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Biomedical subjects

B R Shaw

Publications and source records attributed to B R Shaw.

At least 37 records · Page 2Linked to original sources
PubMed1994

Bisulfite induces tandem double CC-->TT mutations in double-stranded DNA. 2. Kinetics of cytosine deamination.

Deamination of cytosine to uracil in double-stranded DNA (ds DNA) by sodium bisulfite has been monitored with a sensitive genetic assay. In this system, reversion of a mutant in the lacZ alpha gene coding sequence of bacteriophage M13mp2 C141 was detected by employing an ung- bacterial strain defective in the enzyme uracil glycosylase. Within the 4-base target, it is possible to measure the rates of induction of C-->T, C-->A, C-->G, and CC-->TT mutations in DNA that has been incubated at physiological temperature and pH and then transfected into ung+ and ung- E. coli cells, respectively, for amplification and detection of the mutation. For concentrations of bisulfite from 1 to 50 mM, the reversion frequency in ung- cells increased linearly with time of incubation. The most interesting features of the bisulfite reaction were as follow: (1) Mutations were reduced 5-fold in ung+ cells, indicating ung is involved in repair of bisulfite-treated transforming DNA. (2) Sequencing of 157 revertants revealed that C-->T and tandem CC-->TT transition mutations comprised 100% of the mutations scored. (3) A unique finding was that, at the highest concentrations and longest incubation times, almost every mutant obtained in ds DNA exposed to bisulfite was found to be a CC-->TT tandem double mutation. (4) The high frequency of tandem double mutants is inconsistent with two random, independent mutational events and, coupled with the observed ung dependence, lends support to the concept of catalytic deamination, wherein bisulfite induces deamination in contiguous cytosines by a concerted mechanism.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacteriophage M13↗
PubMed1994

Anti-inflammatory and anti-osteoporotic activities of base-boronated nucleosides and phosphate-boronated nucleotides in rodents.

The 2'-deoxyribonucleoside cyanoboranes were effective anti-inflammatory agents in rodents at 2-8 mg/kg; they blocked induced edema, septic shock, and pleurisy. Overall compounds 3',5'-O-(bis- (triisopropylsilyl)-2'-deoxyinosine (1), 3',5'-O-bis(triisopropylsilyl)-2'-deoxycytidine (10), N3-(cyanoboryl)-2'-deoxycytidine (11), N7-(cyanoboryl)-N2-isobutyryl- 3',5'-O-bis(triisopropylsilyl)-2'-deoxyguanosine (20), and N7-(cyanoboryl)-N2- isobutyryl-5'-O-(4,4'-dimethoxytrityl)-3'-O-(triisopropylsilyl)-2' -deoxyguanosine (22) were the most active when all the anti-inflammatory screens are considered. The agents also blocked both local and central pain caused by inflammation. These nucleosides blocked calcium resorption but were less effective compared to other amine carboxyboranes. The inflammation process appeared blocked by these compounds because of their effectiveness in reducing both hydrolytic lysosomal enzyme and proteolytic enzyme activities. The agents were also dual inhibitors of prostaglandin cyclooxygenase and 5'-lipoxygenase activities in leukocytes and macrophages. These agents at 10(-4) M demonstrated no specific organ toxicity to ileum mucosa cells grown in tissue culture.

Animals↗
PubMed1993

Cytosine deamination in mismatched base pairs.

The rate of deamination of cytosine in mismatched base pairs has been determined. Incubation of M13mp2 nicked heteroduplex DNA molecules containing T.C or C.C mispairs in the lacZ alpha-complementation gene results in deamination of cytosine to uracil, producing T.U or C.U mispairs. Strands which have undergone deamination at the target site to produce uracil will yield dark blue plaque revertants, while all other strands yield faint blue or colorless plaque phenotypes upon transfection of an ung- alpha-complementation Escherichia coli host strain. Rate constants were calculated from the reversion frequencies for several different heteroduplexes incubated at either 60 or 37 degrees C. For the 60 degrees C incubations, the hydrolytic deamination rate constants for mispairs in three different local sequence environments ranged from 8 x 10(-10) to 40 x 10(-10) s-1. For incubations at 37 degrees C, the rate constants were between 0.4 x 10(-10) and 1.3 x 10(-10) sec-1. At both temperatures and for all mispairs, these rate constants are significantly greater than deamination rate constants in properly matched Watson-Crick G.C base pairs and are similar to those constants determined for cytosine deamination in single-stranded DNA. Since deamination most likely occurs via a single-stranded intermediate, the data suggest that, at 37 degrees C, the T.C and C.C mispairs exhibit from 20% to 100% single-stranded character. We conclude that cytosine residues involved in a mispair in DNA are 1-2 orders of magnitude more prone to deaminate to uracil than are cytosines in double-stranded DNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacteriophage M13↗
PubMed1993

Kinetics of bisulfite-induced cytosine deamination in single-stranded DNA.

The rate of bisulfite-induced deamination of cytosine to uracil in single-stranded (ss) DNA at physiological temperature and pH was monitored by a sensitive genetic assay. The assay is based on reversion of a mutation in the lacZ alpha gene of bacteriophage M13mp2 and employs ung- (NR9404) and ung+ (MC1061) bacterial strains which are isogenic except for uracil glycosylase activity. For ss DNA incubated with 1-50 mM bisulfite and transfected into an ung- cell strain, the reversion frequency increased linearly with time of incubation and with concentration of bisulfite. Of 54 revertants sequenced, all were C-->T transitions. Reduction in reversion frequency upon transfecting ss DNA into ung+ cells indicated that the majority of mutations were occurring via a uracil intermediate. Assuming that all revertants arose via uracil, the pseudo-first-order rate constant for deamination in 10 mM sodium bisulfite and 10 mM Hepes-NaOH, pH 7.4, at 37 degrees C as measured by transfecting into an ung- cell strain was 3.5 x 10(-10) s-1, as compared to a spontaneous background rate constant of 0.6 x 10(-10) s-1 in buffer alone.

Bacteriophages↗
PubMed1993

Echinomycin, a bis-intercalating agent, induces C-->T mutations via cytosine deamination.

Echinomycin, a bis-intercalating, antitumor drug, has been studied for its ability to induce the deamination of cytosine to uracil (C-->U) in double-stranded DNA. We have employed a sensitive lacZ alpha-complementation reversion assay to detect G.C-->A.T mutations at a number of sites in M13mp2 DNA to determine the extent to which distortions of DNA structure induced by echinomycin may affect C-->U rates. When double-stranded M13mp2 DNA with a 12-base target containing a CpG site was incubated at 37 degrees C, the reversion frequency of the echinomycin-treated DNA increased linearly over time, with a rate constant 3-fold greater than DNA incubated without echinomycin. Of the 11 ways that blue pseudo-revertants can occur in the target, 96% of the observed revertants arose from C-->T and tandem CC-->TT transitions, with 78% attributable to single-base C-->T changes at three sites. Transfection into ung+ cells decreased the reversion frequencies by 85% to near background levels, indicating that the increase in C-->T mutations was due to deamination of C to U. The cytosine deamination rate constants for the entire target at pH 6.0 and 37 degrees C were 1.2 x 10(-11) sec-1 for untreated DNA and 3.5 x 10(-11) sec-1 for echinomycin-treated DNA. The increase in C-->T mutation rates occurred at cytosines both proximal and distal to a CpG echinomycin-binding site. We hypothesize that this increase in deamination rate is due to a more open or single-stranded DNA structure caused by the echinomycin: DNA interaction.

Base Sequence↗
PubMed1993

Hypolipidemic activity of boronated nucleosides and nucleotides in rodents.

Base-boronated nucleoside and phosphate-boronated nucleotides were potent hypolipidemic agents in rodents, lowering both serum cholesterol and triglyceride levels. Rat VLDL and LDL cholesterol levels were generally reduced and HDL cholesterol levels were significantly elevated after 14 days dosing at 8 mg/kg/day. Tissue cholesterol, triglyceride and phospholipid levels were reduced by selected derivatives. Increased fecal excretion of lipids did not appear to be a mechanism by which these derivatives lowered serum lipids in rodents. Rather, the agents suppressed appetite and reduced the activities of rate-limiting enzymes for de novo lipid synthesis, specifically cytoplasmic acetyl CoA synthetase, squalene synthetase, and phosphatidylate phosphohydrolase with IC50 values of approximately 10(-5) m.

Animals↗
PubMed1992

Variable temperature infrared spectroscopy of cytosine-guanine base pairs: tautomerism versus polarization.

This report describes an infrared (IR) spectroscopic study of a model cytosine-guanine base pair. This base pair is part of a self-consistent experimental system based on lipophilic ribose derivatives of cytidine (C), guanosine (G) and O6-methylguanosine (O6MeG) that are soluble in non-aqueous, low dielectric solvents at appreciable concentrations. Previous experiments on this system have revealed different rotation dynamics for the amino bonds within the CG base pair, an observation that could be explained by the presence of rare tautomers (P.O. Lowdin, Reviews of Modern Physics 35,724 (1963)), or by mutual polarization of the base pairs (L.D. Williams, N.G. Williams and B.R. Shaw,J.Am.Chem.Soc. 112,829 (1990)). The IR spectra in the OH and NH stretching region indicate formation of hydrogen-bonded CG base pairs and self associates in 1,2-dichlorobenzene over a temperature range from 10 to 290K. Changes in the lineshapes and intensities of the IR bands with temperature correlate with phase transitions of the solvent, but no evidence is seen for an OH stretching band that would indicate the formation of hydroxyl tautomers within base pairs. Similarly, the relative intensities of the C = O stretching bands of CG in cyclohexane solution remain constant over this same temperature range, confirming that within the base pair, the tautomeric states of the bases remain essentially unperturbed in the 2-amino/6-keto form of G and the 2-keto/4-amino form of C. The spectra of O6-MeG aid in the band assignments, since this molecule is frozen in an equivalent of the 2-amino/6-hydroxyl tautomer, but without the OH group and its associated stretching band. We conclude that the probability of tautomerism does not appear to be sufficient to explain the different rotation dynamics for the two amino bonds of the CG base pair. Rather it is argued that mutual polarization within the base pair, which would increase the bond order of the amino bond of C within the base pair, can explain the results without the formation of unconventional tautomers.

Base Composition↗
PubMed1992

The synthesis and anti-neoplastic activity of N2-isobutyryl-2'-deoxyguanosine-N7-cyanoborane derivatives.

N2-Isobutyryl-2'-deoxyguanosine-N7-cyanoborane derivatives were observed to be potent antineoplastic agents and to be active against a number of human tissue culture tumor cells, e.g. Tmolt3 leukemia, HeLa-S3 uterine carcinoma. Selective agents were active against colon adenocarcinoma, osteosarcoma and glioma growth. These agents preferentially inhibited both DNA and RNA synthesis of L1210 cells. De novo synthesis of purines was significantly inhibited at the regulatory sites of PRPP amido transferase and IMP dehydrogenase. Other sites of inhibition were thymidylate synthetase, OMP decarboxylase and thymidine kinases. The agents also significantly reduced deoxyribonucleotide levels and caused DNA strand scission.

Animals↗
PubMed1990

A sensitive genetic assay for the detection of cytosine deamination: determination of rate constants and the activation energy.

Previously it has not been possible to determine the rate of deamination of cytosine in DNA at 37 degrees C because this reaction occurs so slowly. We describe here a sensitive genetic assay to measure the rate of cytosine deamination in DNA at a single cytosine residue. The assay is based on reversion of a mutant in the lacZ alpha gene coding sequence of bacteriophage M13mp2 and employs ung- bacterial strains lacking the enzyme uracil glycosylase. The assay is sufficiently sensitive to allow us to detect, at a given site, a single deamination event occurring with a background frequency as low as 1 in 200,000. With this assay, we determined cytosine deamination rate constants in single-stranded DNA at temperatures ranging from 30 to 90 degrees C and then calculated that the activation energy for cytosine deamination in single-stranded DNA is 28 +/- 1 kcal/mol. At 80 degrees C, deamination rate constants at six sites varied by less than a factor of 3. At 37 degrees C, the cytosine deamination rate constants for single- and double-stranded DNA at pH 7.4 are 1 x 10(-10) and about 7 x 10(-13) per second, respectively. (In other words, the measured half-life for cytosine in single-stranded DNA at 37 degrees C is ca. 200 years, while in double-stranded DNA it is on the order of 30,000 years.) Thus, cytosine is deaminated approximately 140-fold more slowly when present in the double helix. These and other data indicate that the rate of deamination is strongly dependent upon DNA structure and the degree of protonation of the cytosine. The data suggest that agents which perturb DNA structure or facilitate direct protonation of cytosine may induce deamination at biologically significant rates. The assay provides a means to directly test the hypothesis.

Bacteriophages↗
PubMed1989

Hydrolysis of N3-methyl-2'-deoxycytidine: model compound for reactivity of protonated cytosine residues in DNA.

Protonation of cytosine residues at physiological pH may occur in DNA as a consequence of both alkylation and aberrant base-pair formation. When cytosine derivatives are protonated, they undergo hydrolysis reactions at elevated rates and can either deaminate to form the corresponding uracil derivatives or depyrimidinate generating abasic sites. The kinetic parameters for reaction of protonated cytosine are derived by studying the hydrolysis of N3-methyl-2'-deoxycytidine (m3dC), a cytosine analogue which is predominantly protonated at physiological pH. Both deamination and depyrimidimation reaction rates are shown to be linearly dependent upon the fraction of protonated molecules. We present here thermodynamic parameters which allow determination of hydrolysis rates of m3dC as functions of pH and temperature. Protonation of cytosine residues in DNA, as induced by aberrant base-pair formation or base modification, may accelerate the rate of both deamination and depyrimidation up to several thousand-fold under physiological conditions.

Chemical Phenomena↗
PubMed1987

Base stacking and molecular polarizability: effect of a methyl group in the 5-position of pyrimidines.

Substitution of a methyl group in the 5-position of pyrimidines increases melting temperatures and modifies biological properties of DNA. Increased DNA stability is often attributed to hydrophobic interactions between water and the methyl group. However, we present evidence that the major effect of methyl substitution is to increase the molecular polarizability of the pyrimidine, thereby increasing the base stacking. Experimentally determined base stacking interaction constants for free bases in water are shown to correlate well with calculated molecular polarizability and DNA melting temperatures.

Adenine↗
PubMed1987

Histone modifications accompanying the onset of developmental commitment.

In the sea urchin, Strongylocentrotus purpuratus, three cell types comprise the 16-cell stage embryo: micromeres, macromeres, and mesomeres. We have analyzed these three cell types for nuclear proteins that were synthesized during the earliest stages of embryonic development. The most striking differences in composition of newly synthesized proteins were found between the micromeres, which are the most committed cell type, and the macromeres and mesomeres. First, the micromeres lacked triply modified forms of histone H3; the levels of doubly modified forms of H3 were also greatly reduced. In contrast, micromeres were enriched in a band which migrated at the position of unmodified, unacetylated, histone H3 protein. Second, the overall distribution of H2A histone variants differed among the three cell types. Compared with macromeres and mesomeres, micromeres had a higher ratio of alpha-stage to cleavage-stage (CS) histone H2A; the micromere nuclei were depleted by 50 and 35%, respectively, in embryonically synthesized histone CS-H2A. Third, micromeres displayed different profiles of H1 histones. (a) They contained a cleavage-stage H1 histone which migrated faster than that of macromeres and mesomeres. This protein displays the electrophoretic behavior expected for a protein with reduced levels of posttranslational covalent modification. (b) Micromeres also had reduced levels of an H1 histone (designated H1 alpha a) band found in the alpha-H1 region of macromeres and mesomeres. These changes in chromatin modification correlate with the degree of commitment of cells in the developing embryo; they may reflect differing activities of the chromatin modifying enzymes in the various cell types at the 16-cell stage. Thus, the newly synthesized chromatin proteins of the individual blastomere types already differ in the developing sea urchin by the 16-cell stage. We suggest that variations in histone subtypes and in the levels of activity of chromatin modifying enzymes, e.g., acetylases and phosphorylases, could be involved in commitment and differentiation of different cell types.

Acetylation↗
PubMed1987

DNA base modification: ionized base pairs and mutagenesis.

The nature of hydrogen bonding between normal and modified bases has been re-examined. It is proposed that hydrogen-bonding schemes may involve tautomeric, ionized or conformational forms (syn, anti and wobble). Several important cases are presented or reviewed in which physical evidence indicates the existence of ionized base pairs. When thermodynamic values determined in aqueous solution under physiological conditions are considered, it can be argued that base ionization will contribute substantially to the stability of many biologically relevant base pairs containing modified bases. A significant incidence of ionized bases in DNA may have important kinetic ramifications for the further chemical reactivity of both the modified base and its cross-strand pairing partner. Moreover, DNA structure at and surrounding ionized base pairs may be altered. For this reason, the model presented in this study should be useful as DNA-sequence analysis becomes more commonly applied to the study of mutagenesis.

Base Composition↗
PubMed1987

Protonated base pairs explain the ambiguous pairing properties of O6-methylguanine.

The base-pairing interactions of promutagenic O6-methylguanine (O6-MeGua) with cytosine and thymine in deuterated chloroform were investigated by 1H NMR spectroscopy. Nucleosides were derivatized at hydroxyl positions with triisopropylsilyl groups to obtain solubility in nonaqueous solvents and to prevent the ribose hydroxyls from forming hydrogen bonds. We were able to observe hydrogen-bonding interactions between nucleic acid bases in a solvent of low dielectric constant, a condition that approximates the hydrophobic interior of the DNA helix. O6-MeGua was observed to form a hydrogen-bonded mispair with thymine. Whereas O6-MeGua did not form hydrogen bonds with cytosine (via usual, wobble, or unusual tautomeric structures), it did form a 1:1 hydrogen-bonded complex with protonated cytosine. The pairing of unprotonated cytosine in chloroform is thus consistent with the known preference of O6-MeGua for thymine over cytosine in polymerase reactions. In contrast, the pairing of protonated cytosine is consistent with the greater stability of oligonucleotide duplexes containing cytosine.O6-MeGua as compared with thymine.O6-MeGua base pairs [Gaffney, B. L., Markey, L. A. & Jones, R. A. (1984) Biochemistry 23, 5686-5691]. Our observation that cytosine must be protonated in order to pair with O6-MeGua suggests that the cytosine.O6-MeGua base pair in DNA is stabilized by protonation of cytosine. Through this mechanism, methylation at the O6 position of guanine in double-stranded DNA could promote cross-strand deamination of cytosine (or 5-methylcytosine) to produce uracil (or thymine).

Base Composition↗
PubMed1986

Intraocular pressure elevation after pupillary dilation in open angle glaucoma.

Acute elevation of intraocular pressure frequently follows pupillary dilation in patients with primary open angle glaucoma. A retrospective study of 60 patients (116 eyes) with primary open angle glaucoma was done to assess the frequency and severity of intraocular pressure elevation following dilation with 2.5% phenylephrine hydrochloride (Neo-Synephrine) and 1% tropicamide (Mydriacyl). Significant pressure elevation (greater than 5 mm Hg) occurred in 37 eyes (32%). Marked pressure elevation (greater than 10 mm Hg) occurred in 14 eyes (12%). The only significant risk factor found was treatment with miotics. The change of intraocular pressure one hour after dilation was compared with the one-hour postoperative change in intraocular pressure in those patients (12 patients, 18 eyes) who subsequently underwent argon laser trabeculoplasty. No statistically significant correlation was found. There is a potential hazard of routine dilation of eyes with open angle glaucoma.

Adult↗