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Biomedical subjects

B Singer

Publications and source records attributed to B Singer.

At least 109 records · Page 6Linked to original sources
PubMed1989

Black/white differences in health status and mortality among the elderly.

Grade of membership (GOM) representations are used to characterize and compare the health status of a very heterogeneous sample of blacks and whites in an elderly cohort of 2,806 noninstitutionalized men and women living in New Haven, Connecticut. They were interviewed in 1982 as part of the Established Populations for the Epidemiologic Study of the Elderly (EPESE). Ideal profiles based on functional disabilities, chronic diseases, and selected biomedical and behavioral risk factors are constructed empirically. Each individual in the sample is represented by a set of GOM scores, interpreted as degrees of similarity of his or her health record to each of the profiles. Four profiles emerge from GOM analyses: healthy elderly, elderly with cognitive impairment, elderly with impairment in mobility function and physical performance and with selected chronic conditions, and elderly with major limitations in activities of daily living and multiple chronic conditions. Although elderly blacks and whites generally have similar configurations of profiles, there are important differences, especially when chronic conditions are related to specific types of functional impairments. Questions about and claims for black/white mortality crossovers at older ages, usually addressed with aggregate data, are examined conditional on GOM scores that correspond to diverse combinations of disabilities (or lack thereof) together with housing characteristics of cohort members (e.g., whether they live in public housing for the elderly or in owned or rented housing in the community).

Activities of Daily Living↗
PubMed1988

Formation of interstrand cross-links in chloroacetaldehyde-treated DNA demonstrated by ethidium bromide fluorescence.

Chloroacetaldehyde, the stable metabolite of the human carcinogen vinyl chloride, forms interstrand cross-links in vitro in salmon sperm DNA and in the alternating copolymer, poly(deoxyadenylate-deoxythymidylate) [poly(dA-dT)]. Formation of the cross-link was a function of both time of reaction and concentration of chloroacetaldehyde. Cross-linking in chloroacetaldehyde-treated poly(dA-dT) was detected initially by changes in renaturation hysteresis [Singer et al., Carcinogenesis (Lond.), 5: 1165-1171, 1984]. This has been confirmed and quantitated using the relative fluorescence of ethidium bromide after denaturation and reannealing at 40 degrees C. Three percent cross-linking was detected after 10 min reaction with 20 mM chloroacetaldehyde at 24 degrees C. In DNA the relative fluorescence of ethidium bromide after denaturation and rapid cooling was used to estimate the number of cross-links formed. Three times as much cross-linking occurs in DNA compared to poly(dA-dT) under identical reaction conditions. The postulated structure for an interstrand cross-link in poly(dA-dT) is a hydroxyethyl bridge across the strands between the N6-amino groups of alternate adenine residues. In DNA, other amino groups in the proper configuration can be involved.

Acetaldehyde↗
PubMed1988

Nucleoside adducts are formed by cooperative reaction of acetaldehyde and alcohols: possible mechanism for the role of ethanol in carcinogenesis.

The exocyclic amino groups of ribonucleosides and deoxyribonucleosides react rapidly at ambient temperature with acetaldehyde and alcohols to yield mixed acetals [--NH--CH(CH3)OR]. Nucleotides and nucleoside di- and triphosphates also react. Depending on the nucleoside used and on the relative amounts of aldehyde, alcohol, and water, preparative reactions reach equilibrium with yields up to 75% in a few hours. The structures have been confirmed by fast atom bombardment MS and proton NMR. Half-lives at 37 degrees C have been determined, and maximum stability is in the pH range of 7.5-9.5. In the absence of alcohol, acetaldehyde-nucleoside adducts could be isolated at 4 degrees C, but these were too unstable to characterize except for their UV spectra, also at 4 degrees C. Ethanol is often present in human blood and tissues, and acetaldehyde is its initial metabolic product, as well as being formed by many other metabolic processes. Both chemicals have separately been implicated in carcinogenic and other cytopathologic processes, but no cooperative mechanism has been proposed. The reactions reported here are of biological concern because they also occur in dilute aqueous solution. These findings supply a mechanism by which ethanol can be covalently bound to nucleic acids under physiological conditions.

Alcohols↗
PubMed1987

Comparison of the relative mutagenicities of O-alkylthymines site-specifically incorporated into phi X174 DNA.

The relative mutagenicities of O-alkylthymine-DNA adducts were analyzed in vivo by site-specific mutagenesis. Purified DNA polymerases were used to incorporate O4-methyl (Me)-, O4-ethyl (Et)-, O4-isopropyl (iPr)-, or O2-Me-dTTP onto the 3' terminus of a synthetic oligonucleotide (15-mer) hybridized to phi X174 am3 DNA. The product oligonucleotides were further extended in the presence of unmodified dNTPs to yield 21-mers containing single O-alkylthymine adducts opposite the adenine residue of the bacteriophage amber codon. Polyacrylamide gel electrophoresis and nearest-neighbor analyses confirmed the identities and nucleotide positions of the adducts. Transfection and replication of the site-specifically alkylated DNAs in ada- Escherichia coli (defective in the alkyltransferase capable of repairing O4-alkylthymine-DNA adducts) yielded mutant progeny phage with reversion frequencies of: O4-Me-dThd (19.5 X 10(-6) ) greater than O4-Et-dThd (7.5 X 10(-6) ) greater than O4-iPr-dThd (3.0 X 10(-6) ) greater than or equal to O2-Me-dThd (1.0 X 10(-6) ) approximately equal to dThd (2.0 X 10(-6) ). None of the adducts produced mutations above background following replication in ada+ E. coli. DNA sequence analyses of 40 independently isolated mutant phage derived from the O4-Me- or O4-Et-dThd-containing DNAs showed that all mutants contained guanine residues opposite the original site of the alkylthymines. These data are consistent with a mechanism of mutagenesis involving the formation of O4-alkyl-T.G base pairs during DNA replication in E. coli and suggest that the formation of A.T----G.C transition mutations is characteristic of mutagenesis by O4-Me- and O4-Et-dThds in vivo.

Bacteriophage phi X 174↗
PubMed1987

The vinyl chloride-derived nucleoside, N2,3-ethenoguanosine, is a highly efficient mutagen in transcription.

N2,3-Ethenoguanine (N2,3-epsilon G) was recently identified in the liver of vinyl chloride-exposed rats. We have now synthesized the nucleoside and the 5'-diphosphate which was copolymerized with CDP. The deoxypolynucleotide complement, synthesized by AMV reverse transcriptase contained, in addition to dG, dC and dT. The total pyrimidine content was approximately equivalent to the N2,3-epsilon G content of the template. Incorporation of dC is neither lethal nor mutagenic, while dT incorporation represents a mutagenic event, occurring with approximately 20% frequency. N2,3-epsilon G X dT base pairs can have two hydrogen bonds with minimal helical distortion, as is also the case for N2,3-epsilon G X C base pairs. N2,3-epsilon G is the only derivative formed in vivo by the human carcinogen, vinyl chloride, that can be shown to have a high probability of causing transitions which could initiate malignant transformation.

Base Composition↗
PubMed1987

Lack of demonstrable density-dependent fecundity of schistosomiasis mansoni: analyses of Egyptian quantitative human autopsies.

Conflicting interpretations regarding the fecundity of schistosomes infecting human beings have arisen and are, in part, due to the inability to directly measure the parameters. The inability to experimentally manipulate human beings necessitates the use of surrogate variables, i.e., number of eggs per gram of feces, as an indicator of worm burden. This study reanalyzes data from quantitative autopsies performed in Egypt by Cheever and colleagues on individuals with active Schistosoma mansoni infections. Exploratory regression analysis of the relationship of female worms recovered to eggs/g of feces and of female worms to eggs retained in host tissues suggests a linear relationship in both cases. Over the observed range of female worms recovered from an individual human being, the estimated worm fecundity, as measured by the number of eggs either in feces or retained in tissues per female worm, is not significantly different from a constant value. Hence, density-dependent fecundity of S. mansoni in the human host, as suggested by others, is not demonstrated in these data.

Adolescent↗
PubMed1987

[A case of acute sensory and autonomic neuropathy with regression].

The authors report the case of a 24 year old man with no previous disease who presented with a severe autonomic neuropathy. This included major gastrointestinal dysfunction characterised by decreased peristalsis without distension and paralysis of the gall bladder, and orthostatic hypotension with a normal cardiac tachycardia reflex. There was an associated sensory neuropathy affecting heat sensitivity without motor dysfunction and an increased CSF protein content. The proprioceptive nerve fibre conduction was decreased but another nerve conduction was normal initially. The mesenteric plexuses examined during sigmoidectomy performed for peritonitis due to multiple bowel perforations caused by fecoliths, showed no significant changes. Peripheral nerve biopsy revealed massive rarefaction of myelinated fibres which were of small diameter, and of the unmyelinated fibres, mainly due to axonal degeneration. Only two similar cases with incomplete recovery were found in the literature. In our case, a complete recovery was observed. The cause of the condition is unknown.

Acute Disease↗
PubMed1987

O-Alkyl deoxythymidines are recognized by DNA polymerase I as deoxythymidine or deoxycytidine.

The O2- and O4-methyldeoxythymidine triphosphates (O-alkyl dTTP) can be used to substitute for dTTP in Escherichia coli DNA polymerase I (Pol I)-catalysed synthesis of poly[deoxyadenosine-deoxythymidine] (dA-dT). When incorporated into the polynucleotide, no detectable perturbation of structure occurred with even 20% O-methyldeoxythymidine in place of dT. However, on replication of such polymers with Pol I, significant amounts of deoxyguanosine triphosphate (dGTP) were incorporated, as well as high levels of deoxyadenosine triphosphate (dATP), indicating tautomer-like behaviour. Higher homologues, such as O4-ethyl (e4) dTTP or O4-isopropyl (ip4) dTTP, could also replace dTTP, but with lower efficiency. Nevertheless, their presence, like O4-methyl (m4) dT substitutions, caused transitions as well as inhibiting enzyme digestion with a variety of 3' nucleases, particularly to the 3'----5' exonuclease activity (proofreading) of polymerases. Further proof of mutagenicity comes from site-directed experiments placing m4dT or e4dT in place of dT at position 587 in am3 of phi X174, in which all revertants sequenced had A----G transitions. This implies that, since m4dT and e4dT are poorly repaired in eukaryotes, it is likely that they will remain in the DNA and lead to effects on enzyme activity, as well as mutations which contribute to the carcinogenicity of N-nitroso compounds.

Alkylation↗
PubMed1986

C4-methyldeoxythymidine replacing deoxythymidine in poly[d(A-T)] renders the polymer resistant to the 3'----5' exonuclease activity of the Klenow and T4 DNA polymerases.

We previously reported that O4-alkyl dTTPs could replace, for short times, dTTP in polymer synthesis [Singer et al., PNAS 83, 26-32, 1986]. The reasons for such early termination of synthesis could be either proofreading or the eventual formation of weakly paired primer termini. Utilizing the known 3'----5' exonucleolytic activity of polymerases, in the absence of dNTPs, enabled us to conclude that, in contrast to the digestibility of poly[d(A-T)] which yielded the expected 3'-mononucleotides, the polymerizing enzymes did not digest O4-methyl dT or its neighbors. The presence of the resistant alpha-phosphorothionate linkage did not prevent measurable digestion of poly[d(A-T)] by the Klenow fragment. This, together with evidence that polymerization of O4-methyl dTTP is favored at low temperatures, supports the model proposed by Ollis et al. [Nature 313, 762-766, 1985] showing independent domains for the two activities in the Klenow fragment.

Alkylation↗
PubMed1986

O4-Methyl-, O4-ethyl-, and O4-isopropylthymidine 5'-triphosphates as analogues of thymidine 5'-triphosphate: kinetics of incorporation by Escherichia coli DNA polymerase I.

O4-Methyl-, O4-ethyl-, and O4-isopropylthymidine 5'-triphosphates, which can be formed by N-nitroso carcinogens, were tested for their ability to substitute for thymidine 5'-triphosphate (dTTP) in synthesis catalyzed by Escherichia coli DNA polymerase I (Pol I) by using activated DNA or synthetic polymers as templates. All could substitute for dTTP for short periods, the rate and extent decreasing with the size of the alkyl group. Because the structure of O4-alkylthymidine does not permit normal hydrogen bond formation with deoxyadenosine, it was inferred that eventual formation of a poor or frayed primer end was responsible for termination of synthesis. Synthesis of polymers at temperatures ranging from 0 to 40 degrees C showed that the extent of incorporation using the O4-alkyl-dTTPs was favored, relative to dTTP, when the terminal helical structure was stabilized by low temperatures. Kmapp values were determined for each O4-alkyldeoxynucleoside 5'-triphosphate. These values were 0.7 microM for dTTP, 5 microM for methyl-dTTP, 11 microM for ethyl-dTTP, and 33 microM for isopropyl-dTTP. O4-Alkyl-dTTPs were tested for their ability to inhibit or compete with dTTP incorporation and found to have a minimal effect, even when present at high concentration. These experiments indicated that Pol I can incorporate deoxynucleotides with O4-alkyl substituents into an ordered DNA structure. A postulated base-pairing scheme with deoxyadenosine is described.

Base Composition↗
PubMed1986

O4-Methyl, -ethyl, or -isopropyl substituents on thymidine in poly(dA-dT) all lead to transitions upon replication.

In a previous paper, we reported that O4-methyl dTTP can be incorporated into poly(dA-dT) in place of thymidine without distortion of the helical structure, but on replication it could behave as deoxycytidine and misincorporate dGTP. Only weak interactions are possible for any O4-modified T X A pair. While O4-alkyl T X G pairing should be favored, experiments to detect the ability of Escherichia coli DNA polymerase I (pol I) to utilize the triphosphate as dCTP were ambiguous. dTTPs with larger alkyl groups (ethyl, isopropyl) have now been synthesized and tested for their recognition as dTTP by pol I. Enhanced steric hindrance could be expected, particularly for O4-isopropyl dTTP, which has a three-carbon branched chain. However, both compounds behaved qualitatively like O4-methyl dTTP, being incorporated into poly(dA-dT) and then directing deoxyguanosine misincorporation by pol I. Quantitative comparisons of mutagenicity were not possible because of the finding that, unlike polymers made with O4-methyl dTTP, those made with ethyl or isopropyl dTTP were resistant to hydrolysis by using a variety of nucleases. The frequent misincorporations of dGTP would be expected to produce transitions in vivo. O4-ethyldeoxythymidine is very poorly repaired in vivo, which would also be expected for repair of O4-isopropyldeoxythymidine. Therefore, under suitable conditions, these particular carcinogen products are likely to be initiators of carcinogenesis.

DNA Polymerase I↗
PubMed1986

Mutagenic potential of O4-methylthymine in vivo determined by an enzymatic approach to site-specific mutagenesis.

O4-Alkylthymine-DNA adducts have been implicated as causative lesions in chemical mutagenesis and carcinogenesis. To directly assess the mutagenic potential of these adducts in vivo, we have designed an enzymatic technique for introducing nucleotide analogues at predetermined sites of biologically active DNA. Escherichia coli DNA polymerase I was used in vitro to incorporate a single O4-methylthymine residue at the 3' terminus of an oligonucleotide primer opposite the adenine residue of the amber codon in bacteriophage phi X174 am3 DNA. After further extension of the primer with unmodified nucleotides, the partial-duplex product was transfected into E. coli spheroplasts. Replication of the site-specifically methylated DNA in E. coli deficient in O4-methylthymine-DNA methyltransferase (ada-) yielded 10-fold more mutant progeny phage than replication of nonmethylated DNA; no increase in mutation frequency was observed after replication in repair-proficient (ada+) E. coli. The DNA from 20 independently isolated mutant plaques all contained A.T----G.C transitions at the original site of O4-methylthymine incorporation. These data demonstrate that O4-methylthymine induces base-substitution mutations in E. coli and suggest that this adduct may be involved in mutagenesis by N-nitroso methylating agents. This enzymatic technique for site-specific mutagenesis provides an alternative to the chemical synthesis of oligonucleotides containing altered bases.

Base Sequence↗
PubMed1986

Replication and transcription of polynucleotides containing ethenocytosine, ethenoadenine and their hydrated intermediates.

Replication with Escherichia coli DNA polymerase I (Pol I) and transcription with DNA-dependent RNA polymerase from Escherichia coli or calf thymus, using as templates synthesized ribo- or deoxyribopolynucleotides containing 1,N6-ethenoadenine (epsilon A) or 3,N4-ethenocytosine (epsilon C), showed that only epsilon C could direct significant misincorporation. The hydrated intermediate of epsilon C caused errors only upon transcription, but not upon replication. epsilon A was a very poor mutagen as assessed by replication with Pol I. Transcription of polynucleotides containing epsilon A under error-prone conditions caused frequent A misincorporation which could not be detected in replication assays. It is concluded that epsilon C may lead to point mutations, specifically directing the misincorporation of thymine. The analogous derivative, epsilon A, is bulky and is likely to be bypassed rather than read. This mechanism could cause frameshift mutation, as generally found for other bulky adducts.

Adenosine↗