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Biomedical subjects

B Singer

Publications and source records attributed to B Singer.

At least 55 records · Page 3Linked to original sources
PubMed1996

An unusual mechanism for the major human apurinic/apyrimidinic (AP) endonuclease involving 5' cleavage of DNA containing a benzene-derived exocyclic adduct in the absence of an AP site.

The major human apurinic/apyrimidinic (AP) endonuclease (class II) is known to cleave DNA 5' adjacent to an AP site, which is probably the most common DNA damage produced hydrolytically or by glycosylase-mediated removal of modified bases. p-Benzoquinone (pBQ), one of the major benzene metabolites, reacts with DNA to form bulky exocyclic adducts. Herein we report that the human AP endonuclease directly catalyzes incision in a defined oligonucleotide containing 3,N4-benzetheno-2'-deoxycytidine (pBQ-dC) without prior generation of an AP site. The enzyme incises the oligonucleotide 5' to the adduct and generates 3'-hydroxyl and 5'-phosphoryl termini but leaves the pBQ-dC on the 5' terminus of the cleavage fragment. The AP function of the enzyme is not involved in this action, as no preexisting AP site is present nor is a DNA glycosylase activity involved. Nicking of the pBQ-dC adduct also leads to the same "dangling base" cleavage when two Escherichia coli enzymes, exonuclease III and endonuclease IV, are used. Our finding of this unusual mode of action used by both human and bacterial AP endonucleases raises important questions regarding the requirements for substrate recognition and catalytic active site(s) for this essential cellular repair enzyme. We believe this to be the first instance of the presence of a bulky carcinogen adduct leading to this unusual mode of action.

Amino Acid Sequence↗
PubMed1996

Replication of O4-methylthymine-containing oligonucleotides: effect of 3' and 5' flanking bases on formation and extension of O4-methylthymine . guanine basepairs.

Four 25-mer oligonucleotides containing m4T(T*) with the 3'-->5' sequences A-T*-A, A-T*-C, G-T*-G and G-T*-C, together with four oligonucleotides containing the same sequences with unmodified T, were studied for the effect of the immediate 5' and 3' bases flanking m4T on in vitro replication. Under enzyme-limiting conditions, all m4T-containing oligomers showed a varying sequence-dependent blockage at the base 3' to m4T. However, with time and high concentrations of dNTP's, full replication beyond m4T did occur with Klenow fragment (Kf), although substantial blockage remained, particularly in the case when the sequence was 3'-A-T*-A-5'. The extent of blockage at the base 3' to m4T, compared to complete replication using Kf, followed the order A-T*-A > A-T*-C >> G-T*-C > or = G-T*-G. Two other DNA polymerases, HIV-RT and Sequenase, which, unlike Kf, do not have 3' proofreading activity differ, agreed with data obtained with Kf. We conclude that the extent of complete replication of m4T-containing oligomers is primarily a function of the 3' flanking base with a lesser effect of the 5' base and requires specific dGTP insertion opposite m4T. These data suggest that a 5' G . C pair favors the formation of the following T*-G and this latter step is rate-limiting. However, once a T*-G pair is formed, rapid elongation to full length transcripts occurs.

Autoradiography↗
PubMed1996

DNA damage: chemistry, repair, and mutagenic potential.

This paper is an overview on the chemical and biochemical effects of three different human carcinogens on DNA in vitro in terms of adducts, their effect on replication, in vitro and in vivo, and their repair by enzymes from human cells. The carcinogenic compounds chosen for specific discussion are N-nitroso alkylating agents: the vinyl chloride metabolite, chloroacetaldehyde (CAA), and the benzene metabolite, p-benzoquinone (p-BQ).

Acetaldehyde↗
PubMed1996

1,N6-ethenoadenine and 3,N4-ethenocytosine are excised by separate human DNA glycosylases.

We previously reported our finding that human cells contain glycosylase activity toward all four etheno bases formed in DNA by chloroacetaldehyde and related bi-functional aldehydes. By enzyme purification, including FPLC, we isolated two separate glycosylase activities for 1,N6-ethenoadenine (epsilon A) and for 3,N4-ethenocytosine (epsilon C) respectively, from crude HeLa cell-free extracts, which also contained a number of well-described glycosylases. When Mono-S FPLC purified proteins were assayed against defined oligomers containing either epsilon A or epsilon C, it was found that epsilon A and epsilon C glycosylases were completely separated. It could also be demonstrated that each enzyme bound to and cut only epsilon A- or epsilon C-containing oligomers respectively. There was no overlap in specificity for these two substrates. Several other human glycosylase substrates were also tested and none were cleaved by epsilon C glycosylase. The epsilon C glycosylase activity identified in the present study apparently represents a previously unknown glycosylase. This work also suggests that enzyme recognition of closely related DNA adducts may depend upon subtle changes in local conformation.

Adenine↗
PubMed1996

Psychological well-being: meaning, measurement, and implications for psychotherapy research.

A model of positive psychological functioning that emerges from diverse domains of theory and philosophy is presented. Six key dimensions of wellness are defined, and empirical research summarizing their empirical translation and sociodemographic correlates is presented. Variations in well-being are explored via studies of discrete life events and enduring human experiences. Life histories of the psychologically vulnerable and resilient, defined via the cross-classification of depression and well-being, are summarized. Implications of the focus on positive functioning for research on psychotherapy, quality of life, and mind/body linkages are reviewed.

Adaptation, Psychological↗
PubMed1996

Spatial pooling of contrast in contrast gain control.

We report psychophysical measurements of spatial pooling functions for contrast gain control. We use a nulling technique to measure the dependence of contrast induction on the spatial frequency of a sinusoidal contrast modulation. This dependence on spatial frequency, when transformed, provides the profile of a spatial pooling function. The measured profiles are fitted well by Gaussians. We confirm earlier results that the area over which spatial pooling takes place depends on the scale of the modulated pattern. We also find that pooling functions are similar for achromatic and isoluminant stimuli.

Contrast Sensitivity↗
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

Solitary cystic lymphangioma.

A young girl had a large isolated and localized multicystic lesion of the right humerus, which was revealed by a pathological fracture. MRI and bone biopsy showed liquid-filled bone cysts. The final diagnosis was localized cystic lymphangiomatosis.

Bone Neoplasms↗
PubMed1995

Gender differences in patterns of HPA axis response to challenge: Macarthur studies of successful aging.

Previous research has suggested that women may have greater and more prolonged hypothalamic-pituitary-adrenal response to challenge at older ages. Data on patterns of ACTH and cortisol responses to a "naturalistic" driving simulation challenge were examined to test the hypothesis that older women (aged 70-79 years) would show greater response than a comparable group of older men. Analyses of mean ACTH or cortisol responses in terms of maximal increase, area under the curve and repeated measures ANOVA did not reveal significant gender differences. By contrast, analyses of the joint occurrence of ACTH and cortisol responses above the respective sample medians indicated that women were significantly more likely to respond to the challenge with elevated ACTH and cortisol responses. The lack of significant gender difference in the analyses of mean scores resulted from the sensitivity of this measure of typically (or average response) to outliers in the data, thereby substantially distorting the report of typical (or average) responses for the male population. Analyses of the temporal patterns of ACTH and cortisol response in terms of the stability of the subject's relative rank at each sampling time indicated that: (1) there was considerable stability of rankings based on the initial cortisol response (though not ACTH) through the recovery period; and (2) women exhibited greater instability than men, being more likely to exhibit shifts in rank from high to low over time. These findings suggest that: (1) as a group, older women are more likely to show larger responses than comparably healthy older men to a "naturalistic" driving simulation challenge; (2) there is considerable stability in patterns of cortisol response from initial response at 20 min through the recovery period, indicating that those showing greater initial response continue to experience higher levels of cortisol during the post-challenge recovery period; and (3) instability is more common among women than men.

Adrenocorticotropic Hormone↗
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↗