Triple helix-forming oligonucleotides (TFOs) bind with high affinity and specificity to homopurine-homopyrimidine sequences in DNA and have been shown to inhibit transcription of target genes in various experimental systems. In the present study, we evaluated the ability of 3'-amino-modified phosphodiester TFOs directed to four sites in the c-myc gene to inhibit gene expression and proliferation of human leukemia (CEM, KG-1, and HL-60) and lymphoma (Raji and ST486) cells. GT-rich TFOs were designed to target sequences located either upstream (myc1 and -2) or downstream (myc3 and -4) of the P2 promoter, which is the major c-myc promoter. Myc2, which was directed to a site immediately upstream of this promoter, inhibited c-myc expression and proliferation of CEM cells. The effects of this TFO were sequence- and target-specific, since control oligonucleotides and TFOs directed to other sites were less or not active. Myc2 was also effective in KG-1, HL-60, and Raji cells. In contrast, ST486 cells were more sensitive to myc3, which targets a sequence in intron 1 upstream of the P3 promoter, than myc2. As result of a chromosomal translocation, P3 is the active promoter in ST486 cells. This study demonstrates the activity and specificity of TFOs designed to act as repressors of c-myc gene expression in human leukemia and lymphoma cells. Our results suggest that this is a valid approach to selectively inhibit gene expression and cancer cell growth, and encourage further investigation of its potential applications in cancer therapy.

Although bacterial anaerobic degradation of mono-aromatic compounds has been characterized in depth, the degradation of polycyclic aromatic hydrocarbons (PAHs) such as naphthalene has only started to be understood in sulfate reducing bacteria, and little is known about the anaerobic degradation of PAHs in nitrate reducing bacteria. Starting from a series of environments which had suffered different degrees of hydrocarbon pollution, we used most probable number (MPN) enumeration to detect and quantify the presence of bacterial communities able to degrade several PAHs using nitrate as electron acceptor. We detected the presence of a substantial nitrate reducing community able to degrade naphthalene, 2-methylnaphthalene (2MN), and anthracene in some of the sites. With the aim of isolating strains able to degrade PAHs under denitrifying conditions, we set up a series of enrichment cultures with nitrate as terminal electron acceptor and PAHs as the only carbon source and followed the changes in the bacterial communities throughout the process. Results evidenced changes attributable to the imposed nitrate respiration regime, which in several samples were exacerbated in the presence of the PAHs. The presence of naphthalene or 2MN enriched the community in groups of uncultured and poorly characterized organisms, and notably in the Acidobacteria uncultured group iii1-8, which in some cases was only a minor component of the initial samples. Other phylotypes selected by PAHs in these conditions included Bacilli, which were enriched in naphthalene enrichments. Several nitrate reducing strains showing the capacity to grow on PAHs could be isolated on solid media, although the phenotype could not be reproduced in liquid cultures. Analysis of known PAH anaerobic degradation genes in the original samples and enrichment cultures did not reveal the presence of PAH-related nmsA-like sequences but confirmed the presence of bssA-like genes related to anaerobic toluene degradation. Altogether, our results suggest that PAH degradation by nitrate reducing bacteria may require the contribution of different strains, under culture conditions that still need to be defined.

We aimed to test the hypothesis that the inducible form of nitric oxide synthase (iNOS) contributes to the development of an early subnormal retinal oxygenation response in preclinical models of diabetic retinopathy. In urethane anesthetized Sprague Dawley rats or C57BL/6 mice, functional magnetic resonance imaging was used to noninvasively measure the change in retinal oxygen tension (⌬PO 2 ) during a carbogen-inhalation challenge. In the rat experiments, the retinal ⌬PO 2 of the following groups were compared: control rats (n ‫؍‬ 9), 3-month diabetic rats (n ‫؍‬ 5), and 3-month diabetic rats treated orally with L-N(6)-(1-iminoethyl)lysine 5-tetrazole amide, a prodrug of an inhibitor of iNOS (n ‫؍‬ 6). In addition, the retinal ⌬PO 2 of the following mouse groups were compared: C57BL/6 mice (n ‫؍‬ 20), C57BL/6-Nos2 tm1Lau mice (n ‫؍‬ 10), 4-month diabetic mice (n ‫؍‬ 13), and 4-month diabetic knockout mice (n ‫؍‬ 6). Only the ⌬PO 2 of the superior hemiretina of the diabetic rat and mice groups were significantly subnormal (P < 0.05). The superior ⌬PO 2 of the diabetic rats treated with the prodrug was not significantly (P > 0.05) different from their respective normal controls. In the mice experiments, the superior retinal ⌬PO 2 of the iNOS null mice was not statistically different (P > 0.05) from that of normal control mice. iNOS is required for the development of an early subnormal ⌬PO 2 in experimental diabetic retinopathy. Diabetes 53: [173][174][175][176][177][178] 2004 N itric oxide (NO), a potent regulator of retinal vascular function, is elevated in the vitreous humor of patients with proliferative diabetic retinopathy and with tractional retinal detachment, as well as in the retina of rodents, 2-4 months after the induction of diabetes (1-4). NO synthase (NOS) converts L-arginine to NO and L-citrulline. Excessive NO production by the inducible isoform of NOS (iNOS) in particular has been implicated in the pathogenesis of various ocular diseases (5,6). iNOS is induced in the retina in diabetes, but it is not yet known if iNOS regulates aspects of retinal circulatory pathophysiology associated with diabetes (4).Previously, we developed a novel functional magnetic resonance imaging (MRI) method for measuring the retinal oxygenation response to a hyperoxic inhalation challenge in the newborn and adult rat, rabbit, cat, and human (7-10). In this technique, hyperoxia increases vitreous partial oxygen pressure over room-air values (⌬PO 2 ). Because oxygen is paramagnetic, this ⌬PO 2 will produce an increase in the vitreous signal intensity on a T 1 -weighted image. Furthermore, good agreement is found between the MRI-measured response and that determined by other investigators (11) using an oxygen electrode in normal rat retina under similar conditions. In normal adult and newborn rats, carbogen breathing oxygenated the retina significantly better than pure oxygen breathing (11). Carbogen is a gas mixture of carbon dioxide (5%) and oxygen (95%) that has been used clinically, instead of 100% oxygen, to minim...