Studies on anti-human immunodeficiency virus oligonucleotides that have alternating methylphosphonate/phosphodiester linkages.

Preliminary investigations of the physical properties of oligonucleotide analogs that contain alternating methylphosphonate/phosphodiester linkages are described. An alternating oligo-2'-O-methylribonucleoside methylphosphonate, oligomer 1676, whose sequence is complementary to the upper hairpin region of human immunodeficiency virus TAR RNA, has been synthesized. This 15-mer forms a very stable duplex with its complementary RNA target, whose melting temperature is 71 degrees C. Introduction of two mismatched bases reduces the melting temperature by 16 degrees C. Similar results were obtained with the all-phosphodiester version of oligomer 1676, which demonstrates that introduction of the methylphosphonate linkages does not significantly perturb the ability of the oligo-2'-O-methylribonucleoside methylphosphonate to bind to RNA. Unlike the phosphodiester oligomer, however, oligomer 1676 is completely resistant to hydrolysis by the 3'-exonuclease activity found in mammalian serum. The interactions between nuclease-resistant, 5'-psoralen-derivatized, alternating oligo-2'-deoxypyrimidine methylphosphonates and double-stranded DNA were also studied. A 15-mer that contains thymine, 5-methylcytosine, and 5-propynyl-uracil forms a triplex with a polypurine tract found in the env gene of human immunodeficiency virus proviral DNA with an apparent dissociation constant of 400 nM at 22 degrees C. Maximal triplex formation by these oligomers is observed at approximately 2.5 mM magnesium, whereas maximal triplex formation by the corresponding all-phosphodiester oligomers occurs between 10 and 20 mM magnesium. This reduced magnesium dependence most likely results from reduced charge repulsion between the backbones of the methylphosphonate oligomer and purine strand of the target. The nuclease stability and ability of the methylphosphonate oligomers to form stable complexes with their target nucleic acids suggest that these oligomers are potential candidates for use as antisense or antigene agents in cell culture.

Effects of heme proteins on nitric oxide levels and cell viability in isolated PMNs: a mechanism of toxicity.

Isolated human PMNs served as a model to determine oxyhemoglobin (oxyHb) binding and the effects of oxymyoglobin (oxyMb) or oxyHb on production of both nitric oxide (NO*) and superoxide (O2*-) and the resulting cytotoxicity. Physiologically relevant concentrations of NO* and H2O2 oxidized, to a similar extent, 2,7-dichlorodihydrofluorescein (DCFH) loaded into polymorphonuclear neutrophils (PMNs). Activation of PMNs with phorbol 12-myristate 13-acetate (PMA) markedly increased the internalization of extracellular oxyHb (10-250 microg/mL). OxyMb (10-300 microg/mL) or oxyHb (30-300 microg/mL) enhanced DCFH oxidation by a concentration-dependent mechanism in unstimulated, lipopolysaccharide (LPS) and tumor necrosis factor alpha (TNF-alpha)-, and PMA-stimulated PMNs. This increased DCFH oxidation was eliminated by NO* synthase inhibitors, glutathione and ascorbate, and was reduced by albumin. Nitrite accumulation in PMN filtrates mirrored NO*-induced DCF fluorescence. OxyMb-induced increases in NO* levels paralleled alterations in DNA and cell membrane damage and ATP levels in PMNs and co-cultured lymphocytes, and were attenuated by NO* synthase inhibitors. OxyMb eliminated extracellular O2*- at protein concentrations 100- to 1000-fold above those of superoxide dismutase. These results suggest that heme proteins bind and internalize into PMNs and increase NO*-induced damage in neighboring cells by inhibiting O2*(-)-scavenging of NO*. We propose a mechanism whereby heme protein-induced NO* levels may contribute to immunosuppression and increased infection rates associated with transfusions and cellular damage during inflammation.

Structure-activity relationships in aquatic toxicology.

Relationships among chemical structure, aquatic toxicity, and bioconcentration potential were examined for several classes of organic compounds. Structure-toxicity correlations were based largely on median lethal concentrations (LC50) and toxicant threshold concentrations (LC1) determined in mini-chronic tests with early life stages of fish and amphibians. Exposure was initiated at fertilization and maintained through 4 days posthatching. Bioconcentration potential was assessed using n-octanol/water partition coefficients (log P). In tests with polychlorinated biphenyls (PCB), acute and chronic toxicity generally increased with percent chlorination. In addition, toxicity of specific PCB's appeared to be affected by the ratio of less chlorinated to more highly chlorinated isomers. The toxicity of chlorinated methanes (i.e., methylene chloride, chloroform, carbon tetrachloride) also increased with chlorination. Concerning single ring aromatic compounds, pyridine was much less toxic than benzene and benzene was less toxic than its mono-substituted derivatives, including chlorobenzene, nitrobenzene, toluene, and phenol. However, no consistent order of toxicity was observed for the substituted compounds. Acute toxicity also increased with the number of aromatic rings in a series of nitrogen heterocyclic compounds, and the latter were less toxic than corresponding alicyclic compounds. Within most classes of compounds, a direct correlation was observed between acute toxicity and bioconcentration potential. As observed with PCB compounds, the mini-chronic test described in this study permitted evaluations of structure-activity relationships using both LC50 and LC1 values determined with early life stages. The LC1's compared well with results obtained in life-cycle studies, thus providing an economical and reliable means of estimating chronic values for reproductive impairment.

Triplex formation by psoralen-conjugated chimeric oligonucleoside methylphosphonates.

Interactions between nuclease-resistant, 5'-psoralen-conjugated, chimeric methylphosphonate oligodeoxyribo- or oligo-2'-O-methylribo-triplex-forming oligomers (TFOs) and a purine tract found in the envelope gene of HIV proviral DNA (env-DNA) were investigated by gel mobility shift assays or by photo-cross-linking experiments. These chimeric TFOs contain mixtures of methylphosphonate and phosphodiester internucleotide bonds. A pyrimidine chimeric TFO composed of thymidine and 5-methyl-2'-deoxycytidine (C), d-PS-TpCpTpCpTpCpTpTpTpTpTpTpCpTpC (1mp) where PS is trimethylpsoralen and p is methylphosphonate, forms a stable triplex with env-DNA whose dissociation constant is 1. 3 microM at 22 degrees C and pH 7.0. The dissociation constant of chimeric TFO 2mp, d-PS-UpCpTpCpTpCpTpUpTpUpTpUpCpTpC, decreased to 400 nM when four of the thymidines in 1mp were replaced by 5-propynyl-2'-deoxyuridines (U), a result consistent with the increased stacking interactions and hydrophobic nature of 5-propynyl-U. An even greater decrease, 470 -50 nM, was observed for the all-phosphodiester versions of 1mp and 2mp. The differences in behavior of the chimeric versus the all-phosphodiester oligomers may be related to differences in the conformations between the propynyl-U-substituted versus the nonsubstituted TFOs. Thus, in the chimeric oligomer, the stabilizing effect of the propynyl-U's may be offset by the reduced ability of the methylphosphonate backbone to assume an A-type conformation, a conformation that appears to be preferred by propynyl-U-containing TFOs. A chimeric oligo-2'-O-methylribopyrimidine with the same sequence as 1mp also formed a stable triplex, K(d) = 1.4 microM, with env-DNA. In contrast to the behavior of the pyrimidine TFOs, antiparallel A/G oligomers and parallel or antiparallel T/G oligomers did not form triplexes with env-DNA, even at oligomer concentrations of 10 microM. This lack of binding may be a consequence of the low G content (33%) of the triplex binding site. Irradiation of triplexes formed between the pyrimidine TFOs and env-DNA resulted in formation of photoadducts with either the upper-strand C or the lower-strand T at the 5'-CpA-3' duplex/triplex junction. No interstrand cross-links were observed. The presence of a 5-propynyl-U at the 5'-end of the oligomer caused a reduction in the amount of upper-strand photoadduct but had no effect on photoadduct formation with the lower strand, suggesting that increased stacking interactions caused by the presence of the 5-propynyl-U change the orientation of psoralen with respect to the upper-strand C. The ability of chimeric methylphosphonate TFOs to bind to DNA, combined with their resistance to degradation by serum 3'-exonucleases, suggests that they may have utility in biological experiments.

The effects of chlordane exposure during pre- and postnatal periods at environmentally relevant levels on sex steroid-mediated behaviors and functions in the rat.

Technical chlordane is a mixture of four main isomers (i.e., heptachlor, cis-chlordane, trans-chlordane, and trans-non-achlor) found in meat and dairy products as well as in indoor air of houses treated for termites. These isomers are metabolized to more potent epoxides (heptachlor epoxide and oxychlordane) which accumulate in lipid compartments of tissues and have been shown to reduce chloride influx through GABAA receptor complex channels and to alter steroid levels. However, considering the almost universal human exposure and the potential for accumulation of these agents, very little is known about how chronic, low-level exposures during development affect adult behavior and steroid-mediated processes. Time-pregnant Sprague-Dawley dams (Day 4 of gestation through Day 21 of lactation) and offspring (Day 22 of age through Day 80) were exposed to three levels of technical chlordane (100, 500, or 5000 ng/g) on a daily schedule. The low-exposure level generated heptachlor epoxide and oxychlordane plasma levels in the dam (Day 20) and in the offspring (Day 80) representative of those found in the U.S. populace. Chlordane-dosed offspring exhibited sex- and dose-dependent effects on testosterone levels, behavioral tests, and body weight conducted between postnatal Days 77 and 85. Chlordane-dosed females, but not males, had significant decreases in testosterone levels, significant improvements in spatial abilities (i.e., decreases in Cincinnati maze errors, navigation times, and failures to escape), and significant increases in body weight and in auditory startle-evoked responses. In two other tests, only males were used. These chlordane-dosed males showed significant increases in male-typical mating behaviors and decreases in 36Cl- uptake into brain microsacs. For all behavioral and body weight measurements, dose-response effects were observed for the 100 and 500 ng/g dosed groups. However, the 5000 ng/g dose group responses were closer to those of control values. These results suggest that these cyclodienes masculinize sexually dimorphic functions and behaviors by mimicking sex steroids and/or changing their levels.

Biphasic changes in left ventricular function during hyperdynamic endotoxemia.

Cardiac contractility was studied in a clinically relevant conscious swine model simulating human hemodynamics during endotoxemia. The slope of the end-systolic pressure-volume relationship [end-systolic elastance (EES)] was used as a load-independent contractility index. Chronic instrumentation in 10 pigs included two pairs of endocardial ultrasonic crystals for measuring internal major and minor axial dimensions of the left ventricle, a micromanometer for left ventricular pressure measurement, and a thermodilution pulmonary artery catheter. After a 10-day recovery period, control measurements of cardiac hemodynamic function were obtained. The following week, Escherichia coli endotoxin (10 micrograms . kg-1. h-1) was administered intravenously for 24 h. EES increased 1 h after endotoxin infusion and decreased beyond 7 h. The later hemodynamic changes resembled human cardiovascular performance during endotoxemia more closely than the changes during the acute phase. EES decreased in the later phase. A similar biphasic response of EES has been reported during a tumor necrosis factor-alpha (TNF) challenge. Even though plasma TNF was highest at 1 h and declined thereafter in this study, no consistent relationship between TNF and EES was identified, and TNF levels did not correlate directly with the changes in EES.