Synthesis and Antibody Mediated Detection of 2,4-Dinitrophenyl (DNP) Labelled Oligonucleotides

Abstract

Different DNP phosphoramidites based on non-nucleoside and nucleoside backbone molecules are developed and used in the multiple labelling of oligonucleotides during the solid phase synthesis. It is demonstrated that the antibody mediated detection of DNP labelled oligonucleotides is comparable to that of digoxigenin, biotin and fluorescein.     

In vivo and in vitro phosphorylation of DNA-binding proteins from Escherichia coli.

A deoxyribonucleoprotein (DNP) complex has been isolated from Escherichia coli cells by chromatography on Sephadex G-200. The DNP complex contains phosphoproteins and the content of phosphorus bound to the DNP protein is 3 times higher than in cytoplasmic proteins not bound to DNA. These results have been confirmed by in vivo (32-P-KH2PO4) and in vitro (32-P-ATP) phosphorylation of E. coli DNA-binding proteins isolated by chromatography on DNA--cellulose.     

The abnormal-shaped mitochondria in thymus lymphocytes treated with inhibitors of mitochondrial energetics

The effects of uncouplers (DNP, FCCP), oligomycin, and rotenone on the energetics and mitochondrial ultrastructure in lymphocytes have been studied. We confirmed the previous observations done on Ehrlich ascites and cardiomyocyte culture cells that uncouplers and respiratory inhibitors cause the appearance of ringlike and dumbbell-like mitochondria. It is shown that this effect does not correlate with decrease in ATP concentration, changes in oxygen consumption, or condensation of the mitochondrial matrix. FCCP (2 µM) is more effective in the induction of abnormal-form mitochondria than 240 µM DNP, oligomycin, or rotenone. Combined treatment with DNP, oligomycin, and rotenone or with DNP and rotenone produces an effect as strong as 2 µm FCCP. DNP (240 µM) and FCCP (2 µM) have a similar effect on respiration and intracellular ATP, but only the latter induces condensation of the mitochondrial matrix.     

The mechanism of action of DNP on phospholipid bilayer membranes

Summary The weak acid 2,4-dinitrophenol (DNP) acts as an uncoupler of oxidative phosphorylation in biological systems and, in consonance with the Mitchell hypothesis, also enhances the conductance of phospholipid bilayer membranes. Several models have been proposed in the literature to explain the molecular mechanism by which DNP exerts its electrical effects on the model membranes, none of which accounts for all of the data, and all of which ignore the possibility that the anion of DNP is also binding to the surface of the bilayer and modifying the charge density. Experimental evidence is presented in this report which suggests that when a bilayer membrane is formed from a neutral lipid, DNP does in fact adsorb to its surface and produce a substantial negative surface potential. When this phenomenon is taken into account, the model proposed by Lea and Croghan and by Finkelstein is capable of describing all of the effects of DNP on bilayer membranes. In this model, the permeant species is a negatively charged complex formed from the undissociated acid and its anion.     

A new horizon of DNP technology: application to in-vivo 13C magnetic resonance spectroscopy and imaging

Dynamic nuclear polarization (DNP) is an emerging technique for increasing the sensitivity (>10,000-fold) of magnetic resonance spectroscopy and imaging (MRSI), in particularly for low-γ nuclei. DNP methodology is based on polarizing nuclear spins in an amorphous solid state at low temperature (ca. 1 K) through coupling of the nuclear spins with unpaired electron spins that are added to the sample via an organic free radical. In an amorphous solid state, the high electron spin polarization can be transferred to the nuclear spins by microwave irradiation. While this technique has been utilized in solid-state research for many years, it is only recently that dissolution methods and the required hardware have been developed to produce the high nuclear polarization provided by DNP to produce injectable hyperpolarized solutions suitable for in vivo studies. It has been applied to a number of ¹³C-labeled cell metabolites in biological systems and their real-time metabolic conversion has been imaged. This review focuses briefly on the DNP methodology and the significant molecules investigated to date in preclinical cancer models, in terms of their downstream metabolism in vivo or the biological processes that they can probe. In particular, conversion between hyperpolarized ¹³C-labeled pyruvate and lactate, catalyzed by lactate dehydrogenase, has been shown to have a number of potential applications such as diagnosis, staging tumor grade, and monitoring therapy response. Strategies for making this technique more viable to use in clinical settings have been discussed.     

Dissociation of 5' AMP-activated protein kinase activation and glucose uptake stimulation by mitochondrial uncoupling and hyperosmolar stress: differential sensitivities to intracellular Ca2+ and protein kinase C inhibition

2,4-dinitrophenol (DNP) compromises ATP production within the cell by disrupting the mitochondrial electron transport chain. The resulting loss of ATP leads to an increase in glucose uptake for anaerobic generation of ATP. In L6 skeletal muscle cells, DNP increases the rate of glucose uptake by twofold. We previously showed that DNP increases cell surface levels of glucose transporter 4 (GLUT4) and hexose uptake via a Ca2+-sensitive and conventional protein kinase C (cPKC)-dependent mechanism. Recently, 5' AMP-activated protein kinase (AMPK) has been proposed to mediate the stimulation of glucose uptake by energy stressors such as exercise and hypoxia. Changes in Ca2+ and cPKC have also been invoked in the stimulation of glucose uptake by exercise and hypoxia. Here we examine whether changes in cytosolic Ca2+ or cPKC lead to activation of AMPK. We show that treatment of L6 cells with DNP (0.5 mM) or hyperosmolar stress (mannitol, 0.6 M) increased AMPK activity by 3.5-fold. AMPK activation peaked by 10-15 min prior to maximal stimulation of glucose uptake. Intracellular Ca2+ chelation and cPKC inhibition prior to treatment with DNP and hyperosmolarity significantly reduced cell surface GLUT4 levels and hexose uptake but had no effect on AMPK activation. These results illustrate a break in the relationship between AMPK activation and glucose uptake in skeletal muscle cells. Activation of AMPK does not suffice to stimulate glucose uptake in response to DNP and hyperosmolarity.     

A model system for studying the adsorption of a hydrophobic mutagen to dietary fibre

The adsorption of 1,8-dinitropyrene (DNP) to alpha-cellulose has been studied as a model system for examining the adsorption of a hydrophobic mutagen to dietary fiber. Most of the DNP rapidly disappeared from an aqueous solution and partitioned between the glass wall of the test tube and the alpha-cellulose. Factors affecting DNP distribution included (i) the time of incubation, (ii) the final concentration of the solvent, dimethyl sulphoxide, in which the DNP has been dissolved, and (iii) the relative concentrations of DNP and alpha-cellulose. We suggest that this model system could be applied to other mutagens, and that alpha-cellulose would provide a useful standard fiber to permit inter-laboratory comparisons.     

Novel neuroprotective, neuritogenic and anti-amyloidogenic properties of 2,4-dinitrophenol: the gentle face of Janus

In Roman mythology, Janus was the god of gates, doors, beginnings and endings. He was usually depicted with two faces looking in opposite directions. Janus was frequently used to symbolize change and transitions, such as the progression from past to future or from one viewpoint to another. 2,4-dinitrophenol (DNP) and other nitrophenols have long been known to be toxic at high concentrations (the 'bad' face of DNP), an effect that appears essentially related to interference with cellular energy metabolism due to uncoupling of mitochondrial oxidative phosphorylation. Five years ago, however, we published the first report showing that low concentrations of DNP protect neurons against the toxicity of the amyloid-beta peptide (De Felice et al. (2001) FASEB J. 15:1297 - 1299]. Since then, other studies have provided evidence of beneficial actions of DNP (at low concentrations), including neuroprotection against different types of insult, blockade of amyloid aggregation, stimulation of neurite outgrowth and neuronal differentiation, and even extension of lifespan in certain organisms. Some of these effects appear to be due to mild mitochondrial uncoupling and prevention of cellular oxidative stress, whereas other actions are related to activation of additional intracellular signaling pathways. Thus, a novel and 'gentle' face of DNP is emerging from such studies. In this review, we discuss both toxic and beneficial actions of DNP. The evidence available so far suggests that DNP and other compounds with similar biological activities may be of significant interest to the development of novel therapeutic approaches for neurodegenerative diseases and other neurological disorders.     

Suppression of reaginic antibody formation. III. Relationship between immunogenecity and tolerogenicity of hapten-carrier conjugates.

From the study of the effect of epitope density on the immunogenicity of haptenated ovalbumin (DNP-OA) it was concluded that the lightly haptenated conjugate, DNP0-5-OA, induced, on the one hand, only low titers of anti-DNP hemagglutinating antibody and no reaginic antibodies to the hapten and, on the other, high reaginic and high hemagglutinating antibody responses to the carrier. The conjugate with a slightly higher degree of haptenation, i.e., DNP2.3-OA, induced both reaginic and hemagglutinating antibodies to both the hapten and the carrier. By contrast, the heavily haptenated conjugate, DNP20-OA, elicited reaginic and hemagglutinating antibodies only against the hapten but not against the carrier. Specific suppression of anti-hapten reaginic antibody formation had been achieved by treatment of mice with a tolerogen consisting of the hapten (DNP) conjugated covalently to isologous gamma globulins (MgammaG). The epitope density of the DNPx-MgammaG conjugates was shown to play a dominant role in determining whether or not the conjugate was tolerogenic. Thus, lightly haptenated conjugates (DNP0.5-MgammaG, DNP1.3-MgammaG or DNP1.9-MgammaG) were not tolerogenic, moderately haptenated conjugates (DNP4.2-MgammaG, DNP8-MgammaG, and DNP 14-MgammaG) were tolerogenic, and heavily haptenated conjugates (DNP32-MgammaG and DNP53-MgammaG) were immunogenic, being capable of priming the recipients for the DNP hapten. Further evidence for the nonimmunogenicity of DNP 8-MgammaG conjugate was inferred from its rate of clearance in tolerized and normal mice. Thus, the half-life of 125I-labeled DNP8-MgammaG in circulation was not significantly different for normal and tolerized mice; it was 3.7 and 3.5 days, respectively, which is within the range of data reported for clearance of normal MgammaG. These results suggest that DNP8-MgammaG was catabolized at a rate similar to that of nonconjugated, isologous MgammaG. Moreover, there was no significant difference in the localization of DNP8-MgammaG in identical difference in the localization of DNP8-MgammaG in identical organs (spleen, thymus, kidney, and liver) of normal and tolerized mice. All the multivalent DNPx-MgammaG conjugates were shown to be able to elicit passive cutaneous anaphylaxis (PCA) reaction on i.v. challenge of rats which had been pre-sensitized i.d. with anti-DNP reaginic antibodies.     

Can metal ion complexes be used as polarizing agents for solution DNP? A theoretical discussion

Dynamic nuclear polarization (DNP) can be used to dramatically increase the NMR signal intensities in solutions and solids. DNP is usually performed using nitroxide radicals as polarizing agents, characterized by sharp EPR lines, fast rotation, fast diffusion, and favorable distribution of the unpaired electron. These features make the nitroxide radicals ideally suited for solution DNP. Here, we report some theoretical considerations on the different behavior of some inorganic compounds with respect to nitroxide radicals. The relaxation profiles of slow relaxing paramagnetic metal aqua ions [copper(II), manganese(II), gadolinium(III) and oxovanadium(IV)] and complexes have been re-analyzed according to the standard theory for dipolar and contact relaxation, in order to estimate the coupling factor responsible for the maximum DNP enhancement that can be achieved in solution and its dependence on field, temperature and relative importance of outer-sphere versus inner-sphere relaxation.     

Acute exposure to 2,4-dinitrophenol alters zebrafish swimming performance and whole body triglyceride levels

While swimming endurance (critical swimming speed or U(crit)) and lipid stores have both been reported to acutely decrease after exposure to a variety of toxicants, the relationship between these endpoints has not been clearly established. In order to examine these relationships, adult zebrafish (Danio rerio) were aqueously exposed to solvent control (ethanol) or two nominal concentrations of 2,4-dinitrophenol (DNP), a mitochondrial electron transport chain uncoupler, for a 24-h period. Following exposure, fish were placed in a swim tunnel in clean water for swimming testing or euthanized immediately without testing, followed by analysis of whole body triglyceride levels. U(crit) decreased in both the 6 mg/L and 12 mg/L DNP groups, with 12 mg/L approaching the LC??. A decrease in tail beat frequency was observed without a significant change in tail beat amplitude. In contrast, triglyceride levels were elevated in a concentration-dependent manner in the DNP exposure groups, but only in fish subjected to swimming tests. This increase in triglyceride stores may be due to a direct interference of DNP on lipid catabolism as well as increased triglyceride production when zebrafish were subjected to the co-stressors of swimming and toxicant exposure. Future studies should be directed at determining how acute DNP exposure combines with swimming to cause alterations in triglyceride accumulation.     

Actions of a novel synthetic natriuretic peptide on hemodynamics and ventricular function in the dog

Dendroaspis natriuretic peptide (DNP) is a recently discovered peptide with structural similarity to known natriuretic peptides. DNP has been shown to possess potent renal actions. Our objectives were to define the acute hemodynamic actions of DNP in normal anesthetized dogs and the acute effects of DNP on left ventricular (LV) function in conscious chronically instrumented dogs. In anesthetized dogs, DNP, but not placebo, decreased mean arterial pressure (141 +/- 6 to 109 +/- 7 mmHg, P < 0.05) and pulmonary capillary wedge pressure (5.8 +/- 0.3 to 3.4 +/- 0.2 mmHg, P < 0.05). Cardiac output decreased and systemic vascular resistance increased with DNP and placebo. DNP-like immunoreactivity and guanosine 3',5'-cyclic monophosphate concentration increased without changes in other natriuretic peptides. In conscious dogs, DNP decreased LV end-systolic pressure (120 +/- 7 to 102 +/- 6 mmHg, P < 0.05) and volume (32 +/- 6 to 28 +/- 6 ml, P < 0.05) and LV end-diastolic volume (38 +/- 5 to 31 +/- 4 ml, P < 0.05) but not arterial elastance. LV end-systolic elastance increased (6.1 +/- 0.7 to 7.4 +/- 0.6 mmHg/ml, P < 0.05), and Tau decreased (31 +/- 2 to 27 +/- 1 ms, P < 0.05). The effects on hemodynamics, LV function, and second messenger generation suggest synthetic DNP may have a role as a cardiac unloading and lusitropic peptide.     

Presence of dendroaspis natriuretic peptide and its binding to NPR-A receptor in rabbit kidney

Natriuretic peptides help to maintain sodium and fluid volume homeostasis in a healthy cardio-renal environment. Since the identification of Dendroaspis natriuretic peptide (DNP) as a new member of the natriuretic peptide family, DNP has been considered as an important regulator of natriuresis and dieresis. The present study was undertaken to investigate the presence of immunoreactive Dendroaspis natriuretic peptide (DNP) and its specific receptor in rabbit. DNP was detected in heart, kidney, liver, brain, and plasma by radioimmunoassay (RIA). DNP contents of cardiac atrium and ventricle, renal cortex and medulla, liver, and brain were 1.42 ± 0.15, 1.0 6 ± 0.08, 2.55 ± 0.21, 1.81 ± 0.16, 1.36 ± 0.22, and 0.69 ± 0.15 pg/mg of wet weight, respectively. The concentration of DNP in plasma was 235.44 ± 15.44 pg/ml. By quantitative in vitro receptor autoradiography, specific 12?I-DNP binding sites were revealed in glomeruli, interlobular artery, acuate artery, vasa recta bundle, and inner medulla of the kidney with an apparent dissociation constant (K(d)) of 0.29 ± 0.05, 0.36 ± 0.03, 0.84 ± 0.19, 1.18 ± 0.23, and 10.91 ± 1.59 nM, respectively. Basal rate of 3', 5'-cyclic guanosine monophosphate (cGMP) production by particulate guanylyl cyclase (GC) activation of glomerular membranes was basally 13.40 ± 1.70 pmol/mg protein/min. DNP caused an increment of cGMP production in similar magnitude to that caused by ANP, BNP, and urodilatin, while the production of cGMP by CNP was significantly lower than that by DNP. Our results show that plasma levels of DNP were higher when compared to other tissues. DNP produces cGMP via the NPR-A receptor subtype in the kidney, similarly to ANP and BNP, suggesting that plasma DNP could have similar functions as ANP and BNP.     

Circulatory responses to 2,4-dinitrophenol in dog limb during normoxia and hypoxia

We tested whether blood flow to skeletal muscle would increase in proportion to an increase in O2 uptake caused by 2,4-dinitrophenol (DNP). We further tested the metabolic control in the face of a central challenge, hypoxic hypoxia. Three injections of DNP were made at 30-min intervals into the arterial supply of the left hindlimb in anesthetized dogs. Similar experiments were done on a second group of dogs ventilated with 12% O2-88% N2 (DNP and hypoxia). A third group served as time controls. Limb O2 uptake increased in a linear fashion in the DNP group with each injection. The increase in limb O2 uptake fell off with the second and third injections in the DNP and hypoxia group and appeared to be limited by the hypoxia. Limb blood flow increased only with the last injection in that group and not at all in the DNP group. Limb vascular resistance decreased in both the experimental groups relative to the time-related changes in the control group. This became more marked as the O2 extraction ratio exceeded 0.5. Even in the absence of nerve stimulation and active muscle contractions, both distribution and resistance control vessels responded in a coordinated fashion to an increase in O2 uptake. Mild hypoxia enhanced these responses but also appeared to limit a fraction of O2 uptake that may not have been concerned with maintaining tissue energy levels.     

The Effect of 2,4-Dinitrophenol on Translocation in the Phloem

The effect of 2,4-dinitrophenol (DNP) on sucrose-¹⁴C transport in Soya seedlings has been analysed. The aim was to distinguish between an effect of the inhibitor on sugar movement within the phloem sieve tubes themselves, and on the prior steps of uptake and secretion of sugar into the conducting cells. DNP drastically inhibited sucrose-¹⁴C transport if it was applied to the ¹⁴C-treated leaf immediately before, or during, ¹⁴C supply. Transport was also strongly inhibited if DNP was applied along the translocation path while the ¹⁴C-treated leaflet was still in position on the plant. When, bowever, DNP was applied through the cut petioles of the primary leaves after removal of the ¹⁴C-treated terminal leaflet of the first trifoliate leaf, no inbibition was observed. On the contrary, transport appeared to have been promoted: significantly more ¹⁴C disappeared from the upper regions of DNP-treated plants as compared with controls, while in the lower plant parts more ¹⁴C accumulated. Different rates of synthesis of sucrose-¹⁴C into non-alcohol-soluble compounds could not account for this result. A similar stimulatory effect was observed when DNP was applied to the cut petiole of a primary leaf opposite that treated with ¹⁴C. Several indications were obtained that ¹⁴C which has reached the lower parts of the plant may circulate upwards again through the phloem within about 15 minutes. When sucrose-¹⁴C was introduced into the roots via the xylem, both DNP treatment and prior steam girdling resulted in the apparent accumulation of ¹⁴C in the lower plant parts. the results would be compatible with DNP inhibition of upwards movement in the phloem. DNP might also have affected sugar uptake processes in cells neighbouring the translocation path. It is concluded that the inhibitory effect of DNP on downwards phloem transport reported by earlier workers was probably due to an effect on uptake and/or secretion into the sieve tubes, not to an effect on the conducting cells themselves. Modern theories for phloem transport are discussed in the light of these findings.     

In vitro adsorption of a hydrophobic mutagen to gastrointestinal mucus glycoprotein (mucin) and dietary fibre.

The adsorption of mutagens by some dietary fibres has been suggested as one mechanism by which dietary fibres protect against colorectal cancer. It is thought that these dietary fibres carry the mutagen out of the digestive tract, decreasing the effective mutagen concentration to which epithelial cells are exposed. The ability of gastrointestinal mucin to alter the extent to which the hydrophobic mutagen 1,8-dinitropyrene (DNP) adsorbs in vitro onto the insoluble dietary fibre alpha-cellulose, was investigated. It was found that crude and purified human ileal mucins themselves adsorbed DNP and decreased the adsorption of DNP onto alpha-cellulose. Purified mucin which had been treated with trypsin also adsorbed DNP. These studies suggest that in the digestive tract there would be competition for the adsorption of DNP between mucin and insoluble dietary fibres, such as alpha-cellulose. This factor must be considered in predictions about the distribution of hydrophobic, mutagenic carcinogens in the digestive tract and their role in the etiology of colorectal cancer.     

Synthesis and physical properties of anti-HIV antisense oligonucleotides bearing terminal lipophilic groups.

A number of phosphoramidite monomers have been prepared and used in the synthesis of antisense phosphorothioate oligonucleotides bearing 5'-polyalkyl and cholesterol moieties. Similar groups have also been attached to the 3'-end of oligonucleotides by means of functionalised CPG. Melting temperatures of duplexes formed between phosphorothioate oligonucleotides with lipophilic end-groups and complementary DNA strands were found to be identical to those formed by the equivalent unmodified phosphorothioates.     

Adsorption of a hydrophobic mutagen to dietary fibre from the skin and flesh of potato tubers.

One of the theories to explain the protective action of some dietary fibres against colon cancer is that certain mutagens and/or cancer promoters are adsorbed to these dietary fibres making the mutagens and/or cancer promoters less available to gut mucosal cells. The abilities of 2 contrasting cell wall preparations (dietary fibre preparations) from potato tubers to adsorb in vitro the hydrophobic mutagen, 1,8-dinitropyrene (DNP), were studied using an incubation mixture containing DNP in phosphate-buffered saline (PBS). Walls from potato skins strongly adsorbed DNP and, at the highest wall concentration tested, only a small porportion of the DNP remained in solution. In marked contrast to the skin walls, potato flesh walls adsorbed only a small proportion of the DNP. Unexpectedly, the flesh walls also caused a large increase in the proportion of DNP found in solution. When flesh walls were pre-extracted with PBS, the ability of the extracted walls to bind DNP increased. The material extracted from the flesh walls was able to maintain DNP in solution, when added to the incubation medium in the absence of cell walls. Pectic polysaccharides appear to be the soluble component responsible for maintaining the DNP in solution. Competition between soluble and insoluble fibre components may have major implications for the availability and distribution of hydrophobic mutagens in the alimentary tract.     

Antisense therapeutics: is it as simple as complementary base recognition?

Antisense oligonucleotides provide a simple and efficient approach for developing target-selective drugs because they can modulate gene expression sequence-specifically. Antisense oligonucleotides have also become efficient molecular biological tools to investigate the function of any protein in the cell. As the application of antisense oligonucleotides has expanded, multiple mechanisms of oligonucleotides have been characterized that impede their routine use. Here, we discuss different mechanisms of action of oligonucleotides and the possible ways of minimizing non-antisense-related [corrected] effects to improve their specificity.     

质谱在寡核苷酸药物质量控制中的应用

介绍了质谱在寡核苷酸质量控制方面的应用.合成寡核苷酸及类似物作为反义治疗剂在病毒感染和一些癌症治疗方面有良好的前景.寡核苷酸作为药物,其结构特性必须进行确证.寡核苷酸浓度和纯度的分析可使用色谱或电泳技术,对寡核苷酸的碱基组成、序列、同一性,修饰基团,色谱或电泳分析方法无能为力.质谱的高鉴别能力使其能有效、灵敏、快速和精确地确定寡核苷酸的这些特性.