Uracil Incorporation Research Articles

Permeability of Coxiella burnetii to ribonucleosides.

Knowledge about transport in Coxiella burnetii, an obligate phagolysosomal parasite, is incomplete. The authors investigated the capability of isolated, intact, host-free Coxiella to transport ribonucleosides while incubated at a pH value typical of lysosomes. Because of the low activities and limitations of obtaining experimental quantities of isolated, purified Coxiella, incorporation of substrate into nucleic acid was used as a trap for determination of uptake abilities. Virulent wild-type (phase I) organisms possessed uptake capability for all ribonucleosides. Both phase I and phase II (avirulent) organisms incorporated the purine nucleosides guanosine, adenosine and inosine, and showed a more limited uptake of thymidine and uridine. Both phases were poorly active in cytidine uptake. Neither phase of the organism was capable of transport and incorporation of NTPs, CMP, cytosine or uracil. Water space experiments confirmed that the uptake process concentrated the purine nucleosides within the cytoplasm of both wild-type and phase II Coxiella via a low-pH-dependent mechanism. Comparison of uptake rates in Escherichia coli versus Coxiella verified that the incorporation of ribonucleosides by Coxiella is a slow process. It is concluded that Coxiella possesses some transport pathways consistent with utilization of pools of nucleosides found within its host cell lysosomal pathway.

Site-directed mutagenesis facilitated by DpnI selection on hemimethylated DNA.

AbstractOligonucleotide-based, site-directed mutagenesis (SDM) of cloned DNA has become a fundamental tool of modern molecular biology, used to introduce insertions, deletions, and substitutions into DNA. Current techniques available for performing in vitro mutagenesis fall into three categories: 1. Those employing single-stranded DNA (ssDNA) as template (1,2), originally using phage M13 vector to produce the ssDNA. Several methods were developed to reduce the background of nonmutated parental DNA, including in vivo incorporation of uracil in the parental strand (3,4), and in vitro incorporation of 5-methyl cytidine or thiophosphates in the synthesized strand (5,6). Selection against the parental strand was accomplished during intracellular DNA repair, or by differential restriction endonuclease sensitivity in vitro, respectively. 2. Use of double-stranded DNA (dsDNA) as template. A widely used method called unique site elimination (USE) (7) allows introduction of specific mutations into a target gene or region cloned into a ds plasmid with a unique restriction site. 3. Polymerase chain reaction (PCR)-based methods, in which site-specific mutants are created by introducing mismatches into oligonucleotides used to prime in vitro amplification (8–11). Such methods often include splicing by overlap extension, which involves a two-step PCR procedure, and subsequent cloning. KeywordsHigh Transformation EfficiencyUnique Restriction SiteMutation EfficiencyModern Molecular BiologyParental StrandThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Glycine rich P-loop motif in deoxyuridine pyrophosphatase.

Deoxyuridine pyrophosphatase (dUTPase) cleaves the alpha-beta phosphodiester bond of dUTP to form pyrophosphate and dUMP, preventing incorporation of uracil into DNA and providing the substrate for dTTP synthesis. Similar to other nucleotide binding proteins, dUTPase also consists of a sequence motif rich in glycine residues known as P-loop motif. The P-loop motif of the nucleotide binding proteins are involved in substrate binding, catalysis, recognition and regulation of activity. In dUTPase the function of the P-loop motif is not well understood. One of the main reasons for this limited information is the lack of the three-dimensional structure of a dUTPase enzyme with an ordered Gly-rich P-loop motif with a bound substrate and Mg(2+) ion. This review presents an insight into the role of Gly-rich P-loop motif in the function of dUTPase as revealed from the crystal structure. The analysis reveals the Gly-rich P-loop motif of dUTPase to be the longest in terms of its amino-acid composition as compared to other nucleotide binding proteins and exhibit a high-degree of sequence conservation among spectrum of species. The enzyme utilizes adaptive recognition to bind to the phosphate groups of the nucleotide. In particular, the alpha-beta phosphodiester bond adopts an unfavorable eclipsed conformation in the presence of the Gly-rich P-loop motif. This conformation may be relevant to the mechanism of alpha-beta phosphodiester bond cleavage.

Requirement for uracil-DNA glycosylase during the transition to late-phase cytomegalovirus DNA replication.

Cytomegalovirus gene UL114, a homolog of mammalian uracil-DNA glycosylase (UNG), is required for efficient viral DNA replication. In quiescent fibroblasts, UNG mutant virus replication is delayed for 48 h and follows the virus-induced expression of cellular UNG. In contrast, mutant virus replication proceeds without delay in actively growing fibroblasts that express host cell UNG. In the absence of viral or host cell UNG expression, mutant virus fails to proceed to late-phase DNA replication, characterized by rapid DNA amplification. The data suggest that uracil incorporated early during wild-type viral DNA replication must be removed by virus or host UNG prior to late-phase amplification and encapsidation into progeny virions. The process of uracil incorporation and excision may introduce strand breaks to facilitate the transition from early-phase replication to late-phase amplification.

Methylenetetrahydrofolate reductase C677T polymorphism does not alter folic acid deficiency-induced uracil incorporation into primary human lymphocyte DNA in vitro.

Methylenetetrahydrofolate reductase (MTHFR) is an enzyme which converts 5,10-methylene tetrahydrofolate (5,10-MnTHF) to 5-methyl tetrahydrofolate. A common C to T transition (C677T) in the MTHFR gene is reported to reduce the risk for colorectal cancer and acute lymphocytic leukemia in homozygotes (TTs). It is hypothesized that because TTs have reduced MTHFR activity, more 5,10-MnTHF is available to provide methyl groups for the conversion of uracil to thymidine. Folic acid deficiency causes the intracellular accumulation of dUMP and the subsequent incorporation of uracil into DNA. The removal of uracil from DNA may result in double-stranded DNA breaks, the accumulation of which is a putative risk factor for cancer. We tested whether human lymphocytes taken from TTs (n = 10) were more able to resist uracil incorporation into DNA than controls (n = 14 CCs and 6 CTs) when cultured in medium containing 12-120 nM folic acid for 9 days. DNA uracil content of these lymphocytes was measured by CG-MS. TTs and controls showed a dose-dependent increase in DNA uracil content during folic acid deficiency (P < 0.0001, R2 = 0.23 for TTs and P < 0.0001, R2 = 0.19 for controls). DNA uracil content was not different between the two groups at any of the folic acid concentrations (two-way ANOVA: media [folic acid], P < 0.0001; genotype, P = 0.4). The results show that, in this in vitro system, the MTHFR C677T polymorphism does not affect the cell's ability to resist uracil incorporation into DNA. Chromosome breakage, as measured by micronuclei, was also shown to correlate with folic acid concentration in a preliminary experiment (P < 0.0001). Although the results appear not to support the hypothesis that a reduced risk for certain cancers in TTs is due to diversion of folic acid to thymidine synthesis, differences between the in vivo and in vitro situation make this conclusion not definitive.

In vitro investigation of host resistance to Toxoplasma gondii infection in microglia of BALB/c and CBA/Ca mice.

Toxoplasmic encephalitis (TE) is a life-threatening disease of immunocompromised individuals and has increased in prevalence as a consequence of AIDS. TE has been modeled in inbred mice, with CBA/Ca mice being susceptible and BALB/c mice resistant to the development of TE. To better understand the innate mechanisms in the brain that play a role in resistance to TE, nitric oxide (NO)-dependent and NO-independent mechanisms were examined in microglia from BALB/c and CBA/Ca mice and correlated with the ability of these cells to inhibit Toxoplasma gondii replication. These parameters were measured 48 h after stimulation with lipopolysaccharide (LPS) gamma interferon (IFN-gamma), tumor necrosis factor alpha (TNF-alpha), or combinations of these inducers in T. gondii-infected microglia isolated from newborn mice. CBA/Ca microglia consistently produced less NO than did BALB/c microglia after stimulation with LPS or with IFN-gamma plus TNF-alpha, and they inhibited T. gondii replication significantly less than did BALB/c microglia. Cells of both strains treated with IFN-gamma alone significantly inhibited uracil incorporation by T. gondii, and N(G)-monomethyl-L-arginine (NMMA) treatment did not reverse this effect. In cells treated with IFN-gamma in combination with other inducers, NMMA treatment resulted in only partial recovery of T. gondii replication. This IFN-gamma-dependent inhibition of replication was not due to generation of reactive oxygen species or to increased tryptophan degradation. These data suggest that NO production and an IFN-gamma-dependent mechanism contribute to the inhibition of T. gondii replication after in vitro stimulation with IFN-gamma plus TNF-alpha or with LPS. Differences in NO production but not in IFN-gamma-dependent inhibition of T. gondii replication were observed between CBA/Ca and BALB/c microglia.

Analysis of uracil DNA glycosylase in human colorectal cancer.

Uracil DNA glycosylase (UDG) is responsible for the removal of uracil present in DNA after cytosine deamination or misincorporation during replication. Colorectal cancer is widely treated with 5-FU, which leads to thymidylate synthase inhibition; this accounts for increased dUTP intracellular pools and subsequent uracil incorporation into DNA. Uracil misincorporation has also been implicated in the link between folate deficiency and colorectal cancer risk. As there is no information on UDG in colorectal cancer, this study characterized UDG activity and protein expression in a panel of 20 colorectal tumors and 6 colorectal cell lines. UDG activity in colorectal tissue is widely variable and it is statistically higher in tumor tissue (P=0.013) compared to normal bowel. Tumor versus normal activity ratios ranged from 0.49 to 2.2 (median 1.13). Among the six colorectal cell lines tested, UDG activity varied from 40 to 68 units and was markedly (1.7-fold) higher than in tumor tissue (P<0.0001). In both colorectal tissues and cell lines, UDG was expressed as both 29 kDa and 35 kDa forms. Total protein expression varied 3.2-fold in cell lines; variability was also found between patients and between normal and tumoral tissue for the same patient. This study demonstrates UDG protein and functional activity in human colorectal tumors and cell lines. The high tumor:normal tissue ratio supports further interest in base excision repair, through UDG, as a potential source of fluoropyrimidine resistance in colorectal cancer.

Altered Subunit Communication in Subfamilies of Trimeric dUTPases

The enzyme dUTPase is essential in preventing uracil incorporation into DNA. Design of antagonists against this novel chemotherapeutic target requires identification of species-specific differences in the structure and mechanism of the enzyme. This task is now approached via comparisons of available crystallographic structures of dUTPases from Homo sapiens, Escherichia coli, and retroviruses. The eukaryotic protein uniquely displays polar and charged amino acid residues participating in threefold intersubunit interactions. In bacterial and retroviral dUTPases, threefold interactions are mainly hydrophobic. The residues responsible for this contrast are mapped in multiple sequence alignment to positions differently and characteristically conserved in distinct evolutionary branches. The general feature of this contrast is further strengthened by a second eukaryotic model structure constructed using comparative modeling. The dUTPase cDNA from Drosophila melanogaster was identified, sequenced, and the model structure of the encoded polypeptide displayed a polar hydrogen-bonding network of threefold interactions, identically to the human structure. Results allow clear distinction between two subfamilies of trimeric dUTPases where altered subunit communication may account for a functional difference in the catalytic cycle.

Uracil incorporation into a gene targeting construct reduces the frequency of homologous and nonhomologous recombinants in human cells.

Gene targeting allows the introduction of specific modifications into the eukaryotic genome by homologous recombination, but its efficiency is low in many mammalian systems. We are exploring different ways to increase the efficiency of gene targeting and we report here the effect of uracil incorporation in the targeting construct. Plasmids containing uracil substituting for a fraction of thymine residues are hyperrecombinogenic in some bacterial systems. To test whether a similar stimulation of recombination occurs in mammalian cells, we have prepared a uracil-rich HPRT targeting construct and quantified its homologous and nonhomologous recombination frequencies compared to the same plasmid lacking uracil. The uracil-rich plasmid led to reductions in both homologous and nonhomologous recombination in human cells.

Structure-based design of selective inhibitors of dihydrofolate reductase: synthesis and antiparasitic activity of 2, 4-diaminopteridine analogues with a bridged diarylamine side chain.

As part of a larger search for potent as well as selective inhibitors of dihydrofolate reductase (DHFR) enzymes from opportunistic pathogens found in patients with AIDS and other immune disorders, N-[(2,4-diaminopteridin-6-yl)methyl]dibenz[b,f]azepine (4a) and the corresponding dihydrodibenz[b,f]azepine, dihydroacridine, phenoxazine, phenothiazine, carbazole, and diphenylamine analogues were synthesized from 2, 4-diamino-6-(bromomethyl)pteridine in 50-75% yield by reaction with the sodium salts of the amines in dry tetrahydrofuran at room temperature. The products were tested for the ability to inhibit DHFR from Pneumocystis carinii (pcDHFR), Toxoplasma gondii (tgDHFR), Mycobacterium avium (maDHFR), and rat liver (rlDHFR). The member of the series with the best combination of potency and species selectivity was 4a, with IC(50) values against the four enzymes of 0. 21, 0.043, 0.012, and 4.4 microM, respectively. The dihydroacridine, phenothiazine, and carbazole analogues were also potent, but nonselective. Of the compounds tested, 4a was the only one to successfully combine the potency of trimetrexate with the selectivity of trimethoprim. Molecular docking simulations using published 3D structural coordinates for the crystalline ternary complexes of pcDHFR and hDHFR suggested a possible structural interpretation for the binding selectivity of 4a and the lack of selectivity of the other compounds. According to this model, 4a is selective because of a unique propensity of the seven-membered ring in the dibenz[b,f]azepine moiety to adopt a puckered orientation that allows it to fit more comfortably into the active site of the P. carinii enzyme than into the active site of the human enzyme. Compound 4a was also evaluated for the ability to be taken up into, and retard the growth of, P. carinii and T. gondii in culture. The IC(50) of 4a against P. carinii trophozoites after 7 days of continuous drug treatment was 1.9 microM as compared with previously observed IC(50) values of >340 microM for trimethoprim and 0.27 microM for trimetrexate. In an assay involving [(3)H]uracil incorporation into the nuclear DNA of T. gondii tachyzoites as the surrogate endpoint for growth, the IC(50) of 4a after 5 h of drug exposure was 0.077 microM. The favorable combination of potency and enzyme selectivity shown by 4a suggests that this novel structure may be an interesting lead for structure-activity optimization.

Micronutrient deficiencies. A major cause of DNA damage.

Deficiencies of the vitamins B12, B6, C, E, folate, or niacin, or of iron or zinc mimic radiation in damaging DNA by causing single- and double-strand breaks, oxidative lesions, or both. The percentage of the population of the United States that has a low intake (< 50% of the RDA) for each of these eight micronutrients ranges from 2% to 20+ percent. A level of folate deficiency causing chromosome breaks occurred in approximately 10% of the population of the United States, and in a much higher percentage of the poor. Folate deficiency causes extensive incorporation of uracil into human DNA (4 million/cell), leading to chromosomal breaks. This mechanism is the likely cause of the increased colon cancer risk associated with low folate intake. Some evidence, and mechanistic considerations, suggest that vitamin B12 and B6 deficiencies also cause high uracil and chromosome breaks. Micronutrient deficiency may explain, in good part, why the quarter of the population that eats the fewest fruits and vegetables (five portions a day is advised) has about double the cancer rate for most types of cancer when compared to the quarter with the highest intake. Eighty percent of American children and adolescents and 68% of adults do not eat five portions a day. Common micronutrient deficiencies are likely to damage DNA by the same mechanism as radiation and many chemicals, appear to be orders of magnitude more important, and should be compared for perspective. Remedying micronutrient deficiencies is likely to lead to a major improvement in health and an increase in longevity at low cost.

Flow Cytometric Quantification of Toxoplasma gondii Cellular Infection and Replication

The invasion and replication of Toxoplasma gondii are usually analyzed through either optical microscopy or incorporation of tritiated uracil. A new method has been developed using flow cytometric analysis to examine the entry and replication of T. gondii RH strain in Saimiri brain endothelial cells. After cell fixation and permeabilization using saponin, intracellular T. gondii were labeled with a monoclonal antibody against T. gondii SAG-1 (P30; the major cell-surface antigen) followed by fluorescein-conjugated rabbit anti-mouse IgG. The percentage of infected cells obtained using flow cytometry correlated directly with that obtained by UV light microscopy (r = 0.97). The mean fluorescence intensity of infected cells reflects intracellular P30 and assesses intracellular replication. The distribution of fluorescence per infected cell, considered with the percentage of infected cells, also allows a qualitative analysis of replication. Such a method is rapid, easy, and does not require specialized equipment for radioactive labeling.

The effect of folate deficiency on the hprt mutational spectrum in Chinese hamster ovary cells treated with monofunctional alkylating agents

Folic acid deficiency acts synergistically with alkylating agents to increase DNA strand breaks and mutant frequency at the hprt locus in Chinese hamster ovary (CHO) cells. To elucidate the mechanism of this synergy, molecular analyses of hprt mutants were performed. Recently, our laboratory showed that folate deficiency increased the percentage of clones with intragenic deletions after exposure to ethyl methanesulfonate (EMS) but not N-nitroso- N-ethylurea (ENU) compared to clones recovered from folate replete medium. This report describes molecular analyses of the 37 hprt mutant clones obtained that did not contain deletions. Folate deficient cells treated with EMS had a high frequency of G>A transitions at non-CpG sites on the non-transcribed strand, particularly when these bases were flanked on both sides by G:C base pairs. Thirty-three percent of these mutations were in the run of six G's in exon 3. EMS-treated folate replete cells had a slightly (but not significantly) lower percentage of G>A transitions, and the same sequence specificity. Treatment of folate deficient CHO cells with ENU resulted in predominantly T>A transversions and C>T transitions relative to the non-transcribed strand. These findings suggest a model to explain the synergy between folate deficiency and alkylating agents: (1) folate deficiency causes extensive uracil incorporation into DNA; (2) greatly increased utilization of base excision repair to remove uracil and to correct alkylator damage leads to error-prone DNA repair. In the case of EMS, this results in more intragenic deletions and G:C to A:T mutations due to impaired ligation of single-strand breaks generated during base excision repair and a decreased capacity to remove O 6-ethylguanine. In the case of ENU additional T>A transversions and C>T transitions are seen, perhaps due to mis-pairing of O 2-ethylpyrimidines. Correction of folate deficiency may reduce the frequency of these types of genetic damage during alkylator therapy.

Mechanisms of the Eimeria tenella Growth Inhibitory Activity Induced by Concanavalin A and Reticuloendotheliosis Virus Supernatants with Interferon Gamma Activity in Chicken Macrophages and Fibroblasts

Pretreatment of chicken bone marrow macrophages and embryo fibroblasts with supernatants containing chicken interferon gamma (IFN-gamma) for 24 hr prior to inoculation inhibited intracellular Eimeria tenella replication, measured by [3H] uracil incorporation. The supernatants (Sns) were obtained from culture of lymphoblastoid cells transformed by a reticuloendotheliosis virus (REV) and chicken splenocytes stimulated with concanavalin A (Con A). The mechanisms of the E. tenella growth inhibitory activity induced by Sn REV and Sn Con A in chicken macrophages and fibroblasts were studied. Addition of oxygen scavengers (superoxide dismutase, D-mannitol, DABCO, benzoic acid, L-histidine hydrochloride) was able to overcome the inhibition of E. tenella replication after pretreatment with Sn REV or Sn Con A in macrophage cultures but not in fibroblast cultures. Nitric oxide (NO) synthesis was induced in macrophage culture treated with Sn REV or Sn Con A but not in fibroblast culture. Addition of NG monomethyl-L-arginine, an NO synthase inhibitor together with the supernatants was also able to overcome inhibition of E. tenella replication in macrophage culture. On the other hand, addition of L-tryptophan to Sn REV- or Sn Con A-treated fibroblasts was able to reverse the inhibitory effect on E. tenella replication. In conclusion, production of inorganic NO or toxic oxygen intermediates may be involved in the E. tenella growth inhibitory activity of chicken macrophages pretreated with supernatants containing an IFN-gamma activity, and cellular tryptophan depletion may be involved for chicken fibroblasts, thus matching the mechanisms of the IFN-gamma-induced growth inhibitory activity for protozoans in mammals.

Micronutrients prevent cancer and delay aging

Approximately 40 micronutrients are required in the human diet. Deficiency of vitamins B12, folic acid, B6, niacin, C, or E, or iron, or zinc, appears to mimic radiation in damaging DNA by causing single- and double-strand breaks, oxidative lesions, or both. The percentage of the US population that has a low intake (<50% of the RDA) for each of these eight micronutrients ranges from 2% to ≥20%; half of the population may be deficient in at least one of these micronutrients. Folate deficiency occurs in approximately 10% of the US population, and in a much higher percentage of the poor. Folate deficiency causes extensive incorporation of uracil into human DNA (4 million/cell), leading to chromosomal breaks. This mechanism is the likely cause of the increased cancer risk, and perhaps the cognitive defects associated with low folate intake. Some evidence, and mechanistic considerations, suggest that vitamin B12 and B6 deficiencies also cause high uracil and chromosome breaks. Micronutrient deficiency may explain, in good part, why the quarter of the population that eats the fewest fruits and vegetables (five portions a day is advised) has approximately double the cancer rate for most types of cancer when compared to the quarter with the highest intake. Eighty percent of American children and adolescents and 68% of adults do not eat five portions a day. Common micronutrient deficiencies are likely to damage DNA by the same mechanism as radiation and many chemicals, appear to be orders of magnitude more important, and should be compared for perspective. Remedying micronutrient deficiencies is likely to lead to a major improvement in health and an increase in longevity at low cost.Aging appears to be due, in good part, to the oxidants produced by mitochondria as by-products of normal metabolism. In old rats mitochondrial membrane potential, cardiolipin levels, respiratory control ratio, and overall cellular O2 consumption are lower than in young rats, and the level of oxidants (per unit O2) is higher. The level of mutagenic aldehydes from lipid peroxidation is also increased. Ambulatory activity declines markedly in old rats. Feeding old rats the normal mitochondrial metabolites acetyl carnitine and lipoic acid for a few weeks, restores mitochondrial function, lowers oxidants to the level of a young rat, and increases ambulatory activity. Thus, these two metabolites can be considered necessary for health in old age and are therefore conditional micronutrients. This restoration suggests a plausible mechanism: with age-increased oxidative damage to proteins and lipid membranes causes a deformation of structure of key enzymes, with a consequent lessening of affinity (Km) for the enzyme substrate; an increased level of the substrate restores the velocity of the reaction, and thus restores function.

A method to assess invasion and intracellular replication of Trypanosoma cruzi based on differential uracil incorporation

Screening of candidate trypanocidal compounds or factors affecting invasion of mammalian cells by the infective stages of Trypanosoma cruzi in tissue culture models has primarily involved labor-intensive microscopic counting of the parasites. A very efficient method for quantitating the inhibitory effect of antimicrobial agents or signaling pathways inhibitors on T. cruzi grown in L 6E 9 myoblasts was devised. This assay takes advantage of the selective incorporation of [ 3 H ]uracil into nucleic acids by replicating T. cruzi amastigotes. L 6E 9 rat myoblasts are submitted to gamma irradiation to inhibit their replication. Uracil uptake by uninfected cells is considerably decreased by this method. Nifurtimox, benznidazole, fexinidazole, MK-436, and megazol are drugs known to have activity against T. cruzi and were used in growth inhibition assays. The results demonstrated that [ 3 H ]uracil incorporation in the presence of different concentrations of nifurtimox and benznidazole closely correlated with the number of amastigotes per 100 myoblasts in Giemsa-stained monolayers under the conditions used. This method also has the advantage to differentiate between the effects of the compounds on the invasion and the replication steps of the infection with T. cruzi, as shown by the inhibitory effect of genistein when added in invasion assays.

Diminished adherence and/or ingestion of virulent Mycobacterium tuberculosis by monocyte-derived macrophages from patients with tuberculosis.

The interaction between the macrophage and Mycobacterium tuberculosis is mediated by a variety of macrophage membrane-associated proteins. Complement receptors have been implicated in the adherence of M. tuberculosis to macrophages. In the present work, the adherence and/or ingestion of M. tuberculosis H37Rv to human monocyte-derived macrophages (MDM) from patients with tuberculosis (TB) and healthy controls was measured by microscopical examination, [3H]uracil incorporation, and CFU. The adherence and/or ingestion was enhanced by fresh serum and inhibited by heat inactivation, EDTA treatment, and anti-CR1 and anti-CR3 antibodies. Comparison of MDM from TB patients and healthy controls showed that the former exhibited a significantly decreased capacity to adhere and/or ingest M. tuberculosis, as determined by the number of CFU and 3H incorporation. The expression of CR1 (CD35) and CR3 (CD11b/CD18) on MDM from TB patients and healthy controls, as determined by flow cytometry, did not show significant differences. These results suggest that the lower ingestion of M. tuberculosis by MDM from TB patients is not due to defects in complement receptors, and therefore, there might be other molecules involved in the adherence and/or ingestion process that render MDM from TB patients ingest less mycobacteria than those from healthy controls.

Prevention of fluorodeoxyuridine-induced cytotoxicity and DNA damage in HT29 colon carcinoma cells by conditional expression of wild-type p53 phenotype.

We have examined the effects of conditionally expressing wild-type p53 activity in HT29 cells on DNA damage and cytotoxicity caused by exposure to fluorodeoxyuridine (FdUrd). Expression of wild-type p53 phenotype for 24 hr before FdUrd treatment provided HT29 cells with virtually complete protection from cytotoxicity caused by this drug. In addition, wild-type p53 expression also prevented FdUrd-induced DNA double-strand breaks and, unexpectedly, single-strand breaks in parental (mature) DNA. Temporary expression of wild-type p53 activity in the absence of drug treatment caused some loss of clonogenicity, although the magnitude of this cytotoxic effect was small compared with the level of cell kill obtained by treatment with cytotoxic drugs for similar periods of time, indicating that HT29 cells are not highly sensitive to induction of programmed cell death by wild-type p53. Because these observations conflict with previously suggested models for FdUrd-induced damage to parental DNA, we propose an alternative model to explain how incorporation of uracil into nascent DNA might result in single-strand breaks in the opposite (parental) strand and how these breaks might be converted to the double-strand breaks that produce cell death.

Inhibition of Toxoplasma gondii replication in IFN-gamma-activated human intestinal epithelial cells.

Toxoplasma gondii is an obligate intracellular parasite which is responsible for severe disease after congenital infection and in immunocompromised patients, for which there is no effective therapy. In acquired toxoplasmosis, T. gondii first invade enterocytes and are disseminated throughout the body. Treatment of the adherent human intestinal cell line CaCO2 with recombinant human IFN-gamma inhibited the replication of T. gondii. Growth of the parasite was measured in vitro by [3H]-uracil incorporation assays 18 h after infection. This assay showed that when cells were pretreated with IFN-gamma concentrations ranging from 2.5 to 5000 U/ml, a high degree of inhibition of T. gondii replication could be observed, with the effect being dose dependent. This could be of relevance as a first line of defence against human Toxoplasma infection. Inhibition is due to a different mechanism from that existing in mouse macrophages and human fibroblasts: L-arginine-dependent production of reactive nitrogen intermediates, reactive oxygen intermediates synthesis or production of indoleamine 2.3 dioxygenase were not responsible for inhibition of T. gondii proliferation.

Folate deficiency causes uracil misincorporation into human DNA and chromosome breakage: implications for cancer and neuronal damage.

Folate deficiency causes massive incorporation of uracil into human DNA (4 million per cell) and chromosome breaks. The likely mechanism is the deficient methylation of dUMP to dTMP and subsequent incorporation of uracil into DNA by DNA polymerase. During repair of uracil in DNA, transient nicks are formed; two opposing nicks could lead to chromosome breaks. Both high DNA uracil levels and elevated micronucleus frequency (a measure of chromosome breaks) are reversed by folate administration. A significant proportion of the U.S. population has low folate levels, in the range associated with elevated uracil misincorporation and chromosome breaks. Such breaks could contribute to the increased risk of cancer and cognitive defects associated with folate deficiency in humans.