Hydrodynamic evidence in support of spacer regions in chromatin.

Quasi-elastic light scattering and sedimentation velocity methods were used to study the hydrodynamic properties of purified dimer subunits obtained from partial digestion of chicken erythrocyte chromatin with staphylococcal nuclease. The experimental value of 1.87 +/- 0.08 X 10(-7) gram per second for the friction factor of these dimer subunits in low ionic strength buffer cannot be reasonably interpreted in terms of a contiguous sphere model. Analysis by means of an equivalent dimer method suggests that the spacer region accounts for a maximum of 19 percent of the friction properties of the dimer.

Boranophosphates as mimics of natural phosphodiesters in DNA.

In boranophosphate-oligodeoxynucleosides (BH(3)(-)-ODN) a borane group replaces one of the two non-bridging oxygen atoms in the phosphodiester backbone of the naturally occurring congener. The chemical and biophysical properties of BH(3)(-) ODN are reviewed, as are their interactions with enzymes such as DNA polymerases, exo- and endonucleases, and ribonuclease H. Three approaches to synthesis of BH3- ODN are described: in solution, on solid supports, and by template directed enzymatic polymerization of appropriately modified nucleoside triphosphates. Comparisons are made to other members of the family of phosphorus modified nucleic acids, the phosphorothioates and methyl phos phonates. The potential applications of boranophosphate modified compounds to antisense therapeutics and DNA sequencing are discussed.

Boranophosphate backbone: a mimic of phosphodiesters, phosphorothioates, and methyl phosphonates.

Nucleoside boranophosphates are distinctive in that one of the non-bridging oxygens in the phosphate diester 1 is replaced by a borane moiety (BH3). Although they retain the same net charge, BH3(-)-ODN have unique chemical and biochemical characteristics relative to other analogs. The change in polarity, lipophilicity, nuclease resistance, and the activation of RNase H cleavage of RNA in RNA: boranophosphate hybrids make boranophosphates very attractive for applications in enzymology and molecular biology and as potential antisense agents.

In vitro transport and delivery of antisense oligonucleotides.

A variety of techniques are currently available to enhance the cellular uptake and pharmacological effectiveness of antisense oligonucleotides in the in vitro setting. The choice of technique will depend on the context of investigation, the likelihood of cytotoxity due to the delivery agents, and the ease and convenience of the approach. The considerations for the delivery of antisense molecules in the in vivo setting are likely to be quite different from the cell culture situation emphasized in this article.

Antisense inhibition of P-glycoprotein expression using peptide-oligonucleotide conjugates.

Antisense oligonucleotides are potentially a powerful tool for the therapeutic manipulation of genes associated with cancer. However, pharmacological applications of oligonucleotides have been hindered by the inability to effectively deliver these compounds to their sites of action within cells. In this study, we have prepared peptide-oligonucleotide conjugates with the intent of improving intracellular delivery. The phosphorothioate oligonucleotide component of the conjugates was complementary to a site flanking the AUG of the message for P-glycoprotein, a membrane ATPase associated with multidrug resistance in tumor cells. Two types of peptide-antisense oligonucleotide conjugates, but not mismatched control conjugates, provided substantial inhibition of cell surface expression of P-glycoprotein. Surprisingly, the peptide-oligonucleotide conjugates were more potent in the presence of serum than when used under serum-free conditions; this is in striking contrast to most other approaches for intracellular delivery of nucleic acids. Effective inhibition of P-glycoprotein expression was attained with submicromolar concentrations of antisense conjugates under serum-replete conditions. The combination of relatively modest molecular size and good efficacy in the presence of serum proteins suggests that peptide-antisense oligonucleotide conjugates may have significant promise for in vivo therapeutic applications.

Intraocular pressure elevation after pupillary dilation in open angle glaucoma.

Acute elevation of intraocular pressure frequently follows pupillary dilation in patients with primary open angle glaucoma. A retrospective study of 60 patients (116 eyes) with primary open angle glaucoma was done to assess the frequency and severity of intraocular pressure elevation following dilation with 2.5% phenylephrine hydrochloride (Neo-Synephrine) and 1% tropicamide (Mydriacyl). Significant pressure elevation (greater than 5 mm Hg) occurred in 37 eyes (32%). Marked pressure elevation (greater than 10 mm Hg) occurred in 14 eyes (12%). The only significant risk factor found was treatment with miotics. The change of intraocular pressure one hour after dilation was compared with the one-hour postoperative change in intraocular pressure in those patients (12 patients, 18 eyes) who subsequently underwent argon laser trabeculoplasty. No statistically significant correlation was found. There is a potential hazard of routine dilation of eyes with open angle glaucoma.

Novel 3',5'-cyclic nucleotide analogue: adenosine 3',5'-cyclic boranomonophosphate.

A general procedure for the first synthesis of a 3',5'-cyclic boranomonophosphate was established. Specifically, adenosine 3',5'-cyclic boranomonophosphosphate (cyclic AMPB, 4c), a P-borane (BH(3)) analogue of adenosine 3',5'-cyclic monophosphate (cAMP), was synthesized via a phosphite approach in good yield. The method is also applicable for syntheses of natural cAMP and its phosphorothioate analogue. The two diasteromers of cyclic AMPB 4c were separated, and their chemical structures were established via spectroscopic methods.

The clinical effects of plasticizers, antioxidants, and other contaminants in medical polyvinylchloride tubing during respiratory and non-respiratory exposure.

In 1999, a Blue Ribbon Panel convened to examine the health effects of two commonly used plasticizers present in medical devices and toys. Of particular interest were the plasticizers used in medical tubing. Hospitalized patients can be exposed to a high dose of these chemicals while receiving respiratory therapy or during hemodialysis, and are more likely to be vulnerable to potentially ill effects than healthy individuals. After extensive review of existing research, the Panel concluded that there was not enough evidence of harmful health effects to remove di-(2-ethylhexyl) phthalate (DEHP) and other plasticizers from use in medical tubing. The Panel recognized the importance of plasticizers in enabling tubing such as PVC for respiratory support or in dialysis to be flexible enough for use in a variety of clinical applications that might not be possible with rigid (plasticizer-free) tubing. Nevertheless, we reviewed the literature regarding the health effects for five of the most common compounds, plasticizers and antioxidants found in medical tubing, and suggest that further clinical studies be conducted. We concur with the Panel's recommendation that alternative materials be developed and studied.

Variable temperature infrared spectroscopy of cytosine-guanine base pairs: tautomerism versus polarization.

This report describes an infrared (IR) spectroscopic study of a model cytosine-guanine base pair. This base pair is part of a self-consistent experimental system based on lipophilic ribose derivatives of cytidine (C), guanosine (G) and O6-methylguanosine (O6MeG) that are soluble in non-aqueous, low dielectric solvents at appreciable concentrations. Previous experiments on this system have revealed different rotation dynamics for the amino bonds within the CG base pair, an observation that could be explained by the presence of rare tautomers (P.O. Lowdin, Reviews of Modern Physics 35,724 (1963)), or by mutual polarization of the base pairs (L.D. Williams, N.G. Williams and B.R. Shaw,J.Am.Chem.Soc. 112,829 (1990)). The IR spectra in the OH and NH stretching region indicate formation of hydrogen-bonded CG base pairs and self associates in 1,2-dichlorobenzene over a temperature range from 10 to 290K. Changes in the lineshapes and intensities of the IR bands with temperature correlate with phase transitions of the solvent, but no evidence is seen for an OH stretching band that would indicate the formation of hydroxyl tautomers within base pairs. Similarly, the relative intensities of the C = O stretching bands of CG in cyclohexane solution remain constant over this same temperature range, confirming that within the base pair, the tautomeric states of the bases remain essentially unperturbed in the 2-amino/6-keto form of G and the 2-keto/4-amino form of C. The spectra of O6-MeG aid in the band assignments, since this molecule is frozen in an equivalent of the 2-amino/6-hydroxyl tautomer, but without the OH group and its associated stretching band. We conclude that the probability of tautomerism does not appear to be sufficient to explain the different rotation dynamics for the two amino bonds of the CG base pair. Rather it is argued that mutual polarization within the base pair, which would increase the bond order of the amino bond of C within the base pair, can explain the results without the formation of unconventional tautomers.

Antineoplastic activity of boron-containing thymidine nucleosides in Tmolt3 leukemic cells.

The sugar boronated thymidine nucleoside, 5' -0-[(triphenylphosphine-boryl) carbonyl]-3'-0-acetyl thymidine 1, and the boron-modified nucleoside phosphotriester, 5'-(diethylphosphite- cyanoborane)-3'-acetylthymidine 2, were successfully synthesized. Both compounds demonstrated differential activity when tested against eight cell lines, with significant cytotoxic activity against the growth of human Tmolt3 leukemia, colon adenocarcinoma, HeLa S3 uterine carcinoma, and osteosarcoma cells. In in vivo studies these agents were found to be active against the growth of Ehrlich ascites carcinoma at 8 mg/kg/day I.P. and to be marginally active against the growth of L1210 and Lewis lung cancers in mice. The mode of action of these thymidine derivatives in Tmolt3 cells was the inhibition of DNA and protein synthesis. Compound 2 was highly effective in inhibiting DNA polymerase alpha and m-RNA, r-RNA and t-RNA polymerase activities. Both compounds inhibited ribonucleoside reductase activity. The de novo purine pathway appeared to be the major site of inhibition of the agents, with IMP dehydrogenase, PRPP amido transferase, and dihydrofolate reductase activities being significantly inhibited. In the pyrimidine pathway, carbamyl phosphate synthetase and aspartate transcarbamylase activities were inhibited by 1. As expected, d[NTP] levels were significantly reduced by treatment with the agents. DNA strand scission was evident after incubating Tmolt3 cells for 24 hr with the agents.

The synthesis and antineoplastic activity of 2'-deoxy-nucleoside-cyanoboranes in murine and human culture cells.

The guanine, inosine, adenine and cytidine deoxyriboside cyanoboranes proved to be cytotoxic and possess in vivo antineoplastic activity against murine and human single cells and cultured cells derived from solid tumor lines. The agents preferentially inhibited DNA synthesis in Tmolt3 leukemic cells. The enzyme sites of drug inhibition of two active derivatives were DNA polymerase-alpha and de novo purine synthesis at the regulatory sites, PRPP amido transferase and IMP dehydrogenase. Moderate inhibition by the agents of TDP kinase and ribonucleoside reductase activities also occurred. Kinetic studies showed that IMP dehydrogenase activity was inhibited the earliest of all of the enzymes affected by the drugs. The d(ATP) pools were also reduced by drug treatment. DNA strand scission was evident after 24 hr incubation at 100 microM of drug. The 14C-cytidine-cyanoborane drug was rapidly taken up over 6 hr and most effective uptake was shown by rapidly dividing cells. The drug was bound to DNA, RNA and protein in Tmolt3 leukemic cells.

Hypolipidemic activity of boronated nucleosides and nucleotides in rodents.

Base-boronated nucleoside and phosphate-boronated nucleotides were potent hypolipidemic agents in rodents, lowering both serum cholesterol and triglyceride levels. Rat VLDL and LDL cholesterol levels were generally reduced and HDL cholesterol levels were significantly elevated after 14 days dosing at 8 mg/kg/day. Tissue cholesterol, triglyceride and phospholipid levels were reduced by selected derivatives. Increased fecal excretion of lipids did not appear to be a mechanism by which these derivatives lowered serum lipids in rodents. Rather, the agents suppressed appetite and reduced the activities of rate-limiting enzymes for de novo lipid synthesis, specifically cytoplasmic acetyl CoA synthetase, squalene synthetase, and phosphatidylate phosphohydrolase with IC50 values of approximately 10(-5) m.

Anti-inflammatory and anti-osteoporotic activities of base-boronated nucleosides and phosphate-boronated nucleotides in rodents.

The 2'-deoxyribonucleoside cyanoboranes were effective anti-inflammatory agents in rodents at 2-8 mg/kg; they blocked induced edema, septic shock, and pleurisy. Overall compounds 3',5'-O-(bis- (triisopropylsilyl)-2'-deoxyinosine (1), 3',5'-O-bis(triisopropylsilyl)-2'-deoxycytidine (10), N3-(cyanoboryl)-2'-deoxycytidine (11), N7-(cyanoboryl)-N2-isobutyryl- 3',5'-O-bis(triisopropylsilyl)-2'-deoxyguanosine (20), and N7-(cyanoboryl)-N2- isobutyryl-5'-O-(4,4'-dimethoxytrityl)-3'-O-(triisopropylsilyl)-2' -deoxyguanosine (22) were the most active when all the anti-inflammatory screens are considered. The agents also blocked both local and central pain caused by inflammation. These nucleosides blocked calcium resorption but were less effective compared to other amine carboxyboranes. The inflammation process appeared blocked by these compounds because of their effectiveness in reducing both hydrolytic lysosomal enzyme and proteolytic enzyme activities. The agents were also dual inhibitors of prostaglandin cyclooxygenase and 5'-lipoxygenase activities in leukocytes and macrophages. These agents at 10(-4) M demonstrated no specific organ toxicity to ileum mucosa cells grown in tissue culture.

Echinomycin, a bis-intercalating agent, induces C-->T mutations via cytosine deamination.

Echinomycin, a bis-intercalating, antitumor drug, has been studied for its ability to induce the deamination of cytosine to uracil (C-->U) in double-stranded DNA. We have employed a sensitive lacZ alpha-complementation reversion assay to detect G.C-->A.T mutations at a number of sites in M13mp2 DNA to determine the extent to which distortions of DNA structure induced by echinomycin may affect C-->U rates. When double-stranded M13mp2 DNA with a 12-base target containing a CpG site was incubated at 37 degrees C, the reversion frequency of the echinomycin-treated DNA increased linearly over time, with a rate constant 3-fold greater than DNA incubated without echinomycin. Of the 11 ways that blue pseudo-revertants can occur in the target, 96% of the observed revertants arose from C-->T and tandem CC-->TT transitions, with 78% attributable to single-base C-->T changes at three sites. Transfection into ung+ cells decreased the reversion frequencies by 85% to near background levels, indicating that the increase in C-->T mutations was due to deamination of C to U. The cytosine deamination rate constants for the entire target at pH 6.0 and 37 degrees C were 1.2 x 10(-11) sec-1 for untreated DNA and 3.5 x 10(-11) sec-1 for echinomycin-treated DNA. The increase in C-->T mutation rates occurred at cytosines both proximal and distal to a CpG echinomycin-binding site. We hypothesize that this increase in deamination rate is due to a more open or single-stranded DNA structure caused by the echinomycin: DNA interaction.

Hydrolysis of N3-methyl-2'-deoxycytidine: model compound for reactivity of protonated cytosine residues in DNA.

Protonation of cytosine residues at physiological pH may occur in DNA as a consequence of both alkylation and aberrant base-pair formation. When cytosine derivatives are protonated, they undergo hydrolysis reactions at elevated rates and can either deaminate to form the corresponding uracil derivatives or depyrimidinate generating abasic sites. The kinetic parameters for reaction of protonated cytosine are derived by studying the hydrolysis of N3-methyl-2'-deoxycytidine (m3dC), a cytosine analogue which is predominantly protonated at physiological pH. Both deamination and depyrimidimation reaction rates are shown to be linearly dependent upon the fraction of protonated molecules. We present here thermodynamic parameters which allow determination of hydrolysis rates of m3dC as functions of pH and temperature. Protonation of cytosine residues in DNA, as induced by aberrant base-pair formation or base modification, may accelerate the rate of both deamination and depyrimidation up to several thousand-fold under physiological conditions.

DNA base modification: ionized base pairs and mutagenesis.

The nature of hydrogen bonding between normal and modified bases has been re-examined. It is proposed that hydrogen-bonding schemes may involve tautomeric, ionized or conformational forms (syn, anti and wobble). Several important cases are presented or reviewed in which physical evidence indicates the existence of ionized base pairs. When thermodynamic values determined in aqueous solution under physiological conditions are considered, it can be argued that base ionization will contribute substantially to the stability of many biologically relevant base pairs containing modified bases. A significant incidence of ionized bases in DNA may have important kinetic ramifications for the further chemical reactivity of both the modified base and its cross-strand pairing partner. Moreover, DNA structure at and surrounding ionized base pairs may be altered. For this reason, the model presented in this study should be useful as DNA-sequence analysis becomes more commonly applied to the study of mutagenesis.

The analysis of thebaine in urine for the detection of poppy seed consumption.

The consumption of poppy seeds in various foods may lead to a positive opiate result in urine subjected to testing for drugs of abuse. As a natural constituent of poppy seeds, thebaine was investigated as a possible marker for poppy seed consumption. Poppy seeds were examined for opiate content by gas chromatography-ion trap mass spectrometry (GC-MS) after extraction with methanol. Urine samples spiked with thebaine and urine from subjects given 11 g of poppy seeds were tested for the presence of thebaine, codeine, and morphine. Street heroin, one morphine and one codeine tablet, and urine from individuals who had used heroin were also examined for thebaine. Urine specimens were screened by enzyme immunoassay (EMIT) and confirmed for thebaine by GC-MS using a solid-phase extraction method. The GC-MS assay showed a linear response over a range of 1-100 ng/mL and a limit of detection of 0.5 ng/mL. Thebaine was detectable in the urine of poppy seed eaters in concentrations ranging from 2 to 81 ng/mL. Because thebaine was absent in powdered drugs and the urine of true opiate drug users, thebaine is proposed as a direct marker for poppy seed use.

Synthesis of a novel nucleic acid mimic: P-boranomethylphosphonate.

A new type of non-ionic nucleotide analogue with a doubly modified internucleotide linkage, P-boranomethylphosphonate, has been successfully synthesized and characterized. Dithymidine boranomethylphosphonate 5 is the first example of a P-boranomethylphosphonate compound; it is a highly lipophilic phosphodiester analog, which is almost totally resistant to both snake venom phosphodiesterase (SVPDE) and bovine spleen phosphodiesterase (BSPDE). P-boranomethylphosphonates are expected to be promising candidates for mechanistic, diagnostic and therapeutic applications.

Synthesis of new classes of boron-containing nucleotides.

Four different types of boron-modified nucleotides are reported: P-boranophosphorothioates, P-cyanoboranophosphates, P-boranomethylphosphonates, and P3'-N5'-boranophosphoramidates. Synthesis of dinucleoside borano-phosphorothioates and nucleoside P-borano-P-thiomonophosphates via a lithium sulfide method is described. The Li2S method also provides an alternative way to synthesize phosphorothioates through a dinitrophenyl P(V) phosphotriester precursor. The mechanism of Li2S substitution was investigated.

Synthesis and properties of novel triphosphate analogues: ribonucleoside and deoxyribonucleoside (alpha-P-borano, alpha-P-thio)triphosphates.

The first ribo- and deoxyribo-nucleoside (alpha-P-borano, alpha-P-thio)triphosphates have been synthesized. The chemical and biochemical properties of adenosine (alpha-P-borano, alpha-P-thio)triphosphate and thymidine (alpha-P-borano, alpha-P-thio) triphosphate have been investigated.