergic neurons were not affected significantly by AF64A (P > 0.05). Furthermore, AF64A had no effect on NADPH-diaphorase single labelled neurons in all subgroups of septal complex. These results indicate that: (1) the administration of AF64A has different effects on the cholinergic neurons with or without NOS in different subgroups of the septal complex, and the NADPH-d + ChAT double labelled neurons resist the neurotoxicity of AF64A; (2) in the intermediate subgroup, the cholinergic neurons containing NOS may have projections different from those without NOS.

Alzheimer Disease↗
PubMed1995

Studies on the isolated transcriptionally active and inactive chromatin fractions from rat liver nuclei.

Using mild sonication, nucleoplasmic, nucleolar, and subnucleolar P-3 and S-3 chromatin fractions are isolated from rat liver nuclei. These fractions differ widely (over 80-fold) from each other in transcriptional activity as measured by the chromatin bound engaged RNA polymerases. Chemical analyses indicate that the active chromatin, e.g. P-3 and nucleolar fractions, are rich in RNA and protein as compared to the inactive chromatin, e.g. nucleoplasmic, and S-3 fractions. However, the DNA base content are all the same, showing 40% GC and 60% AT, including P-3 which is enriched in rDNA. Polyacrylamide gel electrophoresis of the 0.25 N HCl extracted proteins shows that all five histones are present in active chromatin. Additionally, the gel reveals two protein bands, one ahead of histone H2B and another ahead of histone H4, that are diminished or missing from the inactive chromatin. On the other hand, there is a fast moving protein band ahead of H4 in the inactive chromatin that is almost absent in the active chromatin. Transcriptional tests using E. coli RNA polymerase and several synthetic DNA templates of known base content and sequence indicate that the 0.25 N HCl soluble protein extracts from active chromatin contain activator proteins which are capable of countering the histone suppressors present in the extracts in a DNA base and sequence specific manner. The data show that although the histone suppressors are able to strongly inhibit the template function of poly[d(A-T)], the protein activators are able to overcome the suppressor activity and stimulate RNA synthesis several-fold when poly(dA).poly(dT) or poly(dT) is used.

Animals↗
PubMed1994

Studies on the binding and transcriptional properties of aflatoxin B1-8,9-epoxide.

[3H]Aflatoxin B1-8,9-epoxide ([3H]AFB1-8,9-epoxide), the putative ultimate carcinogen of AFB1, was synthesized and tested for its binding specificity to and transcriptional effect on several single- and double-stranded DNAs containing cytosine. The test was carried out over a 200-fold concentration range (i.e. 0.1-20 microgram [3H]AFB1-8,9-epoxide per 0.025 A260 units of DNA). The results show: (i) [3H]AFB1-8,9-epoxide bound preferentially to the double-stranded alternating co-polymer poly[d(G-C)] over the double-stranded poly(dG).poly(dC) and single-stranded poly(dG) or poly(dC) homopolymers. (ii) The binding affinity of [3H]AFB1-8,9-epoxide to poly(dC) was essentially the same as the poly(dG). (iii) Under identical conditions, [3H]AFB1-8,9-epoxide bound to poly(dG).poly(dC) 2.5-3 times more than to poly[d(I-C)]; however, poly[d(I-C)]-directed RNA synthesis was clearly more sensitive to [3H]AFB1-8,9-epoxide inhibition than poly(dG).poly(dC). Conversely, the binding affinity of [3H]AFB1-8,9-epoxide to poly(dC) and to poly[d(I-C)] was quite similar, yet poly(dC)-directed RNA synthesis was much more resistant to [3H]AFB1-8,9-epoxide inhibition than poly[d(I-C)]. (iv) After [3H]AFB1-8,9-epoxide was hydrolyzed to [3H]AFB1-8,9-dihydrodiol (0.01 N NaOH, 10 min 23 degrees C), it was no longer able to bind poly[d(G-C)] or to inhibit poly[d(G-C)]-directed RNA synthesis. These results confirm our earlier studies using microsome-activated AFB1 and AFB1-Cl2 that AFB1 after activation is able to bind cytosine in DNA, and the binding is not via AFB1-8,9-dihydrodiol. Furthermore, the results also suggest that AFB1 adducts may not have the same biological effect depending on the base, sequence as well as the conformation of the DNA where the adducts are formed.

Aflatoxin B1↗
PubMed1993

A hypothesis for chemical carcinogen induced chromatin condensation.

A hypothesis for chemical carcinogen induced condensation of nuclear and nucleolar chromatin is proposed. Chromatin condensation is believed to be a result of cascade effects initiated by the inhibition of messenger RNA synthesis after exposure to chemical carcinogen in vivo. Inhibition of messenger RNA synthesis leads to the loss of protein production, which in turn causes the dephosphorylation of histone H1 and triggers the condensation of chromatin in vivo.

Amanitins↗
PubMed1993

Transcriptional specificities of adriamycin.

The transcriptional effect of adriamycin using E. coli RNA polymerase on several single- and double-stranded DNAs of known base content and sequence is studied in vitro. The results show that adriamycin inhibits strongly and with little difference toward both poly[d(A-T)] and poly[d(G-C)] templates, and that it inhibits both single- and double-stranded DNA directed RNA synthesis, albeit the inhibition is clearly preferential to the double-stranded alternating copolymers over the double- and single-stranded homopolymers. Since adriamycin inhibition of RNA synthesis can be totally abolished when assayed in excess amount of DNA, the possibility that adriamycin may also directly inhibit the enzyme RNA polymerase per se is ruled out.

DNA-Directed RNA Polymerases↗
PubMed1991

Transcriptional effect of aflatoxin B1 on cytosine and/or hypoxanthine containing DNAs.

The effect of aflatoxin B1 (AFB1) on the template function for RNA synthesis of several single and double-stranded synthetic DNAs containing cytosine (C) and/or hypoxanthine (H) bases is studied in vitro. The results indicate that AFB1, after liver microsome activation, strongly inhibits the template function of poly[d(I-C)] and has little, if any, effect on polydI.polydC, polydI, or polydC. This conclusion is reached whether rat liver nuclear free RNA polymerase or E. coli RNA polymerase is used for the transcription. The mechanism of this inhibition is believed mainly due to the inhibition of elongation of RNA synthesis, because autoradiography of the [alpha-32 P]GTP labeled RNAs after polyacrylamide gel electrophoresis clearly shows that the size of the RNA from AFB1 treated group is dramatically reduced. The evidence that the selective inhibition of poly[d(I-C)] template function is a direct reflection of the binding of AFB1 to poly[d(I-C)] is provided by the use of radioactive [3H]AFB1 for the binding and by spectrum analysis of the appearance of a broad AFB1-DNA adduct peak between 300 nm and 400 nm right after the typical DNA peak at 260 nm. These data, which are in direct support to our recent report (F.L. Yu, et al., Carcinogenesis, 11, 475-478, 1990), suggest that the binding of AFB1 prefers alternating, double-stranded DNA, and the binding affinity of AFB1 to DNA is greatly reduced when the bases are in either single- or double-stranded homopolymer forms.(ABSTRACT TRUNCATED AT 250 WORDS)

Aflatoxin B1↗
PubMed1991

Evidence for the covalent binding of aflatoxin B1-dichloride to cytosine in DNA.

In vitro studies of the effect of aflatoxin B1-dichloride (AFB1-Cl2) on the template function for RNA synthesis of several single- and double-stranded synthetic DNAs containing cytosine and/or hypoxanthine bases are reported. The results indicate: (i) AFB1-Cl2 strongly inhibits the template function of the single-stranded homopolymer polydC and has no effect on polydI, (ii) the inhibition is stronger when cytosine is in the double-stranded alternating copolymer poly[d(I-C)], and (iii) polydI directed RNA synthesis can be inhibited if it is in the double-stranded homopolymer polydI.polydC, although the template function of the polydC strand is still inhibited to a greater extent. The evidence that the selective inhibition of the DNA template function is a direct reflection of the binding specificities of AFB1-Cl2 is provided by the binding studies of [3H]AFB1-Cl2 to these DNAs. The binding of AFB1-Cl2 to polydC is substantiated by the dose-response template inhibition and by the dose-response template binding studies. Additionally, these results show that AFB1 per se has neither inhibitory nor binding activity. Auto radiography of [alpha-32P]GTP labeled RNAs suggests that the mechanism of inhibition of polydC template function by AFB1-Cl2 is mainly due to the inhibition of the elongation of RNA synthesis. Spectrum measurement of the products of enzyme digestion of the AFB1-Cl2 modified polydC reveals that the deoxycytidine fraction gives a typical cytosine absorption peak at 275 nm followed by a broad peak between 300 and 400 nm with a maximum at 390 nm. High performance liquid chromatography confirms the existence of a cytosine-AFB1 adduct which absorbs strongly in the regions between 250 and 400 nm with peaks identifiable at 260, 350 and 390 nm. These results strongly suggest that AFB1 in the activated form of AFB1-Cl2 is able to covalently bind to cytosine in DNA.

Aflatoxin B1↗
PubMed1990

Base and sequence specificities of aflatoxin B1 binding to single- and double-stranded DNAs.

The inhibitory effect of alfatoxin B1 (AFB1) on the template function for RNA synthesis of several single- and double-stranded DNAs with known base content and sequence was studied in vitro. The results showed that AFB1 strongly inhibits the template function of poly[d(G-C)] and has little, if any, effect on poly[d(A-T)]. Using [3H]AFB1 for the binding, and by spectrum analysis of the appearance of a broad AFB1-DNA adduct peak between 300 and 400 nm right after the typical DNA peak at 260 nm, it is possible to conclude that the binding preference of AFB1 to DNA is: poly[d(G-C)] greater than polydG.polydC greater than polydG greater than polydC, with no detectable binding to poly[d(A-T)]. These studies have therefore provided evidence that the selective inhibition of DNA template function is a direct reflection of the binding specificities of AFB1 to DNA. Furthermore, since there is a 3-fold binding preference of AFB1 for poly[d(G-C)] over polydG.polydC on an equal weight basis, and with very low binding affinity toward either G or C when it is in single-stranded form, these data also suggest: (i) AFB1 binds preferentially to DNA with an alternating G-C sequence compared to DNA with a sequence of contiguous Gs or Cs; and (ii) intercalation may be part of the mechanism for the binding of AFB1 to DNA.

Aflatoxin B1↗
PubMed1990

Actinomycin D binding in vitro: active chromatin preferred.

When [3H] Actinomycin D (Act. D) is used to interact with nuclei and nucleoli in vitro, it binds preferentially to nucleolar chromatin. The preferential binding is no longer detectable, when purified nuclear and nucleolar DNAs are used. In parallel, Act. D preferentially inhibits nucleolar over nuclear RNA synthesis when chromatin templates are used, and the preferential inhibition is lost when purified nuclear and nucleolar DNAs are used. It is concluded: 1) the preferential inhibition of nucleolar over nuclear RNA synthesis by Act. D is a direct reflection of the preferential binding of Act. D to the nucleolar chromatin; and 2) the nucleolar chromosomal proteins, not the nucleolar DNA, confer the preferential binding of Act. D.

Animals↗
PubMed1989

Template specificities of aclacinomycin B on the inhibition of DNA-dependent RNA synthesis in vitro.

The effect of Aclacinomycin B (ACM-B), an anthracycline antitumor antibiotic, on the DNA-dependent RNA synthesis using single- and double-stranded DNAs of known base content and sequence is studied. The data show that ACM-B effectively inhibits the double-stranded DNA-directed RNA synthesis with a preference of poly[d(A-T)] greater than poly[d(G-C)] greater than poly[d(I-C)]. In contrast, it has no inhibitory effect on the template function of single-stranded DNA (e.g. poly dA, poly dT, and poly dC). These results suggest that the mechanism of ACM-B inhibition, like other anthracycline antibiotics, is by intercalation. In addition to the base specificity, there are also dramatic differences in inhibition depending on the base sequence in the DNA template. Thus, ACM-B preferentially inhibits the alternating double-stranded copolymers over the double-stranded homopolymers; e.g. poly[d(A-T)] is inhibited to a greater extent than poly dA.poly dT and poly [d(G-C)] is inhibited more than poly dG.poly dC. Since the inhibition by ACM-B can be totally abolished when assayed in excess amount of DNA, this result suggests that ACM-B inhibition of RNA synthesis is solely on the DNA template (which is in support of the intercalation model), and has ruled out the possibility that ACM-B may also exert an inhibitory effect on the activity of RNA polymerase per se.

Aclarubicin↗
PubMed1988

Correlation studies between the binding of aflatoxin B1 to chromatin components and the inhibition of RNA synthesis.

Aflatoxin B1 (AFB1) is a potent inhibitor of rat liver nuclear and nucleolar RNA synthesis. However, since after activation AFB1 binds to both DNA and chromosomal proteins, the question is which form of binding is responsible for the inhibition of RNA synthesis. Male Sprague-Dawley rats (200 g) were given i.p. injections of 10, 50, 100, 300 and 500 micrograms AFB1 per 100 g body wt containing 50 microCi [3H]AFB1 (sp. act. 25 Ci/mmol), and the animals sacrificed 2 h later. Liver nuclei, nucleoli and P-3 (a transcriptionally active subnucleolar fraction that is 3.4-fold enriched in active rDNA) were isolated and the binding of AFB1 to DNA and protein of each fraction was determined by DNase I digestion and 5% trichloracetic acid (TCA) hydrolysis. We found that the binding of AFB1 to both nuclear and nucleolar DNA plateaus at 300 micrograms AFB1 per 100 g body wt with values around 100 and 400 pmol AFB1 per mg nuclear and nucleolar DNA, respectively. On the other hand, the binding to protein is linear, although with different slopes, for both nuclear and nucleolar fractions even at 500 micrograms AFB1 per 100 g body wt, the highest dose used. Since AFB1 inhibition of nuclear and nucleolar RNA synthesis plateaus respectively at 60% and 90% inhibition levels at the dose of 300 micrograms AFB1 per 100 g body wt, these results suggest the binding of AFB1 to DNA, but not to protein, is responsible for the inhibition of RNA synthesis. Further support for this contention is obtained by comparing the binding and the inhibition data between P-3 and nucleoli. P-3 is three times more transcriptionally active than nucleoli and, as a result, is more sensitive to AFB1 inhibition. This greater sensitivity is reflected by the specific binding activity of AFB1 to P-3 DNA, which is greater than 50% higher than to nucleolar DNA. In contrast, this effect is not reflected by the specific binding activity of AFB1 to protein which is exactly the same for both fractions.

Aflatoxin B1↗
PubMed1988

The binding of aflatoxin B1 to rat liver nuclear proteins and its effect on DNA-dependent RNA synthesis.

This paper reports studies on the binding of aflatoxin B1 (AFB1) to rat liver nuclear proteins in vivo and in vitro, and its effect on RNA synthesis. Two hours after rats (200 g) were given a single i.p. injection of 300 micrograms AFB1 containing 50 microCi [3H]AFB1/100 g body wt, AFB1 was found bound to the free nuclear proteins (29.7 pmol/mg protein), histones (20.3 pmol/mg protein) and chromatin-bound non-histone proteins (13.8 pmol/mg protein). The binding of AFB1 to histones was further studied in vitro. We found that for a given type of histone, the binding level varied greatly depending on the conditions used. Under both in vivo and in vitro conditions, however, H3 was always the most efficient substrate, and H4/H2B always the least efficient substrates for AFB1 binding. These results suggest that the binding preference was mainly related to the intrinsic properties of the histone type, and was little affected by the geometric arrangement of the histones in chromatin. Using nuclear proteins added to the RNA synthesizing system in vitro, we found that only the histone fraction had a strong inhibitory effect. Further studies, however, indicated that this inhibition was not due to histones per se, but rather to poly-ADP-ribosylated histones present in the histone preparations. No detectable difference in effect was found between control and AFB1-bound nuclear proteins on RNA synthesis. Moreover, higher levels of AFB1 binding to histones did not potentiate the inhibitory effect. We therefore conclude, and in direct support to our previous correlation studies (see the preceding paper), that the binding of AFB1 to nuclear proteins has no inhibitory effect on RNA synthesis.

Aflatoxin B1↗