Changing lives, dynamic plans: Prospective assessment of 12-month changes in pregnancy timing intentions and personal circumstances using data from HER Salt Lake.

OBJECTIVES: To explore the association between changes in personal circumstances and shifts in pregnancy intentions. STUDY DESIGN: New start contraceptive clients, who desired to prevent pregnancy for at least one year enrolled in the survey arm of the HER Salt Lake Contraceptive Initiative (September 2015 -March 2017) and responded to the question "What are your future pregnancy plans?" at enrollment and 12-month follow-up. We estimated multivariable binary logistic fixed-effects regressions to examine the association between changes in personal circumstances and a change from never desiring a pregnancy at enrollment to considering one in the future at 12-month follow-up. RESULTS: The majority of the 2825 participants (2246, 79%) maintained their pregnancy timing intention over the 12-month study period. Multivariable analyses of the 208 participants who changed from never desiring a pregnancy to considering pregnancy in the future at 12-month follow-up indicated that entering cohabitation (aOR 3.14, 95% CI 1.30-7.58), increased household income (aOR 1.06, 95% CI 1.00-1.13), and changes from unemployment to full-time employment (aOR 5.94, 95% CI 1.29-27.36) are associated with increased the odds of desiring a future pregnancy after never wanting one a year prior. CONCLUSIONS: Pregnancy intentions are dynamic over twelve months and covary with partner status, household income, and employment status. Pregnancy intentions are linked to changes in life circumstances. Health care providers need to frequently assess pregnancy intentions and resulting contraceptive or preconception needs.

One-year continuation of copper or levonorgestrel intrauterine devices initiated at the time of emergency contraception.

OBJECTIVE(S): This study compares 1-year intrauterine device (IUD) continuation among women presenting for emergency contraception (EC) and initiating the copper (Cu T380A) IUD or the levonorgestrel (LNG) 52 mg IUD plus 1.5 mg oral LNG. STUDY DESIGN: This cohort study enrolled 188 women who presented at a single family planning clinic in Utah between June 2013 and September 2014 and selected either the Cu T380A IUD or LNG 52 mg IUD plus oral LNG for EC. Trained personnel followed participants by phone, text or e-mail for 12 months or until discontinuation occurred. We assessed reasons for discontinuation and used Cox proportional hazard models, Kaplan-Meier estimates and log-rank tests to assess differences in continuation rates between IUDs. RESULTS: One hundred seventy-six women received IUDs; 66 (37%) chose the Cu T380A IUD and 110 (63%) chose the LNG 52 mg IUD plus oral LNG. At 1 year, we accounted for 147 (84%) participants, 33 (22%) had requested removals, 13 (9%) had an expulsion and declined reinsertion, 3 (2%) had a pregnancy with their IUD in place and 98 (67%) were still using their device. Continuation rates did not differ by IUD type; 60% of Cu T380A IUD users and 70% of LNG 52 mg IUD plus oral LNG users were still using their device at 12 months (adjusted hazard ratio 0.72, 95% confidence interval 0.40-1.3). CONCLUSION(S): Two-thirds of women who chose IUD placement at the EC clinical encounter continued use at 1 year. Women initiating Cu T380A IUD and LNG 52 mg IUD had similar 1-year continuation rates. These findings support same-day insertion of IUDs for women who are seeking EC and would like to use a highly effective reversible method going forward. IMPLICATIONS: Providing IUD options for EC users presents an opportunity to increase availability of highly effective contraception.

Targeting the ets binding site of the HER2/neu promoter with pyrrole-imidazole polyamides.

Three DNA binding polyamides () were synthesized that bind with high affinity (K(a) = 8.7. 10(9) m(-1) to 1.4. 10(10) m(-1)) to two 7-base pair sequences overlapping the Ets DNA binding site (EBS; GAGGAA) within the regulatory region of the HER2/neu proximal promoter. As measured by electrophoretic mobility shift assay, polyamides binding to flanking elements upstream () or downstream (2 and 3) of the EBS were one to two orders of magnitude more effective than the natural product distamycin at inhibiting formation of complexes between the purified EBS protein, epithelial restricted with serine box (ESX), and the HER2/neu promoter probe. One polyamide, 2, completely blocked Ets-DNA complex formation at 10 nm ligand concentration, whereas formation of activator protein-2-DNA complexes was unaffected at the activator protein-2 binding site immediately upstream of the HER2/neu EBS, even at 100 nm ligand concentration. At equilibrium, polyamide 1 was equally effective at inhibiting Ets/DNA binding when added before or after in vitro formation of protein-promoter complexes, demonstrating its utility to disrupt endogenous Ets-mediated HER2/neu preinitiation complexes. Polyamide 2, the most potent inhibitor of Ets-DNA complex formation by electrophoretic mobility shift assay, was also the most effective inhibitor of HER2/neu promoter-driven transcription measured in a cell-free system using nuclear extract from an ESX- and HER2/neu-overexpressing human breast cancer cell line, SKBR-3.

Targeting E2F1-DNA complexes with microgonotropen DNA binding agents.

Microgonotropen (MGT) DNA binding drugs, which consist of an A+T-selective DNA minor groove binding tripyrrole peptide and polyamine chains attached to a central pyrrole that extend drug contact into the DNA major groove, were found to be extraordinarily effective inhibitors of E2 factor 1 (E2F1) association with its DNA promoter element (5'-TTTCGCGCCAAA). The most active of these drugs, MGT-6a, was three orders of magnitude more effective than distamycin and inhibited complexes between E2F1 and the dihydrofolate reductase promoter by 50% at 0.00085 microM. A relationship was found between the measured equilibrium constants for binding of MGTs to the A+T region of d(GGCGA3T3GGC)/d(CCGCT3A3CCG) and their inhibition of complex formation between E2F1 and the DNA promoter element. A representative of the potent MGT inhibitors was significantly more active on inhibition of E2F1-DNA complex formation compared with disruption of a preexisting complex.

Kinetics of cleavage of intra- and extracellular simian virus 40 DNA with the enediyne anticancer drug C-1027.

A kinetic analysis of cleavage of simian virus DNA (SV40 DNA) inside and outside green monkey BSC-1 cells by the enediyne-protein antibiotic C-1027 and its free chromophore is described. Information on rate constants was obtained by fitting populations of forms I (closed circular DNA), II (nicked circular DNA) and III (linear DNA) SV40 DNA as a function of drug concentration to a kinetic model which includes: cutting of form I to give form II with rate constant k1, cutting of form I to give form III with rate constant K4, and cutting of form II to give form III with rate constant k2. The ratio of single-strand (ss) to double-strand (ds) cutting for the holoantibiotic and the free chromophore, k1/k4, is approximately 1.8 for extracellular SV40 DNA. For intracellular DNA and extracellular DNA which has been post-treated with putrescine, ds cutting is much more probable, with k4 about four times as large as k1. This observation suggests that amine groups present in the cell are able to convert abasic sites opposite an ss break into a ds break in SV40 chromatin. The overall rate of cleavage of form-I DNA inside the cell is much larger than the rate outside, the sum k1 + k4 being about three times as large for intracellular DNA as for extracellular DNA.

Effects of bizelesin (U-77779), a bifunctional alkylating minor groove agent, on genomic and simian virus 40 DNA.

Bizelesin is a bifunctional covalent minor groove binding agent which forms adducts with 3'-adenines on opposite DNA strands. DNA lesions induced by bizelesin in genomic DNA of BSC-1 cells, as well as intracellular and purified simian virus 40 (SV40) DNA, were examined. Alkaline sucrose sedimentation analysis indicated a nonrandom distribution of heat-labile damage in BSC-1 cell genomic DNA with frequencies of 1-60 lesions/10(6) base pairs (bp) for bizelesin concentrations from 10 to 400 nM, respectively. Extrapolation of these data suggested that, at 0.15 nM bizelesin, approximately 10(2) adducts per cell may be sufficient to inhibit cell growth by 90% (D10). While the frequency of bizelesin adducts in intracellular SV40 DNA was comparable to that in genomic DNA, higher levels of lesion formation are observed with purified SV40 DNA. Chromatin structure has little effect on localization of bizelesin adducts since treatment of either infected cells or purified SV40 DNA reveals a similar pattern of drug-induced damage. Bizelesin adduction sites (mapped on the SV40 genome as thermally-induced strand breaks at 50-100 bp resolution) are found in regions centered at 4200, 3900, 4700, and approximately 5200. The location of these regions of intense bizelesin bonding coincides with the sites of potential cross-links predicted using the 5'-T-(A/T)4-A-3' sequence. The analysis of bizelesin adducts at the sequence level in the 3943-4451 SV40 DNA fragment indicated that 40% of total damage was in potential cross-linking sites and an additional 35% in the 5'-A-(A/T)4-A-3' monoalkylating sites.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of the DNA-damaging enediyne C-1027 on intracellular SV40 and genomic DNA in green monkey kidney BSC-1 cells.

This study describes the selective ability of C-1027 to induce limited double-strand damage in a viral DNA target. The effect of the cellular environment on C-1027 activity was examined by assaying the extent, as well as the specificity, of damage to simian virus 40 (SV40) DNA in lytically infected mammalian BSC-1 cells and in purified SV40 DNA preparations. C-1027 damage to intracellular SV40 DNA was quantitated by topological forms conversion analysis. A gradual decrease in intracellular supercoiled form I accompanied by an increase in form III was observed with C-1027 concentrations from 2 to 100 nM, with a 50% reduction in form I observed at 50 nM. Damage to purified SV40 DNA also was most pronounced between 10 and 100 nM C-1027. When concentrations were expressed as r values (drug/DNA molar ratio), the amount of C-1027 necessary to effect a 50% reduction in form I was lower for intracellular (r = 0.002) than for purified SV40 DNA (r = 0.0035). Double-strand damage was more likely to occur with C-1027 treatment of intracellular compared to purified SV40 DNA. However, with both purified and intracellular DNA, restriction enzyme digestion analysis revealed double-strand damage at a number of specific sites throughout the genome, particularly within the early region of the SV40 genome (e.g., within the coding sequence for large T-antigen). No significant damage was observed in either the origin (ORI) or the termination (TER) regions of SV40 replication. The extent of C-1027 damage to uninfected BSC-1 cell DNA was also quantitated using pulsed-field gel electrophoresis. At 0.1 nM (r = 2.8 x 10(-5), where incorporation of [3H]thymidine was reduced by 80%, 600 rad equiv of damage was detected in uninfected BSC-1 cells. At C-1027 r values from 1 x 10(-4) to 40 x 10(-4), double-strand breaks were from 80- to 40-fold more frequent in SV40 than in BSC-1 cell genomic DNA. By contrast, 50-fold more drug was necessary to inhibit intracellular SV40 DNA accumulation compared to [3H]thymidine incorporation into uninfected BSC-1 cells. Thus, SV40 DNA synthesis appeared to be less sensitive to C-1027-induced lesions than replication in uninfected BSC-1 cells.

CC-1065 bonding to intracellular and purified SV40 DNA: site specificity and functional effects.

CC-1065 is a minor-groove bonding agent capable of forming covalent adducts with the N-3 position of adenines within A-T-rich regions of duplex DNA. By examining the formation and location of CC-1065 adducts within the simian virus 40 (SV40) DNA molecule, the present study marks the first time that the precise sites of CC-1065 lesions have been identified at the level of eukaryotic genomic DNA. In naked DNA preparations, r values (moles of drug/mole of nucleotide base pair) > or = 0.0015 effected, after thermal treatment, a measurable decrease in intact supercoiled form I, as well as increases in forms II and III, indicating that both single-strand and apparent double-strand damage had occurred. A similar pattern of damage was observed in SV40-infected cells, albeit at higher CC-1065 levels. The amount of CC-1065 required to produce a 50% loss in form I was > 2-fold higher in infected cells (r = 0.029) than with purified DNA samples (r = 0.013). The appearance of double-strand damage at low drug levels suggested a high specificity of CC-1065 bonding to localized regions of the genome. The precise location of these CC-1065 adduction sites was examined by three methods: sequence analysis of the entire genome (GenBank), DNA polymerase termination assay of specific fragments of SV40, and restriction enzyme digestion analysis of the entire SV40 molecule. When sequence analysis of the entire genome was performed by examining both strands for the presence of the consensus CC-1065 binding sequence 5'-A/T-A/T-A/T-A/T-A*-3'[Reynolds et al. (1985) Biochemistry 24, 6228-6247], 294 single-strand adduction sites were predicted, compared to 20 sites where CC-1065 should bond to both strands within a 30-base-pair window and at which, when heated, a double-strand break should occur. DNA polymerase termination assay of actual adduction sites was performed on restriction fragments of SV40 DNA pretreated with CC-1065 in infected cells or in purified supercoiled DNA preparations and selected on the basis of the sequence analysis (i.e., regions 2510-2730, 3701-3920, 4400-4659, 4020-4320, and 5163-65). In general, double-strand lesions were detected in similar regions of the genome by the DNA termination assay and by sequence analysis. When restriction enzyme digestion and the DNA polymerase termination assay were compared throughout the genome, nearly identical patterns of adduct formation were observed. Interestingly, similar alkylation patterns were observed with either naked or infected cell DNA.(ABSTRACT TRUNCATED AT 400 WORDS)

Netropsin and bis-netropsin analogs as inhibitors of the catalytic activity of mammalian DNA topoisomerase II and topoisomerase cleavable complexes.

This study examined the ability of netropsin and related minor groove binders to interfere with the actions of DNA topoisomerases II and I. We evaluated a series of netropsin dimers linked with flexible aliphatic chains of different lengths. These agents are potentially able to occupy longer stretches of DNA than the parental drug as a result of bidentate binding. Both netropsin and its dimers were found: (i) to inhibit the catalytic activity of isolated topoisomerase II and (ii) to interfere with the stabilization of the cleavable complexes of topoisomerase II and I in nuclei. Dimers with linkers consisting of 0-4 and 6-9 methylene groups (n) were far more inhibitory than netropsin against isolated enzyme and in the nuclear system. The compound with n = 5 was less active than netropsin in both assays while the dimer with n = 10 inhibited only the isolated enzyme. The comparison of dimers with fixed linker length (n = 2) but varying number of N-methylpyrrole residues (from 1 to 3) revealed that the inhibitory properties were enhanced with increasing number of N-methylpyrrole units. For dimers with varying linker length, drug ability to inhibit catalytic activity of isolated topoisomerase II was positively correlated with calf thymus DNA association constants. In contrast, no such correlation existed in nuclei. However, the inhibitory effects in the nuclear system were correlated with the association constants for poly(dAdT). The results indicate that bidentate binding can significantly enhance anti-topoisomerase activity of netropsin related dimeric minor groove binders. However, other factors such as the length of the linker, the number of pyrrole moieties and the nature of the target (isolated enzyme/DNA versus chromatin in nuclei) also contribute to these activities.

Induction of heat-labile sites in DNA of mammalian cells by the antitumor alkylating drug CC-1065.

CC-1065 is a very potent antitumor antibiotic capable of covalent and noncovalent binding to the minor groove of naked DNA. Upon thermal treatment, covalent adducts formed between CC-1065 and DNA generate strand breaks [Reynolds, R. L., Molineux, I. J., Kaplan, D.J., Swenson, D.H., & Hurley, L.H. (1985) Biochemistry 24, 6228-6237]. We have shown that this molecular damage can be detected following CC-1065 treatment of mammalian whole cells. Using alkaline sucrose gradient analysis, we observe thermally induced breakage of [14C]thymidine-prelabeled DNA from drug-treated African green monkey kidney BSC-1 cells. Very little damage to cellular DNA by CC-1065 can be detected without first heating the drug-treated samples. CC-1065 can also generate heat-labile sites within DNA during cell lysis and heating, subsequent to the exposure of cells to drug, suggesting that a pool of free and noncovalently bound drug is available for posttreatment adduct formation. This effect was controlled for by mixing [3H]thymidine-labeled untreated cells with the [14C]thymidine-labeled drug-treated samples. The lowest drug dose at which heat-labile sites were detected was 3 nM CC-1065 (3 single-stranded breaks/10(6) base pairs). This concentration reduced survival of BSC-1 cells to 0.1% in cytotoxicity assays. The generation of CC-1065-induced lesions in cellular DNA is time dependent (the frequency of lesions caused by a 60 nM treatment reaching a plateau at 2 h) and is not readily reversible. The induction of heat-labile sites in cellular DNA was confirmed by gel electrophoretic analyses of the damage to intracellular simian virus 40 (SV40) DNA in SV40-infected BSC-1 cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Extrachromosomal chromatin: novel target for bleomycin cleavage in cells and solid tumors.

The preference of bleomycin, a DNA strand scission antitumor agent, to damage extrachromosomal (episomal) DNA was investigated. These episomes contain transcriptional promoters, replication origins, and oncogenes from MMTV, BPV, and v-Ha-ras and confer a neoplastic phenotype to a mouse fibroblast cell line. We found that bleomycin induces dose-dependent single- and double-stranded cleavage of intracellular episomes as measured by topological forms conversion. Bleomycin scission of episomes occurs within 1 min, and upon drug removal, damaged episomes are as rapidly repaired. By expressing the episomal and genomic damage as breaks per nucleotide, bleomycin has a 30-50-fold cleavage preference for episomal chromatin compared to genomic DNA. The episomes have preferred regions of the bleomycin-induced damage, particularly within the MMTV LTR and BPV origin of replication. Also, it is possible to assess bleomycin action on episomes in solid tumors in mice. Single intravenous injections of BLM into tumor-bearing mice result in single- and double-stranded cleavage of episomes that are dose related and occur within 1 min. Specific double-stranded breaks occur in the same regulatory regions of episomes in solid tumors and in cultured cells. Finally, we observe that damage to the episomal drug target occurs at therapeutic doses in mice.

[Comparison of the results of simultaneous trabeculectomy and cataract extraction with microscopic evaluation]. / Konfrontacja wyników jednoczesnej trabekulektomii i ekstrakcji zacmy z ocenq mikroskopowq.

The dependence of hydrodynamics on the topography of trabeculectomy performed simultaneously with cataract extraction was checked in 35 eyes with cataract and glaucoma. The eye was opened by a three-surface incision with a broad scleral flap. The specimen were examined in a microscope. In 21 eyes the whole trabeculum together with Schlemm's canal were excised, in 12 eyes the anterior band. In the specimen from 2 eyes no elements of the trabeculum could be found. The dependence of the regulation of the IOP on the geography of the trabeculectomy was established. Accepting the filtering mechanism of the trabeculectomy one may assume that the applied method of incision promotes the formation of the outflow++ ways of the aqueous in a high percentage of cases.

Topoisomerase II-mediated DNA damage of episomes in tumor-bearing mice.

Observations of cells in culture have demonstrated that, for many antitumor agents, topoisomerase II-mediated DNA damage relates to cytotoxicity. However, there is no evidence in tumor-bearing animals to suggest that such agents induce topoisomerase II-mediated damage of DNA in solid tumors or that such damage reflects inhibition of tumor growth. To address this question, a mouse fibroblast cell line neoplastically transformed by an episomal element containing the v-Ha-ras and bovine papillomavirus genes was utilized to measure topoisomerase II-induced DNA damage and growth inhibition of solid tumors derived from this line. Using the topoisomerase II inhibitor amsacrine, the episomal element was found to be a sensitive indicator of topoisomerase II-mediated damage in vivo. The DNA breaks induced by single i.v. injections of amsacrine were protein linked and occurred preferentially in episomal regulatory regions. A strong correlation between suppression of tumor growth and topoisomerase II-mediated damage of the episome was demonstrated.

Bleomycin-induced aggregation of presolubilized and nuclear chromatin from L1210 cells.

These studies have identified a new activity of bleomycin (in addition to the well-known DNA cleavage): drug-induced chromatin aggregation. Bleomycin treatment of presolubilized chromatin from L1210 nuclei resulted in two types of effect as shown by sedimentation analysis of intact nucleoprotein. The first effect was a dose-dependent fragmentation of chromatin to mononucleosomes (12 S) and subnucleosomal fragments (under 5 S). The second effect was aggregation manifested by the generation of large chromatin particles (over 120 S) that sedimented faster than the original material (20-40 S). Bleomycin treatment of nuclei from L1210 cells resulted in a similar, almost bimodal, size distribution of drug-released chromatin fragments. Increasing levels of bleomycin produced a gradual enhancement of the amount of small fragments (under 5 S) accompanied by generation of large, aggregated particles. Aggregation caused by high drug concentrations significantly reduced the overall extent of chromatin solubilization and allowed only the release of the most degraded fragments from nuclei. The aggregation required intact nucleoprotein, since it was not detected after high-salt deproteinization of bleomycin-treated presolubilized or nuclear chromatin. The aggregation phenomenon reflects a novel activity of bleomycin which may contribute to the drug's antiproliferative properties.