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Biomedical subjects

B Singer

Publications and source records attributed to B Singer.

At least 19 recordsLinked to original sources
PubMed1995

The benzene metabolite p-benzoquinone forms adducts with DNA bases that are excised by a repair activity from human cells that differs from an ethenoadenine glycosylase.

Benzene is a ubitiquous human environment mental carcinogen. One of the major metabolites is hydroquinone, which is oxidized in vivo to give p-benzoquinone (p-BQ). Both metabolites are toxic to human cells. p-BQ reacts with DNA to form benzetheno adducts with deoxycytidine, deoxyadenosine, and deoxyguanosine. In this study we have synthesized the exocyclic compounds 3-hydroxy-3-N4-benzetheno-2'-deoxycytidine (p-BQ-dCyd) and 9-hydroxy-1,N6-benzetheno-2'-deoxyadenosine (p-BQ-dAdo), respectively, by reacting deoxycytidine and deoxyadenosine with p-BQ. These were converted to the phosphoamidites, which were then used to prepare site-specific oligonucleotides with either the p-BQ-dCyd or p-BQ-dAdo adduct (pbqC or pbqA in sequences) at two different defined positions. These oligonucleotides were efficiently nicked 5' to the adduct by partially purified HeLa cell extracts--the pbqC-containing oligomer more rapidly than the pbqA-containing oligomer. In contrast to the enzyme binding to derivatives produced by the vinyl chloride metabolite chloroacetaldehyde, the oligonucleotides up to 60-mer containing p-BQ adducts did not bind measurably to the same enzyme preparation in a gel retardation assay. Furthermore, there was no competition for the binding observed between oligonucleotides containing 1,N6-etheno A deoxyadenosine (1,N6-etheno-dAdo; epsilon A in sequences) and these oligomers containing either of the p-BQ adducts, even at 120-fold excess. When highly purified fast protein liquid chromatography (FPLC) enzyme fractions were obtained, there appeared to be two closely eluting nicking activities. One of these enzymes bound and cleaved the epsilon A-containing deoxyoligonucleotide. The other enzyme cleaved the pbqA- and pbqC-containing deoxyoligonucleotides. One additional unexpected fact was that bulk p-BQ-treated salmon sperm DNA did compete effectively with the epsilon A-containing oligonucleotide for protein binding. This raises the possibility that such DNA contains other, as-yet-uncharacterized adducts that are recognized by the same enzyme that recognizes the etheno adducts. In summary, we describe a previously undescribed human DNA repair activity, possibly a glycosylase, that excises from DNA pbqC and pbqA, exocyclic adducts resulting from reaction of deoxycytidine and deoxyadenosine with the benzene metabolite, p-BQ. This glycosylase activity is not identical to the one previously reported from this laboratory as excising the four etheno bases from DNA.

Autoradiography↗
PubMed1995

Large scale synthesis of p-benzoquinone-2'-deoxycytidine and p-benzoquinone-2'-deoxyadenosine adducts and their site-specific incorporation into DNA oligonucleotides.

Benzene is a carcinogen in rodents and a cause of bone marrow toxicity and leukemia in humans. p-Benzoquinone (p-BQ) is one of the stable metabolites of benzene, as well as of a number of drugs and other chemicals. 2'-Deoxycytidine (dC) and 2'-deoxyadenosine (dA) were allowed to react with p-BQ in aqueous solution at pH 7.4 and 4.5. The yields were considerably higher at pH 4.5 than at pH 7.4, as indicated by HPLC analysis. The desired products were isolated by column chromatography on silica gel or cellulose. Identification was done by FAB-MS, 1H NMR, and UV spectroscopy. The reaction of p-BQ with dC and dA at pH 4.5 produced the exocyclic compounds 3-hydroxy-1,N4-benzetheno-2'-deoxycytidine (p-BQ-dC), and 9-hydroxy-1,N6-benzetheno-2'-deoxyadenosine (p-BQ-dA), respectively, in a large scale and high yield. These adducts have been previously made in a microgram scale as the 3'-phosphate for 32P-postlabeling studies of their incidence in DNA. The p-BQ-dC and p-BQ-dA adducts have, in addition to the two hydroxyl groups of deoxyribose, one newly formed hydroxyl group at the C-3 or C-9 of the exocyclic base of each product respectively. Incorporation of these adducts into oligonucleotides as the phosphoramidite requires the protection of all three hydroxyl groups in these compounds. The exocyclic hydroxyl on the base has been successfully protected by acylation after protecting the 5'- and the 3'-hydroxyl groups of the sugar moiety with a 4,4'-dimethoxytrityl group and a cyanoethyl N,N-diisopropylphosphoramidite group, respectively. For the first time, to our knowledge, the fully protected phosphoramidites of p-BQ-dC and p-BQ-dA were prepared and incorporated site-specifically into a series of oligonucleotides. The coupling efficiency was very high (> 98%). However, deprotection of the DNA oligomers with ammonia produced only 50% of the desired oligomers containing the adduct. In contrast, when 10% of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) in methanol at room temperature was used, only the desired oligomers were detected by HPLC. Thus, by deprotecting the oligomers with methoxide ions (DBU/methanol) and avoiding the use of ammonia, a high yield of modified DNA was obtained. After purification of these oligomers by HPLC, they were hydrolyzed enzymatically and analyzed by HPLC, which confirmed the base composition and the incorporation of the adducts. The mass spectroscopic analysis of the DNA oligomers was confirmed by electrospray MS. These oligomers are now under investigation for their biochemical properties.

Base Sequence↗
PubMed1995

Genetic analysis of idiopathic torsion dystonia in Ashkenazi Jews and their recent descent from a small founder population.

We have examined data on six closely linked microsatellite loci on chromosome 9q34 from 59 Ashkenazi Jewish families with idiopathic torsion dystonia (ITD). Our data show that the vast majority (> 90%) of early-onset ITD cases in the Ashkenazi population are due to a single founder mutation, which we estimate first appeared approximately 350 years ago. We also show that carriers preferentially originate from the northern part of the historic Jewish Pale of settlement (Lithuania and Byelorussia). The recent origin of this dominant mutation and its current high frequency (between 1/6,000 and 1/2,000) suggest that the Ashkenazi population descends from a limited group of founders, and emphasize the importance of genetic drift in determining disease allele frequencies in this population.

Alleles↗
PubMed1995

Contrast gain control: a bilinear model for chromatic selectivity.

We report the results of psychophysical experiments on color contrast induction. In earlier work [Vision Res. 34, 3111 (1994)], we showed that modulating the spatial contrast of an annulus in time induces an apparent modulation of the contrast of a central disk, at isoluminance. Here we vary the chromatic properties of disk and annulus systematically in a study of the interactions among the luminance and the color-opponent channels. Results show that induced contrast depends linearly on both disk and annulus contrast, at low and moderate contrast levels. This dependence leads us to propose a bilinear model for color contrast gain control. The model predicts the magnitude and the chromatic properties of induced contrast. In agreement with experimental results, the model displays chromatic selectivity in contrast gain control and a negligible effect of contrast modulation at isoluminance on the appearance of achromatic contrast. We show that the bilinear model for chromatic selectivity may be realized as a feed-forward multiplicative gain control. Data collected at high contrast levels are fit by embellishing the model with saturating nonlinearities in the contrast gain control of each color channel.

Adaptation, Ocular↗
PubMed1995

Medullary lavage reduces embolic phenomena and cardiopulmonary changes during cemented hemiarthroplasty.

We randomised 24 patients before they had a cemented hemiarthroplasty for hip fracture to receive either thorough or minimal saline lavage of the femoral canal. We then determined the effect in each group on the thromboembolic and cardiopulmonary responses to the pressurised insertion of cement, using transoesophageal echocardiography to show the echogenic embolic response. We found a statistically significant reduction in both the duration of the response and the number of large emboli in patients who had had thorough lavage as compared with the control group with minimal lavage. There was also less disturbance of pulmonary function, as assessed by the change in end-tidal CO2 levels and oxygen saturation, in patients who had thorough lavage. Three patients had a significant fall in blood pressure during cement insertion; all had only minimal lavage. We consider that thorough lavage should be an essential part of the preparation of the proximal femur before cement insertion.

Aged↗
PubMed1994

Enzyme-dependent pausing during in vitro replication of O4-methylthymine in a defined oligonucleotide sequence.

We had previously reported that an oligonucleotide containing a site-specifically incorporated O4-methylthymine (m4T) was replicated under kinetic conditions by the Klenow fragment of E. coli DNA polymerase I (Kf) (Dosanjh et al., 1993). Using other polymerases for complete replication, but with limiting enzyme, a pause site before the m4T was observed. In order to investigate whether such a pause could be due to enzyme dissociation or stalling, trapping experiments were designed to aid in differentiating the two mechanisms. Rather than the generally used heparin or sheared DNA trap, these experiments utilized as the acceptor the same oligonucleotide containing unmodified thymine. It was observed that, under enzyme-limiting conditions, the nature of the enzyme played a major role in replication of m4T. With a running start, Kf and calf-thymus polymerase alpha-primase allowed replication beyond the m4T, while Sequenase and T7 showed a strong pause site at the base before m4T. When the oligonucleotide trap was added after different times of replication, it was found that Sequenase remained bound to the template-primer, regardless of whether T or m4T was present. In contrast, Kf dissociated and re-associated rapidly. Thus, m4T appears to be a strong replication block when using limiting amounts of a highly processive enzyme such as Sequenase or T7. This may imply that such enzymes discriminate against forming a poor basepair but remain bound to the primer-template or become inactivated.

Base Sequence↗
PubMed1994

1,N6-ethenoadenine is preferred over 3-methyladenine as substrate by a cloned human N-methylpurine-DNA glycosylase (3-methyladenine-DNA glycosylase).

A lethal DNA adduct induced by methylating agents, 3-methyladenine (m3A), is removed by both the constitutive (Tag) and inducible (AlkA) bacterial m3A-DNA glycosylases. The human 3-methyladenine-DNA glycosylase also releases m3A as well as other methylated bases. The rate of release of m3A from alkylated DNA by the purified or recombinant human m3A glycosylase is much higher than that of the other methylated bases. We now find that a partially purified recombinant human m3A-DNA glycosylase, expressed in Escherichia coli, releases at least 10-fold more 1,N6-ethenoadenine (epsilon A) than m3A from DNA. epsilon A is completely unrelated to m3A since it is a heterocyclic adduct produced by the carcinogen vinyl chloride. The rates of release of epsilon A and m3A were both dependent on protein concentration and time. The differential release of epsilon A and m3A occurs regardless of whether DNA containing each adduct is assayed separately or is assayed in a mixed substrate containing both DNAs. This result raises the question of what structural features are involved in recognition and excision by the human m3A-DNA glycosylase and what may be its primary substrate.

Adenine↗
PubMed1994

All four known cyclic adducts formed in DNA by the vinyl chloride metabolite chloroacetaldehyde are released by a human DNA glycosylase.

We have previously reported that human cells and tissues contain a 1,N6-ethenoadenine (epsilon A) binding protein, which, through glycosylase activity, releases both 3-methyladenine (m3A) and epsilon A from DNA treated with methylating agents or the vinyl chloride metabolite chloroacetaldehyde, respectively. We now find that both the partially purified human epsilon A-binding protein and cell-free extracts containing the cloned human m3A-DNA glycosylase release all four cyclic etheno adducts--namely epsilon A, 3,N4-ethenocytosine (epsilon C), N2,3-ethenoguanine (N2,3-epsilon G), and 1,N2-ethenoguanine (1,N2-epsilon G). Base release was both time and protein concentration dependent. Both epsilon A and epsilon C were excised at similar rates, while 1,N2-epsilon G and N2,3-epsilon G were released much more slowly under identical conditions. The cleavage of glycosyl bonds of several heterocyclic adducts as well as those of simple methylated adducts by the same human glycosylase appears unusual in enzymology. This raises the question of how such a multiple, divergent activity evolved in humans and what may be its primary substrate.

Acetaldehyde↗
PubMed1994

Color contrast induction.

We report the results of psychophysical experiments on the intensive, spatial, temporal and chromatic properties of color contrast induction. Modulating the contrast of an annulus induces an apparent modulation of the color contrast of a central disk, at isoluminance. Results of varying the size of the annulus suggest that mechanisms which control contrast gain are spatially localized. Results of varying the orientations of disk and annulus patterns, with peak spatial frequencies at about 2 c/deg, suggest that the mechanisms are spatially isotropic. Results of varying the rate at which annulus contrast is modulated shows that mechanisms which mediate contrast induction have a low-pass temporal sensitivity that cuts off at about 8 Hz. Results of an experiment on the interocular transfer of color contrast induction suggest that the induction has a cortical locus. Finally, the results of varying the chromatic properties of disk and annulus suggest that the underlying mechanisms are partially, but not fully, chromatically selective.

Color Perception↗
PubMed1994

Synthesis of a 25 base oligonucleotide containing a styrene oxide modification at the O6 position of 2'-deoxyguanosine at a defined site and incorporation studies of the similarly modified 2'-deoxyguanosine-5'-triphosphate.

A diastereomeric mixture of the regioisomers O6-(2-hydroxy-2-phenylethyl)-2'-deoxyguanosine (st6G, beta-isomer) and O6-(2-hydroxy-1-phenylethyl)-2'-deoxyguanosine (alpha-isomer) was site-specifically placed in a 25 base oligonucleotide template 5'-CCGCTAst6GCGGGTACCGAGCTCGAAT-3' using CED phosphoramidite chemistry. Using 32P-post-labeling we found the oligonucleotide to contain 95% of the beta-isomer and 5% of the alpha-isomer of st6G. st6G as the 3'-phosphate was found to be considerably more acid labile than O6-methyl-2'-deoxyguanosine-3'-phosphate, leading to dealkylation during oligonucleotide synthesis. The diastereomeric mixture of O6-(2-hydroxy-2-phenylethyl)-2'-deoxy-guanosine-5'-triphosphate (st6dGTP) was chemically synthesized and used as a substrate for the exonuclease-free Klenow fragment of Escherichia coli DNA polymerase I. This study demonstrated that st6dGTP could be incorporated opposite deoxycytidine and did not completely block replication.

Base Sequence↗
PubMed1994

Osteoblastoma of the odontoid process.

A case of an osteoblastoma located in the odontoid process in a 7 year-old boy who presented with torticollis is reported. CT scan and MRI disclosed the lesion, but diagnosis was established by a CT-guided needle biopsy. Surgery through a transoral approach allowed total excision of the tumor. This is, to our knowledge, the first case reported of an osteoblastoma of the odontoid process.

Bone Transplantation↗
PubMed1994

Attitudinal, circadian, circumstantial, and subject selection explanations of shiftwork effects on health.

The competing explanations for adverse health symptoms commonly observed in night-shift workers can be divided into "correlational" and "causational" groups. This study monitored health claims and costs over 3 years for all shifts in seven industrial plants and administered a survey canvassing a range of behavioral and attitudinal dimensions. Shift workers differed from day workers in symptomatology and health claims, and, in their feelings of support from management, safety attitudes, and practices, felt support from friends and family, alcohol and tobacco use, and other dimensions. Because shiftwork was associated with adverse health effects but these other measured dimensions were not, the explanation that health effects are due to shiftwork itself rather than to its attitudinal or demographic correlates is supported.

Adult↗
PubMed1994

Simultaneous progressive multifocal leukoencephalopathy, Epstein-Barr virus (EBV) latent infection and cerebral parenchymal infiltration during chronic lymphocytic leukemia.

We report a non-HIV patient who had B chronic lymphocytic leukemia (CLL) with progressive multifocal leukoencephalopathy (PML) and diffuse cerebral leukemic parenchymal infiltration in the presence of JC virus and Epstein-Barr virus (EBV) cerebral co-infection. Multiple subcortical hypodensities lining the cortico-subcortical junction were present within the white matter on computerized tomography (CT) scan, with large areas of high signal intensity on T2-weighted sequences on magnetic resonance imaging (MRI). JCV DNA was identified in peripheral blood nuclear cells and cerebrospinal fluid polymerase chain reaction (PCR) DNA/DNA hybridization plus Southern blot analysis. Frontal stereotactic biopsy confirmed the diagnosis of PML by immunocytochemistry, in situ hybridization (ISH) with JC Enzo probe and electron microscopy. Leukemic B cells with the same phenotype as leukemic blood cells were disseminated in the demyelinated areas. They were labeled by anti-latent membrane protein and by BamHl W EBV probe after ISH. Adhesion and activation molecules were positive for CD23. Autopsy showed diffuse visceral leukemic infiltration without acutization. EBV-transformed B lymphocytes would favour JCV penetration and/or intracerebral reactivation of previously latent JCV infection with further development of simultaneous PML and cerebral CLL infiltration in an immunosuppressed patient.

B-Lymphocytes↗
PubMed1993

Development of monoclonal antibodies specific for 1,N2-ethenodeoxyguanosine and N2,3-ethenodeoxyguanosine and their use for quantitation of adducts in G12 cells exposed to chloroacetaldehyde.

Monoclonal antibodies specific for N2,3-ethenodeoxyguanosine (N2,3-epsilon dGuo) and 1,N2-ethenodeoxyguanosine (1,N2-epsilon dGuo) were developed. In a competitive ELISA, 50% inhibition of binding of the N2,3-epsilon dGuo specific antibody (ETH1) was achieved with 18 fmol of N2,3-epsilon dGuo. Fifty per cent inhibition of the 1,N2-epsilon dGuo-specific antibody (ETH2) required 11 pmol 1,N2-epsilon dGuo. Immunoassays for N2,3-epsilon dGuo and 1,N2-epsilon dGuo in single-stranded DNA were developed using these antibodies. The immunoassays could detect as little as 48 fmol of N2,3-epsilon dGuo or 340 fmol 1,N2-epsilon dGUO in 25 micrograms of single stranded DNA. These assays and previously developed immunoassays for 1,N6-ethenodeoxy-adenosine (1,N6-epsilon dAdo) and 3,N4-ethenodeoxycytidine (3,N4-epsilon dCyd) were used to measure etheno adduct levels in DNA of cells exposed to chloroacetaldehyde. The cells used were V79 cells with an inactivated hprt gene and a single copy of the bacterial gpt gene (G12 cells). The most abundant etheno adduct was 1,N6-epsilon dAdo, followed by 3,N4-epsilon dCyd and N2,3-epsilon dGuo. 1,N2-epsilon dGuo was not detected in chloro-acetaldehyde-treated G12 cells. Chloroacetaldehyde was also shown to be mutagenic in these same cells.

Acetaldehyde↗
PubMed1993

Both O4-methylthymine and O4-ethylthymine preferentially form alkyl T.G pairs that do not block in vitro replication in a defined sequence.

The mutagenic potential of O4-methylthymine (m4T) and O4-ethylthymine (e4T) was determined by a primer extension assay on a 25mer oligonucleotide containing a single site-specifically incorporated modified thymine. The e4T-containing oligonucleotide was prepared by using a new synthetic procedure suitable for large alkyl groups on thymine. The second-order rate constants, K(app)m and V(rel)max, permitted calculation of the frequency of formation and extension of modified base pairs compared to Watson-Crick pairing. With both m4T and e4T, the T.G type pairing was formed at least 10-fold more frequently than the nonmutagenic alkyl T.A pairing. However, there was a small but reproducible preference for m4T.G pairing. In both cases T-->C transitions would result. There was no evidence for formation of alkyl T.C or T.T. These data suggest that reported T-->A transversions by ethylation are not likely to result from O4-alkylthymine. In contrast to insertion, extension beyond alkylthymine under kinetic conditions did not occur with alkyl T.A. but only with the alkyl T.G termini. For this latter T.G type pairing, the larger ethyl group did not hinder extension compared to that of the methyl group, in the sequence studied. Under non-limiting conditions of dNTP concentration and time, complete replication could be demonstrated for both methyl- and ethyl-containing oligonucleotides. We conclude that the increase in size of the alkyl group from methyl to ethyl does not significantly affect the mutagenic potential and type of mutations of O4-alkylthymine in vitro.

Adenosine Triphosphate↗
PubMed1992

Both purified human 1,N6-ethenoadenine-binding protein and purified human 3-methyladenine-DNA glycosylase act on 1,N6-ethenoadenine and 3-methyladenine.

We previously described a protein, isolated from human tissues and cells, that bound to a defined double-stranded oligonucleotide containing a single site-specifically placed 1,N6-ethenoadenine. It was further demonstrated that this protein was a glycosylase and released 1,N6-ethenoadenine. We now find that this enzyme also releases 3-methyladenine from methylated DNA and that 3-methyladenine-DNA glycosylase behaves in the same manner, binding to the ethenoadenine-containing oligonucleotide and cleaving both ethenoadenine and 3-methyladenine from DNA containing these adducts. The rate and extent of glycosylase activities toward the two adducts are similar.

Adenine↗
PubMed1992

Partial purification of a human DNA glycosylase acting on the cyclic carcinogen adduct 1,N6-ethenodeoxyadenosine.

We previously reported that a variety of human cells and tissues contained a Mr35,000 DNA-binding protein which selectively recognized a single 1,N6-ethenoadenine in a defined 25-base double-stranded oligonucleotide (B. Rydberg et al., Proc. Natl. Acad. Sci. USA, 88: 6839-6842, 1991). We now demonstrate that incubation of the same duplex with 50-fold partially purified binding protein from human placenta results in release of the free 1,N6-ethenoadenine base, indicative of DNA glycosylase action. This enzyme activity appears unique in that it excises a cyclic adduct resulting from a known human carcinogen.

Adenine↗