Complexation of single strand telomere and telomerase RNA template polyanions by deoxyribonucleic guanidine (DNG) polycations: plausible anticancer agents

Cancerous cell immortality is due to relatively high concentrations of telomerase enzyme which maintains telomere sequence during cell division. Deoxyribonucleic guanidine (DNG) is a positively charged DNA analog in which guanidine replaces the phosphordiester linkage of DNA. Mixed sequences of DNG and DNA oligonucleotides are referred to as chimera. Complexation of DNG and chimeric polycations with the complementary negatively charged non-coding telomere single strand d(5'-TTAGGG-3')(n) and the 11-base telomeric RNA template (5'-CUAACCCUAAC-3') in the active site of telomerase has been studied. Calculated by ensemble sampling simulations in GBMV solvent model, we found that binding of complementary DNG hexamer with telomere is favored over that of DNA-telomere by approximately 10(6)-fold and binding of chimera hexamer is favored by approximately 10(4)-fold. Binding of complementary DNG with telomeric RNA is favored by 43 kcal/mol over telomere substrate binding with telomeric RNA.     

Solid-phase synthesis of oligopurine deoxynucleic guanidine (DNG) and analysis of binding with DNA oligomers

The first stepwise solid-phase synthesis of deoxynucleic guanidine (DNG), a positively charged DNA analog, using controlled pore glass as the solid support is reported. For the first time, purine bases have been incorporated into the DNG oligomer and DNG has been synthesized using a solid-phase method, proceeding in the 3′→5′ direction, that is compatible with the cleavage conditions used in the solid-phase synthesis of DNA. A DNG sequence containing a pentameric tract of adenosine nucleosides has been synthesized and the thermal denaturation temperature of its complexes with complementary thymidyl DNA oligomers was 79°C. Binding of thymidyl DNA oligomers to adenyl DNG oligomers is 2:1, as seen in thymidyl and adenyl DNA triplexes. No binding of adenyl DNG with octameric cytidyl DNA was observed, indicating that the positive charge does not overcome base pairing fidelity.     

Solid-phase synthesis of positively charged deoxynucleic guanidine (DNG) modified oligonucleotides containing neutral urea linkages: effect of charge deletions on binding and fidelity

A solid-phase synthesis for a DNA analogue with a mixed guanidinium and urea backbone is reported. This material is nearly identical in structure to deoxynucleic guanidine (DNG) but the neutral urea internucleoside linkages can be used to attenuate the overall positive charge on the oligomer. The opposite charge attraction between urea containing DNG oligomers (DNGUs) and complimentary DNA can be controlled so that the affinity of DNG for DNA does not overwhelm the base-pairing discrimination necessary for specific binding. Octameric DNGU containing between 1 and 3 urea substitutions covered the range between very tight and very weak bonding. Each deletion of a positive charge reduced the thermal denaturation temperature (Tm) by approximately 5 degrees C. Mismatches in the DNA oligomers reduced the Tm values by 3 to 5 degrees C for each of the DNGU oligomers. DNGUs were found to bind in a 2:1 fashion to complimentary DNA in the same manner as DNG.     

Comparison of dienogest effects upon 3,3′–diindolylmethane supplementation in models of endometriosis and clinical cases

Dienogest (DNG) administration is a well-established treatment for endometriosis but bleeding irregularities remain its main disadvantage. Changes in diet, mainly to vegetable consumption, are beneficial in the treatment of estrogen-related pathologies but their use for endometriosis has been poorly studied. In this study, addition of the phytochemical 3,3′-diindolylmethane (DIM) to DNG therapy has been investigated in in vitro and ex vivo models for endometriosis and in a small cohort of women with endometriosis. Endometrial Ishikawa cells were treated with DNG or DIM at dosages from 10^?10?M to 10^?5?M for up to 72?h. Cell proliferation was measured by assessing BrdU incorporation. Endometrial tissue from women with endometriosis and controls was incubated with DNG or a combination of DNG and DIM. Tissue viability was determined using a modified colorimetric MTS assay. 17β-estradiol secretion was quantified by an electro-chemiluminescence immunoassay. Finally, DNG as monotherapy or in combination with DIM was randomly administered to women with endometriosis (n?=?8) over 3 months. Bleeding patterns and associated pelvic pain were assessed by Visual Analogue Scale (VAS). DNG and DIM significantly reduced cell proliferation in Ishikawa cells. Ex vivo, DIM reduced viability and estradiol secretion specifically in endometriotic but not in normal endometrial tissue. This effect was enhanced by combination with DNG. Endometriosis associated pelvic pain was significantly reduced in patients taking the DNG-DIM combination therapy compared to those taking DNG alone. Bleeding pattern (number and duration of episodes) was significantly improved by addition of DIM to the DNG treatment. In conclusion, addition of DIM enhances effects of DNG ex vivo and may ameliorate bleeding patterns in endometriosis patients.     

Comparison of positively charged DNG with DNA duplexes: a computational approach

Molecular dynamics is used to investigate the structural properties of the cationic DNA analogue deoxynucleic guanidine (DNG), in which a guanidinium group replaces the phosphate moiety of DNA. This study examines the DNG duplex dodecamers d(Ag)(12).d(Tg)(12) and d(Gg)(12).d(Cg)(12), as well as their DNA counterparts. Watson-Crick base-pairing is maintained in the solvated DNG duplex models during the 5ns simulations. The idealized DNG dodecamers assume many parameters characteristic of the corresponding native DNA, assuming B-DNA conformations. Several helical parameters are rather unique to DNG, including buckle, slide, inclination, propeller, and X-displacement. Fewer transitions in backbone torsions occur in the DNG duplexes compared to those of the DNA, which may result from the greater rigidity of the sp(2) hybridized guanidinium group verses the flexible sp(3) phosphate group. The DNG helices have exceptionally shallow major grooves and very deep minor grooves. The major and minor groove widths of DNG are narrower than those of the respective DNA counterparts.     

Solid-phase synthesis of positively charged deoxynucleic guanidine (DNG) oligonucleotide incorporating 7-deazaguanine bases

DNG nucleotides represent a positively charged DNA analog in which the negatively charged phosphodiester linkages of DNA are replaced by positively charged guanidinium linkages. We report herein the synthesis of 3'-end, middle, and 5'-end monomers required for the synthesis of a DNG sequence in which the natural guanine base is replaced by 7-deazaguanine (c(7)G). 7-Deazaguanine nucleobase was chosen because of their unique glycoside bond stability and their ability to prevent G-quartet formation. A facile and high yield two-step synthesis of xylo-7-deazaguanine 7, a key intermediate for introducing 3'-amino functionality, is carried out under Mitsunobu conditions. Subsequently, the 3'-Fmoc-protected thiourea monomers 13 and 19 were prepared from 7 via their corresponding 3'-amino-7-deazaguanines 11 and 18, respectively. The smooth coupling of these thiourea monomers with monomethoxytrityl (MMTr)-protected 3'-end monomer 25, prepared from 5, occurred on solid phase in 3'-->5' direction. The resultant trimeric HO-c(7)Ggc(7)Ggc(7)G-OH (1) has been designed to be included into DNA using standard DNA synthesis technology. The combination of C-c(7)G base pairing and electrostatic association of phosphodiester and guanidinium backbone allows the small synthesized DNG trimer 1 to form 1:1 complex with DNA-C pentamer.     

DNA fragmentation, DNA repair and apoptosis induced in primary rat hepatocytes by dienogest, dydrogesterone and 1,4,6-androstatriene-17beta-ol-3-one acetate

Four steroids that share the 17-hydroxy-3-oxopregna-4,6-diene structure - cyproterone acetate, chlormadinone acetate, megestrol acetate, and potassium canrenoate - have been shown previously to behave with different potency as liver-specific genotoxic agents, the response being markedly higher in female than in male rats, but similar in humans of both genders. In this study, performed to better define the relationship between chemical structure and genotoxicity, dydrogesterone (DGT) with double bonds C4=C5 and C6=C7, dienogest (DNG) with double bonds C4=C5 and C9=C10, and 1,4,6-androstatriene-17beta-ol-3-one acetate (ADT) with double bonds C1=C2, C4=C5 and C6=C7, were compared with cyproterone acetate (CPA) for their ability to induce DNA fragmentation and DNA repair synthesis in primary cultures of hepatocytes from three rats of each sex. At subtoxic concentrations, ranging from 10 to 90 microM, all four steroids consistently induced a dose-dependent increase of DNA fragmentation, which in all cases was higher in females than in males; their DNA damaging potency decreased in the order CPA > DNG > ADT > DGT. Under the same experimental conditions, the responses provided by the DNA repair-synthesis assay were positive or inconclusive in hepatocytes from female rats and consistently negative in hepatocytes from male rats. In the induction of apoptotic cells, examined in primary hepatocytes from female rats, CPA was more active than ADT and DGT, and DNG was inactive. Considered as a whole these findings suggest that a liver-specific genotoxic effect more marked in female than in male rats might be a common property of steroids with two or three double bonds.     

Deoxynucleic guanidine; synthesis and incorporation of purine nucleosides into positively charged DNG oligonucleotides

The synthesis of purine nucleosides capable of making the guanidinium linkage is described for the first time starting from the corresponding 2'-deoxynucleosides. The positively charged mixed base DNG oligomer containing guanine was synthesized on solid-phase using CPG as support from 3' to 5' direction using the precursor building block nucleosides.     

An investigation of the ionic and solvation patterns of dsDNG versus dsDNA by use of molecular dynamics simulations

Molecular dynamics has been employed to analyze the counterion and water atmospheres around the deoxynucleic guanidine (DNG) duplex G(12)-C(12). These features are compared to those of DNA G(12)-C(12). The chloride counterions of cationic DNG demonstrate fewer penetrations and only fleeting residence times in the minor groove, as opposed to the multi-nanosecond visits seen by sodium ions in DNA minor grooves. The 10 ns simulations also show the differences in hydration patterns between the DNG and DNA duplexes.     

Skeletal muscle basal AMP-activated protein kinase activity is chronically elevated in alloxan-diabetic dogs: impact of exercise.

The effect of diabetes and exercise on skeletal muscle (SkM) AMP-activated protein kinase (AMPK)alpha1 and -alpha2 activities and site-specific phosphorylation of acetyl-CoA carboxylase was examined in the same six dogs before alloxan (35 mg/kg)-induced diabetes (C) and after 4-5 wk of suboptimally controlled hyperglycemic and hypoinsulinemic diabetes (DHG) in the presence and absence of 300-min phlorizin (50 microg.kg-1.min-1)-induced "normoglycemia" (DNG). In each study, the dog underwent a 150-min [3-3H]glucose infusion period, followed by a 30-min treadmill exercise test (60-70% maximal oxygen capacity) to measure the rate of glucose disposal into peripheral tissues (Rdtissue). SkM biopsies were taken from the thigh (vastus lateralis) before and immediately after exercise. In the C and DHG states, the rise in plasma free fatty acids (FFA) with exercise ( approximately 40%) was similar. In the DNG group, preexercise FFA were significantly higher, but the absolute rise in FFA with exercise was similar. However, the exercise-induced increment in Rdtissue was significantly blunted (by approximately 40-50%) in the DNG group compared with the other states. In SkM, preexercise AMPKalpha1 and -alpha2 activities were significantly elevated (by approximately 60-125%) in both diabetic states, but unlike the C group these activities did not rise further with exercise. Additionally, preexercise acetyl-CoA carboxylase phosphorylation in both diabetic states was elevated by approximately 70-80%, but the increases with exercise were similar to the C group. Preexercise AMPKalpha1 and -alpha2 activities were negatively correlated with Rdtissue during exercise for the combined groups (both P < 0.02). In conclusion, the elevated preexercise SkM AMPKalpha1 and -alpha2 activities contribute to the ongoing basal supply of glucose and fatty acid metabolism in suboptimally controlled hypoinsulinemic diabetic dogs; but whether they also play a permissive role in the metabolic stress response to exercise remains uncertain.     

The effect of progesterone and synthetic progestins on serum- and estradiol-stimulated proliferation of human breast cancer cells.

The results from the Women's Health Initiative study on enhanced breast cancer risk in postmenopausal women using an estrogen/progestin combination clearly indicate the need for a comparison of different progestins with regard to cancer risk. To shed some light on this issue, we have investigated the influence of progesterone and various synthetic C19- and C21-progestins on cell proliferation of a human breast cancer cell line in vitro. Of special interested was the comparison of two different regimens commonly used in HRT, sequential and continuous combination with estradiol. We used the human breast cancer cell line MCF-7 as a model. Progesterone (P), chlormadinone acetate (CMA), dienogest (DNG), gestodene (GSD), 3-ketodesogestrel (KDG), levonorgestrel (LNG), medroxyprogesterone acetate (MPA), and norethisterone (NET) were investigated in the range of 0.01nm to 10 micro M alone and in combination with 10 nM estradiol. Cell proliferation was measured after 7 days using the ATP chemosensitivity test. Tested alone, CMA, DNG, GSD, KDG, MPA and NET significantly stimulated cell proliferation, but only at high dosages. Sequentially combined with estradiol, only CMA inhibited cell proliferation over the whole concentration range. Slight effects were found for DNG, GSD and KDG, whereas P and MPA only showed an effect at the highest concentration. NET had no significant effect on cell proliferation. Continuously combined, all progestins exhibited an inhibitory effect over the whole concentration range. The most prominent effects were found for P, CMA, GSD, and KDG. Only slight effects were found for DNG, MPA and NET. Our in vitro results indicate that the influence on breast cancer risk using HRT in postmenopausal women might depend on the type of progestin used as well as on the regimen applied. However, the net inhibitory in vitro effect of the progestins at clinically relevant dosages is rather minimal, and whether progestins in general can reduce breast cancer risk in long-term treatment remains uncertain. Further clinical trials are urgently needed to clarify this issue.     

Modern Theory for Electromagnetic Metamaterials

Plasmonics - The pressure of radiation in double-negative materials (DNG) with simultaneously negative permittivity ε eff and permeability μ eff predicted by Veselago in the 1960s is less...     

Ribonucleic guanidine demonstrates an unexpected marked preference for complementary DNA rather than RNA

Replacement of the phosphodiester linkages of DNA and RNA by guanidinium linkages provides DNG and RNG. We report here the order of stability of mixed duplexes (RNG-U5.DNA-A5>RNA-U5.RNA-A5>RNG-U5.RNA-A5>RNA-U5.DNA-A5>DNA-T5.DNA-A5). The considerable stability of RNG.DNA compared to RNG.RNA is shown to be due to the rigid backbone of RNG existing only in B-form and therefore lowering its affinity for A-RNA. RNG oligomers are putative antigene agents which are specific for DNA and would have minimal competitive binding to ncRNA.     

Structural properties of hybrid triplex of polycation deoxyribonucleic S-methylthiourea (DNmt) strands with a complementary DNA strand, probed by nanosecond molecular dynamics

The replacement of phosphodiester linkages of the polyanion DNA with S-methylthiourea linkers provides the polycation deoxyribonucleic S-methylthiourea (DNmt). Molecular dynamics studies to 1,220 ps of the hybrid triplex formed from octameric DNmt strands d(Tmt)8 with a complementary DNA oligomer strand d(Ap)8 have been carried out with explicit water solvent and Na+Cl- counterions under periodic boundary conditions using the CHARMM force field and the Ewald summation method. The Watson-Crick and Hoogsteen hydrogen-bonding patterns of the A/T tracts remained intact without any structural restraints for triplex structures throughout the simulation. The duplex portion of the triplex structure equilibrated at a B-DNA conformation in terms of the helical rise and other helical parameters. The dynamic structures of the DNmt x DNA x DNmt triplex were determined by examining histograms from the last 800 ps of the dynamics run. These included the hydrogen-bonding pattern (sequence recognition), three-centered bifurcating occurrences, minor groove width variations, and bending of tracts for the hybrid triplex structures. Together with the Watson-Crick hydrogen-bondings, the strong Hoogsteen hydrogen-bondings, the partially maintained three-centered bifurcatings in the Watson-Crick pair, and the medium-strength three-centered bifurcatings in the Hoogsteen pair suggest that the hybrid triplex is energetically favorable as compared to a duplex with similar base stacking, van der Waals interactions, and helical parameters. This is in agreement with our previously reported thermodynamic study, in which only triplex structures were observed in solution. The bending angle measured between the local axis vectors of the first and last helical axis segments is about 20 degrees for the Watson-Crick portion of the averaged structure. Propeller twist (associated with three-centered hydrogen-bonding) up to -30 degrees, native to DNA AT base pairing, was also observed for the triplex structure. The sugar pseudorotation phase angles and the ring rotation angles for the DNA strand are within the C3'-endo domain and C2'-endo domain for the DNmt strand. Water spines are observed in both minor and major grooves throughout the dynamics run. The molecular dynamics simulations of the structural properties of DNmt x DNA x DNmt hybrid triplex is compared to the DNG x DNA x DNG hybrid triplex (In DNG the -O-(PO2-)-O- linkers in DNA is replaced by -NH-C(=N+H2)-NH-).     

An overview of the development of combined oral contraceptives containing estradiol: focus on estradiol valerate/dienogest

Natural estrogens such as estradiol (E(2)) or its valerate ester (E(2)V) offer an alternative to ethinyl estradiol (EE). E(2)-containing combined oral contraceptives (COCs) have demonstrated sufficient ovulation inhibition and acceptable contraceptive efficacy. However, earlier formulations were generally associated with unacceptable bleeding profiles. Two E(2)V-containing preparations have been approved to date for contraceptive use: E(2)V/cyproterone acetate (CPA) (Femilar(?); only approved in Finland and only in women >40 years or women aged 35-40 years in whom a COC containing EE is not appropriate) and E(2)V/dienogest (DNG; Qlaira(?)/Natazia(?)). The objective of the current review is to provide an overview of the development of COCs containing natural estrogen, highlighting past issues and challenges faced by earlier formulations, as well as the current status and future directions. The majority of information to date pertains to the development of E(2)V/DNG.     

DNG cytidine: synthesis and binding properties of octameric guanidinium-linked deoxycytidine oligomer

The synthesis of guanidinium-linked cytidyl oligomer (DNG-C(8)), a cationic DNA analog, and the corresponding cytidine monomers is described. The DNG monomer synthesis was streamlined to produce a shorter route to the final monomer than previously reported for thymidine and subsequent solid-phase synthesis produced an octameric cytidyl DNG strand. Because octameric deoxyguanosine would be used as the complementary strand in our studies, it was necessary to investigate guanosine self-association. Singular value decomposition was used to mathematically deconvolve the spectral data and confirm the presence of transitions due to DNA-G(8) self-association. Job plots show the binding stoichiometry of DNG-C(8) with DNA-G(8) to be 1:1. Thermal denaturation studies of the DNG-C(8).DNA-G(8) duplex established a T(m) > or = 90 degrees and a DeltaG degree = -13.3 kcal mol(-1), indicating the DNG-C(8).DNA-G(8) duplex is over 1000 times more stable than that of DNA-C(8).DNA-G(8).     

Binding properties of positively charged deoxynucleic guanidine (DNG), AgTgAgTgAgT and DNG/DNA chimeras to DNA

The melting properties of hexameric oligonucleotide AgTgAgTgAgT, in which the phosphodiester linkages of the DNA have been replaced by guanidium linkages, have been evaluated. Using the juvenile esterase gene as a target, the binding of a 20-mer DNG/DNA chimera that includes AgTgAgTgAgT is more than 10(5.7) stronger than the binding of 20-mer composed solely of DNA.     

Hydrolytic enzyme substrates. I. Chemical synthesis and characterization

The synthesis and characterization of a number of new phosphate, sulfate and acetate esters of 3-(p-nitrophenoxy)-1,2-propanediol (PNG); 3-(2,4-dinitrophenoxy)-1,2-propanediol (DNG); 4-(p-nitrophenoxy)-1,2-butanediol (PNB) and 4-(2,4-dinitrophenoxy)-1,2-butanediol (DNB) are described. These esters were prepared to serve as substrates for their corresponding hydrolytic enzymes. The assay system used to measure enzyme hydrolysis requires periodate oxidation of the diol formed after hydrolysis of the ester. Base treatment of the resulting aldehyde yields either p-nitrophenolate ion or the 2,4-dinitrophenolate ion depending upon the substrate. In the presence of high concentrations of methylamine and excess periodate the oxidation and elimination reactions can be carried out simultaneously at pH 7.5. The reactions leading to these results are described.     

Progestins and estrogens and Alzheimer's disease

Sex-specific incidence rates for Alzheimer's disease (AD) are higher in women than men. Many fundamental researches and some clinical investigations have reported therapeutic and preventive effects of estrogens on AD. But WHIMS [S.A. Shumaker, C. Legault, S.R. Rapp, L. Thal, R.B. Wallace, J.K. Ockene, S.L. Hendrix, B.N. Jones IIIrd, A.R. Assaf, R.D. Jackson, J.M. Kotchen, S. Wabertheil-Smoller, J. Wactawsk-Wende, WHIMS investigators, Estrogen plus progestin and the incidence of dementia and mild cognitive impairment in postmenopausal women. The women's health initiative memory study: a randomized controlled trial, JAMA 289 (2003) 2651–2662], which used daily continuous hormone replacement therapy (HRT), reported that the hazard ratio of the HRT for probable dementia was 2.05.

Effect of progestins, and continuous (not cyclically) HRT, even only with estrogen should be reconsidered.

In our clinical study, conjugated equine estrogen (CEE) alone showed good changes of psychiatric tests for AD on the 3rd week, but addition of medroxyprogesterone acetate (MPA) or norethindrone since 4th week suppressed these tests. Using human umbilical vein epithelial cell (HUVEC), levonorgestrel (LNG), norethindrone acetate (NETA), MPA increased intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion melocule-1 (VCAM-1) and E-secretin but dienogest (DNG) showed no effect. In vitro flow system, estradiol (E₂), suppressed adhesion of white cell, but LNG, NETA, MPA increased the adhesions. DNG showed less effect.

Non-feminizing estrogen J 861, which has Δ8,9 double bond and straight in its structure and has less effect on sexual organs. J 861 has shown ameliorative effects on central nervous system (CNS) (increasing of cholineacetyltransferase immunoreactive cells in substantia innominata (SI), etc.) like E₂.

More investigations about progestins and estrogens and AD should be done.