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Biomedical subjects

S Y Chiang

Publications and source records attributed to S Y Chiang.

At least 19 recordsLinked to original sources
PubMed2001

The use of antibody D8/17 to identify B cells in adults with obsessive-compulsive disorder.

Compared with healthy control subjects, individuals with childhood-onset obsessive-compulsive disorder (OCD) have been reported to have a higher percentage of B cells that react with the monoclonal antibody D8/17, a marker for rheumatic fever. This study sought to replicate these findings in adults with OCD. Double-blind analyses of blood samples from 29 consecutive adults with primary OCD and 26 healthy control subjects were conducted to determine the percentage of B cells identified by D8/17. Using a standard criterion of > or =12% labeled B cells to denote positivity, rates of D8/17 positive individuals did not significantly differ between the OCD (58.6%) and control (42.3%) groups. Early age of onset was not a predictor of D8/17 positivity in the OCD group. The percentage of B cells identified by the monoclonal antibody marker D8/17 did not distinguish adults with OCD from control subjects, nor did it distinguish a sub-group of adults with OCD who described pre-pubertal onset of their OCD symptoms.

Adolescent↗
PubMed2001

Anticonvulsive and free radical scavenging activities of Gastrodia elata Bl. in kainic acid-treated rats.

Gastrodia elata Bl. (GE) is a traditional Chinese herb that is commonly used in Chinese communities to treat convulsive disorders such as epilepsy. The purpose of the present study was to determine the anticonvulsive and free radical activities of GE in rats. In vitro studies were conducted by using brain tissue from 6 male Sprague-Dawley (SD) rats treated with 120 microg/ml of kainic acid (KA), with or without the addition of various concentrations of GE. In vivo studies were conducted in a total of 30 male SD rats divided into 5 groups of 6 rats which were treated as follows: 1) the normal group received an intraperitoneal injection (i.p.) of PBS (Phosphate buffer saline, 1 ml/kg); 2) the control group received KA (12 mg/kg) i.p.; 3) the GE 1.0 group received oral administration of GE 1.0 g/kg 30 min prior to KA administration; 4) the GE 0.5 group received oral administration of GE 0.5 g/kg 30 min prior to KA administration; 5) the PH group received oral administration of phenytoin 20 mg/kg 30 min prior to KA administration. Seizures were verified by behavioral observations, electroencephalograph (EEG) and electromyography (EMG). Lipid peroxide levels in the rat brain, luminol chemiluminescence (CL) and lucigenin-CL in the peripheral blood were measured simultaneously after behavioral observations. The results indicate that GE administration significantly reduced KA-induced lipid peroxide levels in vitro. Oral administration of GE 1.0 g/kg and phenytoin 20 mg/kg significantly reduced counts of wet dog shakes (WDS), paw tremor (PT) and facial myoclonia (FM) in KA-treated rats. In addition, oral administration of GE 1.0 g/kg significantly delayed the onset of WDS, from 30 min in the control group to 46 min in the 0.5 g/kg group, and 63 min in the GE 1.0 g/kg group. A significantly reduced level of lipid peroxides in the rat brain was found in the GE 1.0 g/kg, 0.5 g/kg, and phenytoin 20 mg/kg groups. The GE 1.0 g/kg group showed significant reduction of luminol-CL and lucigenin-CL counts in the peripheral blood compared to the control group. The results of the present study demonstrate that GE has anticonvulsive and free radical scavenging activities. Further studies are needed to determine the clinical effectiveness of GE as an anticonvulsant in humans.

Analysis of Variance↗
PubMed2001

Effect of Rhizoma Corydalis on focal cerebral infarct in ischemia-reperfusion injured rats.

AIM: To investigate the effect of Rhizoma Corydalis (RC) on focal cerebral infarct. METHODS: A total of 30 Sprague-Dawley (SD) rats were studied. Focal cerebral infarct was established b y occluding the bilateral common carotid arteries and the right middle cerebral artery for 90 min. After 24 h reperfusion, the neurological status was evaluated and then the rats were killed and the brain tissue was stained with 2,3,5-triphenyl-tetrazolium chloride. The neurological status and the changes in the area of cerebral infarct were used as an index to evaluate the effect of RC on cerebral infarct. In addition, the whole blood was examined 24 h after RC treatment in the other 24 SD rats. RESULTS: Pretreatment with RC 100 mg/kg can improve neurological status and also can reduce the area of cerebral infarct in ischemia-reperfusion injured rats. The counts of erythrocyte and the amount of hematocrit increased in whole blood of RC-treated rats. CONCLUSION: RC can improve neurological status and reduce the area of cerebral infarct in ischemia-reperfusion injured rats.

Animals↗
PubMed2000

Targeting the ets binding site of the HER2/neu promoter with pyrrole-imidazole polyamides.

Three DNA binding polyamides () were synthesized that bind with high affinity (K(a) = 8.7. 10(9) m(-1) to 1.4. 10(10) m(-1)) to two 7-base pair sequences overlapping the Ets DNA binding site (EBS; GAGGAA) within the regulatory region of the HER2/neu proximal promoter. As measured by electrophoretic mobility shift assay, polyamides binding to flanking elements upstream () or downstream (2 and 3) of the EBS were one to two orders of magnitude more effective than the natural product distamycin at inhibiting formation of complexes between the purified EBS protein, epithelial restricted with serine box (ESX), and the HER2/neu promoter probe. One polyamide, 2, completely blocked Ets-DNA complex formation at 10 nm ligand concentration, whereas formation of activator protein-2-DNA complexes was unaffected at the activator protein-2 binding site immediately upstream of the HER2/neu EBS, even at 100 nm ligand concentration. At equilibrium, polyamide 1 was equally effective at inhibiting Ets/DNA binding when added before or after in vitro formation of protein-promoter complexes, demonstrating its utility to disrupt endogenous Ets-mediated HER2/neu preinitiation complexes. Polyamide 2, the most potent inhibitor of Ets-DNA complex formation by electrophoretic mobility shift assay, was also the most effective inhibitor of HER2/neu promoter-driven transcription measured in a cell-free system using nuclear extract from an ESX- and HER2/neu-overexpressing human breast cancer cell line, SKBR-3.

Amides↗
PubMed2000

Anticonvulsive and free radical scavenging activities of vanillyl alcohol in ferric chloride-induced epileptic seizures in Sprague-Dawley rats.

Vanillyl alcohol (VA) is a component of Gastrodia elata Bl. (GE), which is a traditional Chinese herb widely used to treat convulsive disorders or dizziness. This study examined the role of VA in the anticonvulsive properties of GE in a Sprague-Dawley rat model of epilepsy. The anticonvulsive and free radical scavenging activities of VA were examined after intracortical injection of ferric chloride (100 mM, 8 microl) to induce epileptic seizures. These seizures were verified by behavioral observations and electroencephalographic (EEG) and electromyographic (EMG) recordings. Ferric chloride injection resulted in increased lipid peroxide levels in the ipsilateral and contralateral cerebral cortex, and increased luminol-chemiluminescence (CL) and lucigenin-CL counts in the peripheral blood. Intraperitoneal injection (i.p.) of VA (200 mg/kg or 100 mg/kg) or phenytoin 10 mg/kg prior to ferric chloride administration significantly inhibited wet dog shakes (WDS) and lipid peroxide levels in the bilateral cerebral cortex. VA 200 mg/kg also significantly reduced luminol-CL and lucigenin-CL counts in the peripheral blood, but no significant effect was observed following administration of VA 100 mg/kg or phenytoin. These data indicate that VA has both anticonvulsive and suppressive effects on seizures and lipid peroxidation induced by ferric chloride in rats. Data from the present study also demonstrate that VA has free radical scavenging activities, which may be responsible for its anticonvulsive propertics. This finding is consistent with the results from previous studies that generation of superoxide radical evoked by injection of iron salt into rat brain plays a critical role in ferric chloride-induced seizures. In addition, the results of the present study suggest that the anticonvulsive effect of GE may be attributable, at least in part, to its VA component.

Acridines↗
PubMed2000

Adeno-associated virus major Rep78 protein disrupts binding of TATA-binding protein to the p97 promoter of human papillomavirus type 16.

Adeno-associated virus type 2 (AAV) is known to inhibit the promoter activities of several oncogenes and viral genes, including the human papillomavirus type 16 (HPV-16) E6 and E7 transforming genes. However, the target elements of AAV on the long control region (LCR) upstream of E6 and E7 oncogenes are elusive. A chloramphenicol acetyltransferase assay was performed to study the effect of AAV on the transcription activity of the HPV-16 LCR in SiHa (HPV-positive) and C-33A (HPV-negative) cells. The results reveal that (i) AAV inhibited HPV-16 LCR activity in a dose-dependent manner, (ii) AAV-mediated inhibition did not require the HPV gene products, and (iii) the AAV replication gene product Rep78 was involved in the inhibition. Deletion mutation analyses of the HPV-16 LCR showed that regulatory elements outside the core promoter region of the LCR may not be direct targets of AAV-mediated inhibition. Further study with the electrophoretic mobility shift assay demonstrated that Rep78 interfered with the binding of TATA-binding protein (TBP) to the TATA box of the p97 core promoter more significantly than it disrupted the preformed TBP-TATA complex. These data thus suggest that Rep78 may inhibit transcription initiation of the HPV-16 LCR by disrupting the interaction between TBP and the TATA box of the p97 core promoter.

Cell Line↗
PubMed1999

Anticonvulsive and free radical scavenging actions of two herbs, Uncaria rhynchophylla (MIQ) Jack and Gastrodia elata Bl., in kainic acid-treated rats.

Uncaria rhynchophylla (Miq.) Jack (UR) and Gastrodia elata BI. (GE) are traditional Chinese herbs that are usually used in combination to treat convulsive disorders, such as epilepsy, in China. The aim of this study was to compare the anticonvulsive and free radical scavenging activities of UR alone and UR in combination with GE in rats. For the in vitro studies, brain tissues from 6 male Sprague-Dawley (SD) rats were treated with 120 microg/ml kainic acid (KA), with or without varied concentrations of UR or UR plus GE. For the in vivo studies, male SD rats (6 per group) received intraperitoneal (i.p.) injection of KA 12 mg/kg to induce epileptic seizures and generation of free radicals, with or without oral administration of UR 1 g/kg alone or UR 1 g/kg plus GE 1 g/kg. Epileptic seizures were verified by behavioral observations, and electroencephalography (EEG) and electromyography (EMG) recordings. These results showed that UR alone decreased KA-induced lipid peroxide levels in vitro, whereas UR plus GE did not produce a greater effect than UR alone. UR significantly reduced counts of wet dog shakes (WDS), paw tremor (PT) and facial myoclonia (FM) in KA-treated rats and significantly delayed the onset time of WDS, from 27 min in the control group to 40 min in the UR group. UR plus GE did not inhibit seizures more effectively than UR alone, but did further prolong the onset time of WDS to 63 min (P < 0.05 vs. UR alone). UR alone reduced the levels of free radicals in vivo, as measured by lipid peroxidation in the brain and luminol-chemiluminescence (CL) counts and lucigenin-CL counts in the peripheral whole blood, but the combination of GE and UR did not reduce free radical levels more markedly than UR alone. In conclusion, our results indicate that UR has anticonvulsive and free radical scavenging activities, and UR combined with GE exhibit greater inhibition on the onset time of WDS than UR alone. These findings suggest that the anticonvulsive effects of UR and GE may be synergistic. However, the mechanism of interaction between UR and GE remains unknown.

Animals↗
PubMed1998

A comparison of DNA-binding drugs as inhibitors of E2F1- and Sp1-DNA complexes and associated gene expression.

In this study, we examined how DNA-binding drugs prevented formation of transcription factor-DNA complexes and influenced gene transcription from the hamster dihydrofolate reductase promoter, which is regulated by E2F1 and Sp1. Gel mobility shift assay data showed that GC-binding drugs (e.g., mitoxantrone) inhibited the DNA binding of both E2F1 and Sp1. In contrast, AT-binding drugs (e.g., distamycin) interfered only with E2F1-DNA complex formation. In an in vitro transcription assay using HeLa nuclear extracts, inhibition of transcription was observed when mitoxantrone or distamycin was added either before or after assembly of the transcription complex on the DNA, although for the latter, higher drug concentrations were needed. Mitoxantrone, which was a stronger inhibitor of transcription factor-DNA complex, was more effective than distamycin at preventing transcript formation. Time course transcription in a cell-free assay with addition of various drug concentrations indicated that high drug concentrations of either mitoxantrone or distamycin completely blocked transcription, while low drug concentrations could delay the synthesis of transcripts.

Animals↗
PubMed1998

Highly sensitive apurinic/apyrimidinic site assay can detect spontaneous and chemically induced depurination under physiological conditions.

One of the most prevalent lesions in DNA is the apurinic/apyrimidinic (AP) site, which is derived from the cleavage of the N-glycosyl bond by DNA glycosylase or by spontaneous depurination. AP sites are repaired by AP endonucleases during the process of base excision repair; however, an imbalance in this DNA repair system may cause mutations as well as cell death. We have established a sensitive and convenient slot-blot method to detect AP sites in genomic DNA using a novel aldehyde reactive probe (ARP), which reacts with the aldehydic group of ring-opened AP sites. The reaction of 1 mM of ARP with 15 microg of genomic DNA containing AP sites at 37 degrees C was completed within 1 min. The AP site-ARP complex was remarkably stable during incubation in TE buffer, even at 100 degrees C for 60 min. The sensitivity of this assay enables detection of 2.4 AP sites per 10(7) bases. By using this ARP-slot-blot assay, the rate of spontaneous depurination of calf thymus DNA was determined. Under physiological conditions, AP sites were increased at 1.54 AP sites/10(6) nucleotides/day (9000 AP sites/cell/day). This highly sensitive assay allows us to determine the endogenous level of AP sites in genomic DNA, as well as to investigate whether DNA-damaging agents cause imbalances of base excision/AP endonuclease repair in vivo and in vitro.

B-Lymphocytes↗
PubMed1998

Quantitative analysis of 1,3-butadiene-induced DNA adducts in vivo and in vitro using liquid chromatography electrospray ionization tandem mass spectrometry.

1,3-Butadiene (BD) is a high volume industrial chemical which is known as a multi-site rodent carcinogen and is classified as a probable human carcinogen. Covalent interactions of the reactive epoxy metabolites of BD with DNA lead to the formation of DNA adducts which may cause mutations and tumor formation. In the present work, liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) was employed for analyses of BD-induced DNA adducts in vitro and in vivo. Selected reaction monitoring (SRM) using the fragmentation of the [M + H]+ ions of the adducts to the corresponding protonated nucleobases under collision-induced dissociation was performed. Quantitation was based on isotope dilution with 13C- and 15N-labeled internal standards. The methods were applied in vitro [calf thymus DNA and TK6 cell cultures treated with epoxy metabolites of BD, 3,4-epoxy-1-butene (EB) and diepoxybutane (DEB)] and in vivo [DNA isolated from tissues of BD-exposed laboratory animals]. Two regioisomers of N-7-EB-guanine adducts, N-7-(2-hydroxy-3-buten-1-yl)guanine (N-7-EB-Gua I) and N-7-(1-hydroxy-3-buten-2-yl)guanine (N-7-EB-Gua II) and two N-3-EB-adenine isomers, N-3-(2-hydroxy-3-buten-1-yl)adenine and N-3-(1-hydroxy-3-buten-2-yl)adenine (N-3-EB-Ade I and II), were found in EB-exposed samples. N-7-(2',3',4'-trihydroxybut-1'-yl)guanine (N-7-THB-Gua), N6-(2',3',4'-trihydroxybut-1'-yl)adenine (N6-THB-Ade), and N-3-(2',3',4'-trihydroxybut-1'-yl)adenine (N-3-THB-Ade) were detected in DEB-treated DNA. DNA isolated from liver and lung of rats and mice exposed to 1250 ppm BD for 2 weeks contained both regioisomers of N-7-EB-Gua and N-3-EB-Ade, as well as N-7-THB-Gua and N6-THB-Ade. The methods developed in this work provide the means to study accumulation, repair and dose-response relationships of BD-DNA adducts in vivo. Although less sensitive than gas chromatography/electron capture negative ionization high-resolution mass spectrometry (GC/ECNI-HRMS), LC/ESI(+)-MS/MS in the SRM mode is extremely useful for analysis of BD-DNA adducts, which are not amenable to GC and derivatization owing to the presence of several adjacent polar functional groups. Using LC/ESI-MS/MS and isotope dilution, multiple structurally diverse BD-DNA adducts can be analyzed simultaneously in the same sample with minimal sample preparation.

Animals↗
PubMed1997

Targeting E2F1-DNA complexes with microgonotropen DNA binding agents.

Microgonotropen (MGT) DNA binding drugs, which consist of an A+T-selective DNA minor groove binding tripyrrole peptide and polyamine chains attached to a central pyrrole that extend drug contact into the DNA major groove, were found to be extraordinarily effective inhibitors of E2 factor 1 (E2F1) association with its DNA promoter element (5'-TTTCGCGCCAAA). The most active of these drugs, MGT-6a, was three orders of magnitude more effective than distamycin and inhibited complexes between E2F1 and the dihydrofolate reductase promoter by 50% at 0.00085 microM. A relationship was found between the measured equilibrium constants for binding of MGTs to the A+T region of d(GGCGA3T3GGC)/d(CCGCT3A3CCG) and their inhibition of complex formation between E2F1 and the DNA promoter element. A representative of the potent MGT inhibitors was significantly more active on inhibition of E2F1-DNA complex formation compared with disruption of a preexisting complex.

Animals↗
PubMed1997

A microgonotropen branched decaaza decabutylamine and its DNA and DNA/transcription factor interactions.

The central pyrrole of a site-selective DNA minor groove binding tripyrrole peptide 1 has been attached to a branched decaaza decabutylamine via a -(CH-2)3-NHCO-(CH2)-3 linker to provide the decaaza-microgonotropen (8). The decaaza decabutylamine moiety of 8 was designed to have a much greater affinity to the phosphodiester linkages of the backbone of DNA. Employing Hoechst 33258 (Ht) as a fluorescent titrant, the equilibrium constants for the binding for of 8 to the hexadecameric duplex d(GGCGCA3T3GGCGG)/d(CCGCCA3T3GCGCC) and to calf thymus DNA were determined. The log of the product of equilibrium constants (log Kl1Kl2) for 1:1 and 1:2 complexes formation at A3T3 is 17 (35 degrees C). Results of studies of the inhibition of the binding of several proteins to target DNA are discussed. Binding of the E2F1 transcription factor to its DNA target is 50% inhibited at approximately 2 nM concentration of 8.

Animals↗
PubMed1997

Mutagenicity of vinyl chloride and its reactive metabolites, chloroethylene oxide and chloroacetaldehyde, in a metabolically competent human B-lymphoblastoid line.

Vinyl chloride (VC), a known human and rodent carcinogen, is metabolically activated by cytochrome P450 to chloroethylene oxide (CEO), which can rearrange to chloroacetaldehyde (CAA) or undergo hydrolysis. To further understand the roles of CEO and CAA in VC mutagenesis, the types and frequencies of mutations induced at the hypoxanthine (guanine) phosphoribosyl-transferase (hprt) locus were examined in a human B-lymphoblastoid line constitutively expressing human cytochrome P450 2E1 (H2E1 cells). VC was toxic and mutagenic to H2E1 cells as a function of incubation time; exposure to 7.5% VC in air resulted in 75% survival and an hprt mutant frequency of 42 x 10(-6) after 48 h, compared to 5.7 +/- 2.7 x 10(-6) for unexposed cells. The exposure of H2E1 cells to 0.8 to 15.0% VC in air produced similar mutant frequencies without a clear dose-response relationship, suggesting saturation of metabolic activation. Both CEO and CAA exhibited dose-dependent increases in cell killing and mutant frequency in H2E1 cells. Treatment with 16 microM CEO for 24 h resulted in 75% survival and an induced mutant frequency of 23 x 10(-6), while 16 microM CAA produced 5% survival and an induced mutant frequency of 20 x 10(-6). Structural alterations at the hprt locus in independent thioguanine-resistant clones were examined by Southern blot analysis of Pst I-digested DNA with a full-length human hprt cDNA probe. Ten percent (5/50) of VC-induced and 18% (7/38) of CEO-induced mutants showed detectable deletions, compared with 45% (9/20) of CAA-induced mutants. Thus, VC and CEO displayed similar toxicity/mutation profiles and a similar frequency of large deletions, whereas CAA displayed greater toxicity and a larger frequency of deletion mutations. These results suggest that the majority of mutations induced by VC occur through its metabolite, CEO.

Acetaldehyde↗
PubMed1997

Repression of the HSV-1 latency-associated transcript (LAT) promoter by the early growth response (EGR) proteins: involvement of a binding site immediately downstream of the TATA box.

During herpes simplex virus (HSV) latency, in neurons of the nervous system, a single family of viral transcripts (the Latency-Associated Transcripts or LATs) are synthesized. Within the LAT promoter region, we have identified a consensus sequence for the EGR proteins in an unusual position immediately downstream of the TATA box. The early growth response (EGR) proteins are rapidly induced in cells by stimuli which also induce HSV to reactivate from latency. In order to determine if EGR proteins play any role in control of LAT transcription, we have analyzed the interactions between EGR proteins and the LAT promoter. Gel retardation and DNase I protection assays demonstrated that EGR1 zinc finger protein bound specifically to the LAT promoter region EGR consensus sequence. To determine if EGR proteins could modulate transcription through the LAT promoter, cotransfection assays were performed using chloramphenicol acetyltransferase (CAT) reporter constructs driven by either the wild-type LAT promoter or a LAT promoter with a mutated EGR binding site. Contransfection of the wild-type LAT promoter construct with EGR expression plasmids resulted in inhibition of the basal level of CAT activity with EGR-2 but not EGR-1 or 3. However, normal levels of CAT activity were observed in cotransfections using the mutant LAT promoter CAT construct suggesting that repression was mediated by the binding of EGR-2 proteins to the LAT promoter. Furthermore, data from combination binding assays using EGR1 and TATA binding protein (TBP) in vitro support the hypothesis that binding of EGR proteins to the LAT promoter prevents binding of TBP and thus suppresses transcription. These results may provide a link between stress responses in neurons of the CNS which activate the EGR family of proteins and HSV reactivation from latency due to the same stress response.

Animals↗
PubMed1996

Effect of DNA-binding drugs on early growth response factor-1 and TATA box-binding protein complex formation with the herpes simplex virus latency promoter.

Adjacent binding sites for early growth response factor-1 (EGR1) and TATA box-binding protein (TBP) were identified on the herpes simplex virus latency promoter in previous work. The binding of EGR1 to the GC-rich region prevented TBP binding to the AT-rich region. With the simultaneous addition of both EGR1 and TBP, the intercalator nogalamycin prevented EGR1 complex formation, resulting in a dose-dependent increase of the TBP.DNA complex. The minor groove binder chromomycin A3 inhibited EGR1 complex formation but resulted in a smaller increase of the TBP complex. In contrast, an alkylating intercalator hedamycin strongly inhibited binding of both proteins. The ability of these GC-binding drugs to prevent EGR1.DNA complex formation was in the following order: hedamycin > nogalamycin > chromomycin A3, and the specificity was nogalamycin > chromomycin A3 > hedamycin. With transcription factor IIA (TFIIA) in the assay, TBP was able to bind the promoter whereas formation of the EGR1.DNA complex was reduced. An AT minor groove-binding drug, distamycin A, disrupted the TBP.TFIIA.DNA complex and restored the EGR1.DNA complex. We conclude that the binding motif and sequence preference of DNA-interactive drugs are manifested in their ability to inhibit the transcription factor-DNA complexes.

Alkylating Agents↗
PubMed1994

Effects of minor groove binding drugs on the interaction of TATA box binding protein and TFIIA with DNA.

TBP (TATA box binding protein), a general transcription factor required for proper initiation of gene expression by RNA polymerase II, and minor groove binding drugs (MGBs) both interact with DNA within the minor groove at AT sites. This study has evaluated MGBs as inhibitors of DNA/TBP complex formation by gel mobility shift assays. Our results demonstrate that reversible MGBs (DAPI, distamycin A, Hoechst 33258, and netropsin) are effective inhibitors of the formation of DNA/TBP complex and that distamycin A is the most potent (0.16 microM inhibits TBP complex formation by 50%). CC-1065, a drug that covalently binds to DNA in the minor groove, is even more active than distamycin A (0.00085 microM inhibits TBP complex formation by 50%). Significantly more CC-1065 (0.009 microM) is required to break up preformed DNA/TBP complex compared to the drug concentration needed to prevent complex formation. In comparison, the order of drug addition has little influence on the ability of reversible MGBs to disrupt DNA/TBP complex. In the presence of TFIIA, a factor that enhances TBP association with DNA, greater drug concentrations (distamycin A and CC-1065, respectively) are needed to disrupt a preformed complex of DNA/TBP/TFIIA. In comparison to MGBs, drugs capable of binding to DNA by intercalation are generally weaker at blocking TBP complex formation except for hedamycin, which can intercalate and irreversibly bind to DNA and is as effective as reversible MGBs.

Base Sequence↗
PubMed1994

A comparison of two ultrasensitive methods for measuring 1,N6-etheno-2'-deoxyadenosine and 3,N4-etheno-2'-deoxycytidine in cellular DNA.

1,N6-Ethenodeoxyadenosine (edA) and 3,N4-ethenodeoxycytidine (edC) are two mutagenic adducts associated with exposure to ethyl carbamate (urethane) and vinyl chloride. We have recently developed two ultrasensitive methods for determining the molecular dose of these adducts in cellular DNA. In both methods, purified DNA was first enzymatically digested to 2c-deoxyribonucleotide 3c-monophosphates. Etheno-modified nucleotides were then separated from normal nucleotides in one of two ways: either by reverse phase, ion-pair HPLC coupled with 260 nm UV detection, or by immunoaffinity chromatography using reusable microcolumns containing specific monoclonal antibodies coupled to Protein A-Sepharose. Fractions enriched for the adducted nucleotides were labeled using T4 poly-nucleotide kinase and [32P]ATP, and individual nucleotides were subsequently resolved by two-dimensional TLC, visualized by autoradiography, and quantified by liquid scintillation counting. When used to analyze the same sample of etheno-modified calf thymus DNA, both assays produced similar results. However, when both methods were used to analyze rat liver DNA 'spiked' with known amounts of etheno nucleotide standards, the immuno-affinity/32P TLC procedure proved to be more sensitive and more reproducible than the HPLC/32P TLC method: while the detection limit of the immunoaffinity/32P TLC technique was < 4 etheno adducts/10(9) parent deoxynucleotides, the HPLC/32P TLC method often failed to detect adducts at concentrations < 2/10(8). In other experiments, the immunoaffinity/32P TLC method was used to demonstrate formation of edA and edC in cells treated with vinyl chloride monomer. Because of its exquisite sensitivity, the immunoaffinity/32P TLC method promises to be extremely useful for measuring both background and induced levels of etheno adducts, making it possible to examine the role of these adducts in inducing mutations and/or carcinogenesis.

Cell Line↗