Elevated total peripheral leukocyte count may identify risk for neurological disability in asphyxiated term neonates.

OBJECTIVE: The present study investigated the relationship between neurologic outcome and total circulating white blood cell (WBC) and absolute neutrophil counts (ANCs) in the first week of life in term infants with hypoxic-ischemic encephalopathy (HIE). STUDY DESIGN: Long-term neurologic outcome at 18 months was measured retrospectively in 30 term neonates with HIE using the Pediatric Cerebral Performance Category Scale (PCPCS) score with outcomes dichotomized as either good or poor. We then compared white blood cell and ANC levels during the first 4 days of life and magnetic resonance imaging (MRI) obtained within the first month life between the two PCPCS groups. MRI was quantified using a validated scoring system. RESULTS: Neonates with good long-term outcomes had significantly lower MRI scores (indicating lesser injury) than neonates with poor outcomes. More importantly, neonates with poor outcomes had significantly higher WBC and ANC levels as early as12 h after birth and up to 96 h after birth compared to those with good outcomes. These data suggest that elevated peripheral neutrophil counts in the first 96 h of life may signal or predict adverse long-term outcome. CONCLUSIONS: Our findings suggest that elevated peripheral neutrophil counts in the first 96 h of life in term infants with HIE may contribute to abnormal neurodevelopmental outcome.

Examination of the DNA substrate selectivity of DNA cytosine methyltransferases using mass tagging.

The biological significance of cytosine methylation is as yet incompletely understood, but substantial and growing evidence strongly suggests that perturbation of methylation patterns, resulting from the infidelity of DNA cytosine methyltransferase, is an important component of the development of human cancer. We have developed a novel in vitro assay that allows us to quantitatively determine the DNA substrate preferences of cytosine methylases. This approach, which we call mass tagging, involves the labeling of target cytosine residues in synthetic DNA duplexes with stable isotopes, such as (15)N. Methylation is then measured by the formation of 5-methylcytosine (5mC) by gas chromatography/mass spectrometry. The DNA substrate selectivity is determined from the mass spectrum of the product 5mC. With the non-symmetrical duplex DNA substrate examined in this study we find that the bacterial methyltransferase HPA:II (duplex DNA recognition sequence CCGG) methylates the one methylatable cytosine of each strand similarly. Introduction of an A-C mispair at the methylation site shifts methylation exclusively to the mispaired cytosine residue. In direct competition assays with HPA:II methylase we observe that the mispaired substrate is methylated more extensively than the fully complementary, normal substrate, although both have one HPA:II methylation site. Through the use of this approach we will be able to learn more about the mechanisms by which methylation patterns can become altered.

Base-pair induced shifts in the tautomeric equilibrium of a modified DNA base.

We have examined the base-pairing properties of N4-methoxycytosine (mo4C), a mutagenic base analog, in DNA by nuclear magnetic resonance spectroscopy. Unlike standard bases, the tautomeric equilibrium of mo4C could be strongly influenced by base-pair formation. Paired with A, mo4C is found predominantly in the imino configuration in Watson-Crick geometry. However, when paired with G, two structurally distinct configurations are observed in equilibrium with one another. In one configuration, mo4C is in the amino form paired with G in Watson-Crick geometry. In the second species, mo4C is in the imino configuration paired with G in a wobble geometry. This is the first demonstration of basepair induced tautomeric shifts in DNA and supports the hypothesis that rare tautomeric forms may be involved in mutagenesis.

Ionized and wobble base-pairing for bromouracil-guanine in equilibrium under physiological conditions. A nuclear magnetic resonance study on an oligonucleotide containing a bromouracil-guanine base-pair as a function of pH.

A one and two-dimensional nuclear magnetic resonance study of a non-selfcomplementary oligonucleotide containing a central 5-bromouracil-guanine pair is reported. For these two bases three types of hydrogen bonding schemes could exist; wobble, rare tautomer and ionized. The two-dimensional spectra of non-exchangeable protons together with one-dimensional spectra recorded in water show that at pH 7.0 the predominant species is a right-handed B-form DNA in which the brU.G pair has wobble geometry. On raising the pH we observe a transition monitored by proton chemical shift changes for the brU.G and adjacent base-pairs. The mid-point of the transition was observed at pH 8.6. Spectra recorded at pH 9.8 show that the helix remains intact with B form conformation. It is shown that this high pH form has an ionized brU.G base-pair now in Watson-Crick geometry. Thus under physiological conditions an equilibrium exists between wobble and ionized structures.

The abasic site as a challenge to DNA polymerase. A nuclear magnetic resonance study of G, C and T opposite a model abasic site.

An abasic site in DNA creates a strong block to DNA polymerase and is a mutagenic base lesion. In this study, we present structural and dynamic properties of duplex oligodeoxynucleotides containing G, C and T opposite a model abasic site studied by one and two-dimensional nuclear magnetic resonance spectroscopy. We have demonstrated that A opposite the abasic site was positioned within the helix as if paired with T, and that the A residue melted co-operatively with the surrounding helix. We report here that G opposite the abasic site is also observed to be predominantly intrahelical in a normal anti conformation at low temperature. With increasing temperature, the mobility of the G residue increases rapidly and apparently is in a "melted state" well before denaturation of the helix. At low temperature, two species are found for T opposite the abasic site; one, intrahelical, one extrahelical. These species are in slow exchange with one another on a proton nuclear magnetic resonance time-scale. The two species then move into fast exchange with increasing temperature and the proportion of the extra-helical form increases. When C is positioned opposite the abasic site, both the C residue and the abasic sugar are extrahelical, the helix collapses, and the adjacent G.C base-pairs stack over one another. On the basis of these observations, we propose a model that explains why the abasic site acts to block DNA replication. Further, we suggest an explanation for the observed polymerase preference for base selection at abasic sites.

The occurrence and consequences of deoxyuridine in DNA.

Deoxyuridine can become resident in the DNA of prokaryotic and eukaryotic cells via two general mechanisms - deamination of cytosine to uracil, and nucleotide pool changes that lead to misincorporation of deoxyuridine in place of thymidine. In this paper we have examined the chemical basis of deamination reactions in DNA and discussed a possible mechanism for an increased rate of deamination by means of cross-strand protonation of cytosine by alkylated guanine. In addition, we have examined the genetic and drug-induced conditions that lead to dUMP misincorporation into DNA in place of thymidine and have presented experimental evidence indicating that the antifolate-induced lesion is a general drug-dose dependent lesion of human blood cells. Finally, the toxic and genetic impact of this lesion has been evaluated within the context of a review of the repair mechanisms elicited by dUMP in DNA.

Methoxyamine attack on cytosine produces ambivalent base pairing properties of the modified base.

We report the solution structure of two heptanucleotides each containing a central N4-methoxycytosine, in one case with adenine on the opposite strand and in the other with guanine. For the N4-methoxycytosine-adenine pair only the imino form of the N4-methoxycytosine residue is observed and base pairing is in Watson-Crick geometry. However, rotation of the methoxy group about the N-OCH3 bond is not constrained to a particular orientation although it must be anti to the N3 of N4-methoxycytosine. The slow exchange on a proton NMR time scale between the single strand and double strand forms is attributed to the strong preference of the syn conformation of the OCH3 group in the single strand which inhibits base pair formation. For N4-methoxycytosine base paired with guanosine we observe the N4-methoxycytosine base in the amino form in Watson-Crick geometry and a slow exchange of this species with an imino form base paired in wobble geometry. The amino form is predominant at low temperature whereas the imino form predominates above 40 degrees C. Our results point to preferential replacement of dTTP by N4-methoxycytosine in primer elongation.

15N-enriched 5-fluorocytosine as a probe for examining unusual DNA structures.

A new method is presented for the synthesis of oligonucleotides containing 15N-enriched 5-fluorocytosine (FC). Due to the reduced pK of FC, the amino protons of an unpaired FC residue may be observed at lower values of solution pH. The labeled FC residue has been placed as a template base at a model DNA replication fork. The amino protons of the FC residue have been identified in isotope-edited NMR spectra. Data is presented for a template FC residue unpaired, paired with guanine, and mispaired with adenine. These studies demonstrate the utility of labeled FC in examining unusual DNA structures.

Structures of oligonucleotides containing 5-(methoxymethyl)-2'-deoxyuridine determined by NMR spectroscopy.

Base pairing of 5-(methoxymethyl)-2'-deoxyuridine (MMdU) opposite either adenine or guanine in a seven base pair oligonucleotide duplex has been studied by NMR spectroscopy. When paired with A, we observe that the MMdU.A base pair adopts Watson-Crick geometry. The methoxymethyl substituent is not held in a fixed conformation and may rotate around the C5-CH2 and CH2-O bonds. Examination of the potential energy as a function of rotation around these bonds indicates the presence of four low energy conformations. No hydrogen bonding is indicated for the methoxymethyl substituent, and the four potential minima result from reduced steric clash. For the MMdU.G base pair, the two bases adopt a wobble geometry which does not change with increasing solvent pH. Similarly, we find four low energy conformations for the methoxymethyl substituent in the major groove of the DNA helix.

Structural and dynamic properties of a bromouracil-adenine base pair in DNA studied by proton NMR.

We have synthesized and studied by proton NMR a duplex heptaoligonucleotide containing a 5-bromouracil (brU)-adenine base pair. This represents the first structural characterization of a B-form DNA containing brU. The brU.A base pair is Watson-Crick rather than Hoogsteen as seen for the monomers in the crystalline state. From analysis of the NOESY sepctra at very short mixing times evidence is presented that substitution of brU for T induces significant conformational changes from that of a normal B DNA. The helix twist between brU4.A11 and G3.C12 is ca. 15 degrees and for both brU4 and G3 the glycosyl torsion angles are significantly changed. The imino proton of the bru.A base pair shows a pH insensitive line with which shows that the pK of brU in this base pair is very much higher than that of the monomer.

Molecular basis of cisplatin resistance in human carcinomas: model systems and patients.

In this review, cisplatin resistance will be summarized. We will discuss characteristics of potential mechanisms of resistance in tissues from patients with ovarian, colon and breast carcinoma. Secondly, a new molecular biology assay will be discussed which detects changes in gene expression in cisplatin-resistant cells. Finally, based on the biochemical and molecular properties of cisplatin-resistant tumors, new strategies to exploit or circumvent cisplatin resistance in human tumors will be entertained.

A proposed mechanism for the mutagenicity of 5-formyluracil.

5-Formyluracil is a mutagenic base formed in DNA by oxidation of the thymine methyl group. Whereas the thymine methyl group is electron donating, the formyl group is electron withdrawing, predicting increased ionization of the N-3 imino proton under physiological conditions. The pKa values of a series of 5-substituted uracil and deoxyuridine derivatives have been measured. A linear relationship is observed between the electronic inductive property of the 5-substituent and the pKa value of the corresponding imino proton. The pKa value of 5-formyl-2'-deoxyuridine is close to that of the mutagenic nucleoside analogue 5-bromo-2'-deoxyuridine. In analogy with BrU, it is proposed that the mutagenicity of 5-formyluracil results from enhanced mispairing of the ionized form with guanine during DNA replication.

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.

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.

Collateral sensitivity to azidothymidine in methotrexate resistant human leukemia cells.

Resistance to methotrexate (MTX) is a well established clinical problem and strategies to circumvent this would obviously be beneficial. The human leukemia cell line, CCRF-CEM, was grown in folinic acid to study MTX resistance-reflecting more in vivo conditions. A 15.8-fold resistant subline, CEM/MTX, was evolved by stepwise increases in MTX exposure. The MTX resistant cell line exhibited both increased dihydrofolate reductase (DHFR) activity due to gene amplification as well as impaired MTX uptake. An additional mechanism of resistance to MTX was a 2-fold increase in thymidine kinase (TK). As a result of this increased TK activity, the CEM/MTX cells were collaterally sensitive to the nucleoside analogue, azidothymidine (AZT). CEM cells resistant to MTX possess properties that can be exploited by AZT and these studies may have clinical implications.

Effect of mode of birth on purine and malondialdehyde in umbilical arterial plasma in normal term newborns.

OBJECTIVE: To examine the effect of mode of birth on plasma purine and malondialdehyde levels in normal term infants. STUDY DESIGN: Umbilical arterial cord blood was obtained immediately after birth from a convenience sample of 119 normal term newborns born by vaginal delivery, with or without oxytocin augmentation or by elective cesarean delivery. Plasma was analyzed for purine and/or malondialdehyde levels. Numeric data were analyzed utilizing independent samples t-test and ordinal data were analyzed using Mann-Whitney test. Correlation coefficients were obtained using Spearman's rho. RESULT: Uric acid levels were significantly elevated (P<0.001) in neonates undergoing vaginal birth, compared to neonates born by elective cesarean delivery. When the effect of oxytocin and length of labor was analyzed, neonates born to mothers on oxytocin had lower hypoxanthine, significantly lower xanthine (P=0.05) and higher uric acid levels. In addition, malondialdehyde levels were significantly higher (P<0.006) in neonates born to mothers who received oxytocin compared to neonates born to mothers without oxytocin augmentation. We also found significant correlations between malondialdehyde (MDA) and hypoxanthine (r=-0.465, P<0.039) and between MDA and xanthine (r=-0.753, P=0.003) in neonates born via oxytocin-augmented birth. Mode of birth had no statistically significant effect on clinical outcomes, although infants born by elective cesarean had higher incidence of acute respiratory distress and transient tachypnea of the newborn compared to those born vaginally. CONCLUSION: Neonates born by elective cesarean had the lowest purine levels in cord blood compared to neonates born vaginally. Oxytocin augmentation is associated with some degree of uterine hyperstimulation which may enhance the ATP degradation pathway resulting in the rapid conversion of hypoxanthine to xanthine and xanthine to uric acid. Significantly higher MDA levels in neonates whose mothers received oxytocin as well as significant correlation between MDA and the purines hypoxanthine and xanthine, suggest free-radical production, most likely due to xanthine oxidase activation. However, despite differences in plasma purine and malondialdehyde levels, no significant differences were seen in neonatal outcome. Further studies are required to fully characterize the effect of mode of birth on purine metabolism and free-radical production.

An unexpectedly high excision capacity for mispaired 5-hydroxymethyluracil in human cell extracts.

The oxidation of thymine in DNA can generate a base pair between 5-hydroxymethyluracil (HmU) and adenine, whereas the oxidation and deamination of 5-methylcytosine (5mC) in DNA can generate a base pair between HmU and guanine. Using synthetic oligonucleotides containing HmU at a defined site, HmU-DNA glycosylase activities in HeLa cell and human fibroblast cell extracts have been observed. An HmU-DNA glycosylase activity that removes HmU mispaired with guanine has been measured. Surprisingly, the HmU:G excision activity is 60 times greater than the corresponding HmU:A activity, even though the expected rate of formation of the HmU:A base pair exceeds that of the HmU:G base pair by a factor of 10(7). The HmU:G mispair would arise from the 5mC:G base pair, and, if unrepaired, would give rise to a transition mutation. The observation of an unexpectedly high HmU:G glycosylase activity suggests that human cells may encounter the HmU:G mispair much more frequently than expected. The conversion of 5mC to HmU must be considered as a potential pathway for the generation of 5mC to T transition mutations, which are often found in human tumors.

Photochemical deamination and demethylation of 5-methylcytosine.

Cytosine methylation is believed to play a pivotal role in eucaryotic cellular development as well as in viral latency. We have been investigating chemical mechanisms for the perturbation of methylation patterns, including the effects of ultraviolet radiation. We observed that, upon exposure to UV light, 5-methylcytosine (5mC) was converted to thymine, cytosine, and a series of 5-substituted cytosine derivatives as analyzed by gas chromatography/mass spectrometry. Deamination of 5mC to thymine proceeds via formation of the intermediate photohydrate. Formation of 5-substituted cytosine derivatives results from oxidation of the 5-methyl group with initial formation of 5-(hydroxymethyl)cytosine (hmC). Upon exposure to UV light, hmC is converted to cytosine. The conversion of hmC to cytosine likely results from photohydration and elimination of formaldehyde. It is proposed that endogenous oxidation and hydrolysis could result in demethylation of 5mC residues in DNA. Whereas hydrolytic deamination of 5mC to thymine has been widely discussed, demethylation of 5mC has not as yet been described.

The paradoxical influence of thymine analogues on restriction endonuclease cleavage of oligodeoxynucleotides.

Thymine residues in the DNA of eucaryotes may be replaced occasionally by uracil (U) or 5-(hydroxymethyl)uracil (H) as consequences of dUMP misincorporation or thymine oxidation, respectively. In this study, we constructed a series of 44-base oligonucleotides containing site-specific U or H residues and 5'-fluorescein labels in order to probe the influence of such modifications on sequence-specific DNA-protein interactions using several type II restriction endonucleases. We find that substitution within the recognition sites of several restriction endonucleases increases initial cleavage velocity by up to an order of magnitude. These results contrast dramatically with several previous studies which demonstrated that U substitution in short oligonucleotides inhibits or prevents nuclease cleavage. We propose that this apparent paradox results because the rate-limiting step in the cleavage of longer oligonucleotides is product release whereas for shorter oligonucleotides substrate binding is most probably rate-limiting. For longer oligonucleotides and DNA, more rapid release of the cleaved, substituted oligonucleotides results in more rapid turnover and a faster apparent cleavage rate. The sequence length at which the transition in rate-limiting step occurs likely corresponds to the size of the enzyme footprint on its DNA recognition site. We conclude that both U and H do perturb sequence-specific DNA-protein interactions, and the magnitude of this effect is site-dependent.

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.