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Biomedical subjects

J Maybaum

Publications and source records attributed to J Maybaum.

51 records · Page 3Linked to original sources
PubMed1987

Analysis of bromodeoxyuridine incorporation into DNA: comparison of gas chromatographic/mass spectrometric, CsCl gradient sedimentation, and specific radioactivity methods.

A sensitive new method for the quantitation of 5-bromodeoxyuridine (BrdUrd) incorporated into DNA by GC/MS analysis of enzymatically released Thy and bromouracil (BrUra) is presented. The hydrolysis procedure was characterized and found to give uniform results when sample size was 1-10 micrograms DNA and incubation time for DNA digestion was between 40 min and 16 h. Samples of DNA containing 3H-labeled BrdUrd were analyzed in parallel by the GC/MS technique and by specific radioactivity and buoyant density measurements, in order to compare the three methods. The GC/MS procedure gave values for percentage replacement of Thy by BrUra which were higher than those obtained by specific activity and lower than those obtained by buoyant density. This GC/MS method can detect 1% replacement in a 1-microgram DNA sample, equivalent to approximately 10(5) cells or 0.1 mg tissue, and will permit sensitive and quantitative analysis of the presence of this chemotherapeutic agent/radiosensitizer in cellular DNA from biopsy samples of normal or tumor tissue.

Animals↗
PubMed1987

Effects of incorporation of 6-thioguanine into SV40 DNA.

The antileukemic agent 6-thioguanine (TG) is thought to inhibit DNA synthesis as a result of its incorporation into DNA. In the present study we have examined the nature of this inhibition, using replication of SV40 viral DNA as a model system. Addition of TG to SV40-infected CV1P cells from 22 to 24 hr post infection causes a dose-dependent inhibition of viral DNA synthesis. This inhibition plateaus between 250 and 2500 microM TG, resulting in a maximum decrease of viral DNA synthesis of about 50%. Pulse-chase experiments showed no detectable slowing of elongation of nascent DNA chains, whereas measurement of the conversion of incorporated 3H-dThd into supercoiled viral DNA suggested that elongation might be slightly inhibited, but by no more than 20%. Since inhibition of elongation could not account for the total depression of DNA synthesis, we hypothesized that inhibition of initiation of DNA replication takes place. This hypothesis was tested by radioactively labeling newly synthesized viral DNA and then assessing the ability of these molecules to reenter the replicating pool by density labeling with bromodeoxyuridine. The fraction of TG-containing molecules able to re-initiate replication was decreased 15%, compared to control. This effect, which was dependent on the concentration of TG added to the medium, was closely correlated to the extent of TG incorporation into the viral genome. We concluded that a portion of SV40 viral DNA synthesis inhibited by TG is due to an effect on initiation, and hypothesized that this effect may be caused by the substitution of TG for guanine in critical recognition sequences at the origin of replication. We proceeded to test this hypothesis by constructing SV40 origin sequences containing TG and then measuring their ability to bind T-antigen in vitro. The necessary deoxynucleoside triphosphate, TdGTP, was obtained by chemical phosphorylation of thiodeoxyguanosine. In order to selectively place TG within the desired region, a plasmid containing the T-antigen binding sequences was linearized so as to place these sequences at one end of the molecule, and then digested briefly with exonuclease III. The excised strand was resynthesized by use of the Klenow fragment of DNA polymerase I along with various nucleotide mixtures. Although resynthesis with mixtures containing TdGTP in place of dGTP was impeded somewhat, it was possible to achieve complete resynthesis with this analog.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1986

Concurrent unilateral chromatid damage and DNA strand breakage in response to 6-thioguanine treatment.

The delayed cytotoxicity of 6-thioguanine (TG) may relate to the arrest of cells in G2 upon completion of one cell cycle after drug exposure. In Chinese hamster ovary (CHO) cells, both the unilateral chromatid damage in G2 chromosomes, determined by induction of premature condensed chromosome condensation [Maybaum and Mandel, Cancer Res. 43, 3852 (1983)], and incorporation of TG into DNA resulting in DNA strand breakage [Christie et al., Cancer Res. 44, 3665 (1984)] were correlated with cytotoxicity. We have studied the correlation between strand breakage and unilateral chromatid damage in L1210 cells. DNA breaks were detected only when cells were treated with TG (0.25 microM) for one cell cycle time (12 hr) followed by 12 hr in drug-free medium containing [3H]thymidine (TdR) to label the DNA. After simultaneous incubation of cells with drug and label during the first or second 12-hr period, strand breaks were not found. Strand breaks increased with dose, which correlated with greater cytotoxicity (0.01 to 0.25 microM). Treatment of cells with 0.25 microM TG for 12 hr, and transfer to drug-free medium for 12 hr prior to making prematurely condensed chromosomes (PCC), resulted in unilateral chromatid damage. Prominent curving of G2 chromosomes with gapping and diffuse staining of one of the sister chromatids occurred. The 4-fold increase in the percentage of cells in G2 compared with control cells suggested G2 arrest. When cells were treated with TG for 12 hr and PCC made immediately, neither the arrest of cells in G2 nor unilateral chromatid damage was observed. These data suggest that strand breaks and unilateral chromatid damage occur in the second cell cycle after TG exposure and that this damage may be important in TG-delayed cytotoxicity.

Animals↗
PubMed1986

Simultaneous determination of thymine and 5-bromouracil in DNA hydrolysates using gas chromatography-mass spectrometry with selected-ion monitoring.

The gas chromatographic-mass spectrometric method using selected-ion monitoring (GC-MS-SIM) described here quantitatively determines the amount of DNA thymine replacement by 5-bromouracil (BU) after exposure to 5-bromo-2'-deoxyuridine (BUDR) in as few as 10(5) cells. DNA is extracted, enzymatically hydrolyzed, the nucleic acid bases (with added internal standards, 5-iodouracil and 5-chlorouracil) are extracted into ethyl acetate, concentrated and derivatized with bis(trimethylsilyl)trifluoroacetamide. Thymine and BU are then quantitated by GC-MS-SIM. Response is linear to thymine over the range of 100-2000 ng per sample and BU of 1.3-52 ng per sample with a coefficient of variation of less than 10% and an accuracy for seeded samples within 8% of theoretical value. With V79 cells in culture, exposure to increasing BUDR concentrations (0.03-1.0 microM) results in increasing thymine substitution by BU over a range of 1-28%. Other important applications of this technique are mentioned.

Bromodeoxyuridine↗
PubMed1985

Dissimilar actions of 6-mercaptopurine and 6-thioguanine in Chinese hamster ovary cells.

The actions of 6-thioguanine (TG) and 6-mercaptopurine (MP) were compared in Chinese hamster ovary (CHO) cells. Several differences were noted between these two agents. TG caused a greater maximal loss of clonogenicity, leaving about one log fewer survivors than did MP, although the cells killed by MP appeared to succumb much more rapidly than those killed by TG. MP-treated populations experienced a G1 or G1/S arrest which was quickly reversed upon drug removal, while TG-treated cells were arrested in late S/G2, after some delay. Although TG induced a gross chromosome deformation [unilateral chromatid damage, as described earlier in Maybaum and Mandel, Cancer Res. 43, 3852 (1983)] MP caused little or no such deformation. Addition of 4-amino-5-imidazolecarboxamide (AIC) to MP treatments antagonized MP-induced loss of clonogenicity, while AIC caused a dose-dependent potentiation of TG-induced loss of clonogenicity. The interaction between TG and AIC does not seem to represent an increase in either purine starvation or incorporation of TG into DNA, suggesting that a third mechanism is involved. We suggest that this additional mechanism may possibly be related to the induction of differentiation by TG that has been reported in other systems.

Aminoimidazole Carboxamide↗
PubMed1983

Unilateral chromatid damage: a new basis for 6-thioguanine cytotoxicity.

Using the technique of premature chromosome condensation, which permits the visual inspection of interphase chromatin, we have shown previously that 28 hr after exposure to 6-thioguanine (TG) specific and drastic morphological changes in the chromosomes of Chinese hamster ovary fibroblasts in the G2 phase of the cell cycle become evident. In this paper, we demonstrate that this damage is a dose-related effect, appearing as sharp curling or "kinking" at lower TG concentrations and as unilateral chromatid damage and gross chromosome disruption at higher TG concentrations. With the use of a scoring system for quantitating the severity of this specific damage, the threshold concentrations for the appearance of unilateral chromatid damage and for loss of colony-forming ability were shown to be identical. Since the appearance of unilateral chromatid damage paralleled the appearance of TG-induced cytotoxicity in terms of time and dose, and since the severe disruption of G2 prematurely condensed chromosomes is consistent with TG-induced G2 arrest seen in this and other systems, we conclude that unilateral chromatid damage is centrally involved in the delayed cytotoxicity of TG in Chinese hamster ovary cells.

Animals↗
PubMed1981

Guanine nucleotide depletion and toxicity in mouse T lymphoma (S-49) cells.

Incubation of mouse T lymphoma (S-49) cells with the inosinate dehydrogenase inhibitor mycophenolic acid produced a depletion of both GTP and dGTP, and resulted in growth inhibition, partial reduction in RNA synthesis, and drastic inhibition of DNA synthesis. Similar results suggested to others that the depletion of dGTP is primarily responsible for toxicity. However, guanosine was as effective as deoxyguanosine at preventing mycophenolic acid toxicity although deoxyguanosine was more effective at elevating dGTP levels. Moreover, in hypoxanthine-guanine phosphoribosyltransferase-deficient mutants of S-49 (6MPR-3-3) deoxyguanosine was unable to prevent mycophenolic acid toxicity or to re-establish normal DNA synthesis, although it returned cellular dGTP but not GTP levels to normal. No other nucleotide levels changed in a way which could account for the toxicity. Incubation of cells with a combination of deoxyadenosine, deoxycytidine, and erythro-9-(2-hydroxy-3-nonyl)adenine produced a selective depletion of dGTP to levels similar to that produced by mycophenolic acid, but did not affect cell growth. Studies with cells synchronized by centrifugal elutriation show that the toxicity of mycophenolic acid is specific to the S-phase of the cell cycle. Addition of actinomycin D at a concentration that inhibited RNA synthesis increased the availability of GTP and re-established normal DNA synthesis in mycophenolic acid-treated S-49 cells. These results suggest that the depletion of GTP rather than that of dGTP produces toxic effects in S-49 cells and that GTP is required for DNA synthesis.

Animals↗
PubMed1980

Analysis of purine ribonucleotides and deoxyribonucleotides in cell extracts by high-performance liquid chromatography.

A high-performance liquid chromatographic (HPLC) assay for the fourteen major cellular purine ribonucleotides and 2'-deoxyribonucleotides is presented. Following an initial separation by anion-exchange HPLC, the nucleotides are hydrolyzed to their respective nucleosides by alkaline phosphatase and quantified by reversed-phase HPLC and UV absorbance detection. The assay is reproducible, specific, and has a detection limit of 10 pmol/sample. The recovery of nucleosides derived from nucleotides is 85%. Purine nucleotide pool sizes have been measured in cultured mouse T lymphoma (S-49) cells before and after treatment with 2.0 microM mycophenolic acid, an inhibitor of the enzyme IMP dehydrogenase, for 3 h. Control nucleotide levels obtained by this method are consistent with those reported for S-49 cells using other methods, and the observed decrease in guanine nucleotides and increase in IMP after treatment with mycophenolic acid agree with previous reports.

Animals↗
PubMed1980

Determination of pyrimidine ribotide and deoxyribotide pools in cultured cells and mouse liver by high-performance liquid chromatography.

High-performance liquid chromatographic (HPLC) assay for determining tissue pools of uridine, deoxyuridine, cytidine, deoxycytidine, and thymidine mono-, di-, and triphospates is presented. The method utilizes anion-exchange and, after conversion of nucleotides to nucleosides by acid phosphatase, reversed-phase chromatography on a preparative column with UV detection at 254 and 280 nm. The yield of this procedure is 80 +/- 2% with a sensitivity limit of 100 pmole nucleotide per sample. A sensitivity of 10 pmole can be achieved for each compound by rechromatographing appropriate nucleoside fractions on analytical columns. The recovery, including this step, is 66 +/- 7%. The assay is reproducible and highly selective, with a lower sensitivity limit of approximately 0.1 muM using 150--250 mg (wet weight) tissue samples. Nucleotide pools have been determined in Balb/c mouse liver and in mouse lymphoma (S-49) cell culture, the latter with and without addition of 5-fluorouracil (5-FUra) to the medium. Data obtained with this assay are similar to those using alternative methodologies. Observed depletion of dTXP pools and expansion of dUMP and dCXP pools after 5-FUra treatment are in agreement with published observations. Pools of dUDP and dUTP were not detectable (less than 10 pmole/10(8) cells) in any tissue sample. These data illustrate the utility of the present method in studying actions of pyrimidine antimetabolites.

Animals↗
PubMed1980

Regulation of RNA- and DNA-directed actions of 5-fluoropyrimidines in mouse T-lymphoma (S-49) cells.

The mouse T-lymphoma (S-49) cell line is useful for individually studying RNA- and DNA-directed effects of 5-fluoropyrimidines. On the basis of their metabolic activation, biochemical effects on pyrimidine nucleotide metabolism, and biological toxicity, we hve established that incubation of S-49 cells with 5-fluorodeoxyuridine produces only DNA-directed toxicity (thymidylate synthetase inhibition), incubation with 5-fluorouracil (FUra) + thymidine only RNA-directed toxicity, and incubation with FUra alone produces both DNA- and RNA-directed toxicity. The DNA component of 5-fluoropyrimidine toxicity causes immediate growth inhibition of asynchronous S-49 cell cultures, which is self-limited within 12 hr both by the accumulation of intracellular deoxyuridine 5'-monophosphate competing for thymidylate synthetase binding and by the excretion of deoxyuridine into the cell medium which competes with 5-fluorodeoxyuridine uptake. The RNA-directed component causes growth inhibition and cell kill after a delay of 1 doubling time in asynchronous cultures. Studies with cells synchronized by centrifugal elutriation indicate that the RNA-directed FUra effects are expressed only in the G1 phase of the cell cycle and cause rapid cell lysis, while the DNA-directed component is specific to the S phase. Experiments using continuous exposure of synchronized cells to FUra alone demonstrate that the activities of the RNA- and DNA-directed components interact with each other. Specifically, DNA-directed toxicity arrests cells in S phase, preventing them from progressing into G1 where RNA-directed toxicity is expressed, which may account for the augmentation of FUra toxicity by thymidine as reported in other systems.

Animals↗
PubMedDate not supplied

Recognition and resolution of potential workplace violence.

OSHA realizes the significance of providing a safe and healthy environment. Its objective is to eliminate or diminish employee exposure to workplace violence by establishing a preventive management program comprised of effective security mechanisms, administrative work practices and other safety control measures. In addition to creating a safer work environment, these systems strengthen employee confidence and productivity and reduce employer workers' compensation fees.

Dental Offices↗