Crosstalk between G-Quadruplexes and Dnmt3a-Mediated Methylation of the c-MYC Oncogene Promoter.

The methylation of cytosines at CpG sites in DNA, carried out de novo by DNA methyltransferase Dnmt3a, is a basic epigenetic modification involved in gene regulation and genome stability. Aberrant CpG methylation in gene promoters leads to oncogenesis. In oncogene promoters, CpG sites often colocalize with guanine-rich sequences capable of folding into G-quadruplexes (G4s). Our in vitro study aimed to investigate how parallel G4s formed by a sequence derived from the c-MYC oncogene promoter region affect the activity of the Dnmt3a catalytic domain (Dnmt3a-CD). For this purpose, we designed synthetic oligonucleotide constructs: a c-MYC G4-forming oligonucleotide and linear double-stranded DNA containing an embedded stable extrahelical c-MYC G4. The topology and thermal stability of G4 structures in these DNA models were analyzed using physicochemical techniques. We showed that Dnmt3a-CD specifically binds to an oligonucleotide containing c-MYC G4, resulting in inhibition of its methylation activity. c-MYC G4 formation in a double-stranded context significantly reduces Dnmt3a-CD-induced methylation of a CpG site located in close proximity to the quadruplex structure; this effect depends on the distance between the non-canonical structure and the specific CpG site. One would expect DNA hypomethylation near the G4 structure, while regions distant from this non-canonical form would maintain a regular pattern of high methylation levels. We hypothesize that the G4 structure sequesters the Dnmt3a-CD and impedes its proper binding to B-DNA, resulting in hypomethylation and activation of c-MYC transcription.

Impact of G-Quadruplex Structures on Methylation of Model Substrates by DNA Methyltransferase Dnmt3a.

In mammals, de novo methylation of cytosines in DNA CpG sites is performed by DNA methyltransferase Dnmt3a. Changes in the methylation status of CpG islands are critical for gene regulation and for the progression of some cancers. Recently, the potential involvement of DNA G-quadruplexes (G4s) in methylation control has been found. Here, we provide evidence for a link between G4 formation and the function of murine DNA methyltransferase Dnmt3a and its individual domains. As DNA models, we used (i) an isolated G4 formed by oligonucleotide capable of folding into parallel quadruplex and (ii) the same G4 inserted into a double-stranded DNA bearing several CpG sites. Using electrophoretic mobility shift and fluorescence polarization assays, we showed that the Dnmt3a catalytic domain (Dnmt3a-CD), in contrast to regulatory PWWP domain, effectively binds the G4 structure formed in both DNA models. The G4-forming oligonucleotide displaced the DNA substrate from its complex with Dnmt3a-CD, resulting in a dramatic suppression of the enzyme activity. In addition, a direct impact of G4 inserted into the DNA duplex on the methylation of a specific CpG site was revealed. Possible mechanisms of G4-mediated epigenetic regulation may include Dnmt3a sequestration at G4 and/or disruption of Dnmt3a oligomerization on the DNA surface.

Measurement of segmental lumbar spine flexion and extension using ultrasound imaging.

STUDY DESIGN: Clinical measurement, technical note. OBJECTIVES: To describe a technique to measure interspinous process distance using ultrasound (US) imaging, to assess the reliability of the technique, and to compare the US imaging measurements to magnetic resonance imaging (MRI) measurements in 3 different positions of the lumbar spine. BACKGROUND: Segmental spinal motion has been assessed using various imaging techniques, as well as surgically inserted pins. However, some imaging techniques are costly (MRI) and some require ionizing radiation (radiographs and fluoroscopy), and surgical procedures have limited use because of the invasive nature of the technique. Therefore, it is important to have an easily accessible and inexpensive technique for measuring lumbar segmental motion to more fully understand spine motion in vivo, to evaluate the changes that occur with various interventions, and to be able to accurately relate the changes in symptoms to changes in motion of individual vertebral segments. METHODS: Six asymptomatic subjects participated. The distance between spinous processes at each lumbar segment (L1-2, L2-3, L3-4, L4-5) was measured digitally using MRI and US imaging. The interspinous distance was measured with subjects supine and the lumbar spine in 3 different positions (resting, lumbar flexion, and lumbar extension) for both MRI and US imaging. The differences in distance from neutral to extension, neutral to flexion, and extension to flexion were calculated. RESULTS: The measurement methods had excellent reliability for US imaging (intraclass correlation coefficient [ICC3,3] = 0.94; 95% confidence interval: 0.85, 0.97) and MRI (ICC3,3 = 0.98; 95% confidence interval: 0.95, 0.99). The distance measured was similar between US imaging and MRI (P>.05), except at L3-4 flexion-extension (P = .003). On average, the MRI measurements were 1.3 mm greater than the US imaging measurements. CONCLUSION: This study describes a new method for the measurement of lumbar spine segmental flexion and extension motion using US imaging. The US method may offer an alternative to other imaging techniques to monitor clinical outcomes because of its ease of use and the consistency of measurements compared to MRI.

Hospitalization rates for coronary heart disease in relation to residence near areas contaminated with persistent organic pollutants and other pollutants.

Exposure to environmental pollutants may contribute to the development of coronary heart disease (CHD). We determined the ZIP codes containing or abutting each of the approximately 900 hazardous waste sites in New York and identified the major contaminants in each. Three categories of ZIP codes were then distinguished: those containing or abutting sites contaminated with persistent organic pollutants (POPs), those containing only other types of wastes ("other waste"), and those not containing any identified hazardous waste site ("clean"). Effects of residence in each of these ZIP codes on CHD and acute myocardial infarction (AMI) hospital discharge rates were assessed with a negative binomial model, adjusting for age, sex, race, income, and health insurance coverage. Patients living in ZIP codes contaminated with POPs had a statistically significant 15.0% elevation in CHD hospital discharge rates and a 20.0% elevation in AMI discharge rates compared with clean ZIP codes. In neither of the comparisons were rates in other-waste sites significantly greater than in clean sites. In a subset of POP ZIP codes along the Hudson River, where average income is higher and there is less smoking, better diet, and more exercise, the rate of hospitalization for CHD was 35.8% greater and for AMI 39.1% greater than in clean sites. Although the cross-sectional design of the study prevents definite conclusions on causal inference, the results indirectly support the hypothesis that living near a POP-contaminated site constitutes a risk of exposure and of development of CHD and AMI.

Analysis of Immunomodulating Properties of Retinoids.

In this work analysis of immunomodulating properties of retinoids and carotinoids, mainly beta-carotene is presented. The main attention is concentrated on the latest investigations of the role of retinoid receptors in realization of retinoid influence on differentiation of hematopoietic elements and the apoptosis induction. The conclusion concerning the principal advantages of beta-carotene as immunomodulator comparing to other existing immunomodulating agents has been made.