The aim of this study was to characterize self-assembled structures of guanosine derivatives in aqueous solutions by vibrational circular dichroism (VCD) and electronic circular dichroism (ECD). Three guanosine derivatives were studied [5'-guanosine monophosphate (GMP), diphosphate (GDP), and triphosphate (GTP)] using a broad range of concentrations and various metal/guanosine ratios. VCD was used for the first time in this field and showed itself to be a powerful method for obtaining specific structural information in solution. It can also help to determine the impact that the cations have, when added to the solution, on the versatile structures of guanine derivatives in terms of their association and disassociation. Based on the markedly different intensities and signs of the VCD signals observed for different concentrations of guanosine derivatives, we propose various structures based on guanine quartets for high guanosine concentrations and high K(+)/guanosine ratios (i.e., columnar helical organization of the quartets, which are rearranged into a continuous helix). We performed a degenerate coupled oscillator (DCO) calculation to interpret the VCD spectra obtained and how they vary during the assembly of guanosine derivatives. The calculations correctly predicted the VCD spectra and enabled us to identify the structures of the metal cation/guanosine monophosphate aggregates. ECD in the ultraviolet region was used as a diagnostic tool to characterize the studied systems and as a contact point between the previously defined structures of the guanine derivative assemblies and the molecular systems studied here. These studies revealed that the VCD technique is a powerful new method for determining the structures of optically active guanosine motifs.
The (3)J(C8-H1'), (3)J(C4-H1'), (1)J(C8-H8), (1)J(C1'-H1'), (1)J(C2'-H2'), and (1)J(C2'-H2'2) indirect scalar coupling constants were calculated with the density functional theory in the deoxyguanosine and riboguanosine molecules. The following geometry descriptors were considered in analysis of the structural dependence of the six J couplings: the glycosidic torsion angle chi and conformation of the hydroxymethyl group at the C4' carbon of sugar mimicking the backbone residue and the sugar pucker (C2'-, C3'-endo). The (3)J(C8-H1') and (3)J(C4-H1') couplings, which are typically assigned to the chi torsion, also depended on the sugar pucker, although the calculated dependence of the latter coupling on sugar pucker was nearly negligible. New parametrization of the Karplus equations, taking into account the stereoinversion effect at the glycosidic nitrogen atom and solvent effects, was calculated for the (3)J(C8-H1') and (3)J(C4-H1') coupling assigned to the chi torsion. The calculated phase shift of chi torsion angle in these new Karplus equations was larger by approximately 10 degrees compared to its commonly accepted value of 60 degrees (Wijmenga, S. S.; van Buuren, B. N. M. Prog. NMR Spectrosc. 1998, 32, 287.). The calculated (1)J(C2'-H2') and (1)J(C2'-H2'2) coupling dominantly depended on the sugar type (deoxyribose or ribose) and its pucker, while the (1)J(C1'-H1') and (1)J(C8-H8) coupling dominantly depended on the glycosidic torsion angle, although quantitatively, all four (1)J couplings depended on both geometry parameters. The dependences of j-couplings on the torsion angle chi calculated in isolated nucleosides were compared with those taking into account the effect of base pairing occurring in the WC/SE RNA base pair family, which appeared to be minor. The calculated (3)J couplings agreed well with available experimental data similarly as the (1)J couplings, although lack of experimental data diminished more reliable validation of the later couplings.
- MeSH
- Deoxyguanosine chemistry MeSH
- DNA chemistry MeSH
- Glycosides chemistry MeSH
- Guanosine MeSH
- Quantum Theory MeSH
- Models, Molecular MeSH
- Base Pairing MeSH
- Solvents chemistry MeSH
- Carbohydrates chemistry MeSH
- Water chemistry MeSH
- DNA, Z-Form chemistry MeSH
- Publication type
- Research Support, Non-U.S. Gov't MeSH
BACKGROUND: Recent results indicate a key role for cyclic guanosine monophosphate (cGMP) in the regulation of oocyte meiotic arrest in preovulatory mammalian follicles. The aim of our study was to determine whether the resumption of oocyte meiosis and expansion of cumulus cells in isolated pig cumulus-oocyte complexes (COCs) can be blocked by a high intracellular concentration of cGMP, and whether this effect is mediated by a cGMP-dependent inhibition of mitogen-activated protein kinase 3/1 (MAPK3/1). METHODS: The COCs were isolated from ovaries of slaughtered gilts and cultured in vitro in M199 supplemented with 5% fetal calf serum. The expression levels of the C-type natriuretic peptide (CNP) precursor (NPPC) and its receptor (NPR2) mRNAs during the culture of COCs were determined by real-time RT-PCR. To control the intracellular concentration of cGMP in the COCs, the culture medium was further supplemented with CNP or various concentrations of synthetic cGMP analogues; the concentration of cGMP in COCs was then assessed by ELISA. The effect of the drugs on oocyte maturation was assessed after 24 and 44 h of culture by determining nuclear maturation. The expansion of cumulus cells was assessed by light microscopy and the expression of cumulus expansion-related genes by real-time RT-PCR. A possible effect of cGMP on FSH-induced activation of MAPK3/1 was assessed by immunoblotting the COC proteins with phospho-specific and total anti-Erk1/2 antibodies. RESULTS: The COCs expressed NPPC and NPR2, the key components of cGMP synthesis, and produced a large amount of cGMP upon stimulation with exogenous CNP, which lead to a significant (P < 0.05) delay in oocyte meiotic resumption. The COCs also responded to cGMP analogues by inhibiting the resumption of oocyte meiosis. The inhibitory effect of cGMP on meiotic resumption was reversed by stimulating the COCs with FSH. However, high concentration of intracellular cGMP was not able to suppress FSH-induced activation of MAPK3/1 in cumulus cells, cumulus expansion and expression of expansion-related genes (P > 0.05). CONCLUSIONS: The findings of this study indicate that high cGMP concentrations inhibit the maturation of pig oocytes in vitro but the inhibitory mechanism does not involve the suppression of MAPK3/1 activation in cumulus cells.
- MeSH
- Enzyme Activation drug effects MeSH
- Gonadotropins pharmacology MeSH
- Cyclic GMP pharmacology MeSH
- Cells, Cultured MeSH
- Cumulus Cells drug effects physiology MeSH
- Meiosis drug effects MeSH
- Mitogen-Activated Protein Kinase 3 metabolism MeSH
- Oocytes drug effects physiology MeSH
- Oogenesis drug effects MeSH
- Sus scrofa MeSH
- Animals MeSH
- Check Tag
- Female MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
We investigated peroxidase-mediated oxidation of and the formation of the (deoxy)guanosine adduct by 1-phenylazo-2-hydroxynaphthalene (Solvent Yellow 14, Sudan I), a liver and urinary bladder carcinogen for rodents and a potent contact allergen and sensitizer for humans. Using thin layer chromatography (TLC) and/or high performance liquid chromatography (HPLC) combined with mass and/or nuclear magnetic resonance (NMR) spectrometry, we characterized the structures of two major peroxidase-mediated Sudan I metabolites and those of the adducts of (deoxy)guanosine that are formed during Sudan I oxidation. Peroxidase oxidizes Sudan I to radical species that react with another Sudan I radical to form the Sudan I dimer, or in the presence of (deoxy)guanosine, the oxidized Sudan I can attack the exocyclic amino group of guanine, forming the 4-[(deoxy)guanosin-N(2)-yl]Sudan I adduct. The reaction product with a second Sudan I radical results in a dimer where the oxygen 2 radical of Sudan I reacted with carbon 1 in the second Sudan I skeleton. The Sudan I dimer is unstable and decomposes spontaneously to the second oxidation product. This compound consists of the 4-oxo-Sudan I skeleton connected via the oxygen of its 2-hydroxyl group and nitrogen of its azo group with carbon 1 of 2-oxonaphthalene, having a unique spironaphthooxadiazine structure. If (deoxy)guanosine is present during the formation of this Sudan I metabolite, an adduct, in which this Sudan I metabolite is bound to the exocyclic amino group of guanine, is generated. This (deoxy)guanosine adduct is again unstable and decomposes spontaneously to the same adduct that is formed by the direct reaction of oxidized Sudan I, the 4-[(deoxy)guanosin-N(2)-yl]Sudan I adduct. The results presented here are the first structural characterization of Sudan I-(deoxy)guanosine adducts formed during the oxidation of this carcinogen by peroxidase.
- MeSH
- DNA Adducts chemistry MeSH
- Coloring Agents chemistry metabolism toxicity MeSH
- Biocatalysis MeSH
- Deoxyguanosine chemistry metabolism MeSH
- Carcinogens chemistry metabolism toxicity MeSH
- Naphthols chemistry metabolism toxicity MeSH
- Oxidation-Reduction MeSH
- Peroxidase metabolism MeSH
- Publication type
- Research Support, Non-U.S. Gov't MeSH
- MeSH
- Muscarinic Agonists pharmacology MeSH
- Alcuronium pharmacology MeSH
- Guanosine 5'-O-(3-Thiotriphosphate) metabolism drug effects MeSH
- Humans MeSH
- Liposomes chemistry metabolism MeSH
- GTP-Binding Proteins metabolism MeSH
- Receptors, Muscarinic metabolism drug effects MeSH
- In Vitro Techniques MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Animals MeSH
OBJECTIVES: We sought to identify factors that discriminate heart failure (HF) patients with normal and elevated pulmonary vascular resistance (PVR) and to elucidate the role of cyclic guanosine monophosphate (cGMP)-dependent vasodilation. BACKGROUND: Mechanisms of PVR increase in patients with chronic HF are incompletely understood. METHODS: Twenty-two HF patients with high pulmonary vascular resistance (H-PVR) (>200 dyn.s.cm(-5)) were compared with 24 matched low pulmonary vascular resistance (L-PVR) patients of similar age, sex, body size, HF severity, and volume status who were undergoing invasive hemodynamic study. Pulmonary arterial (PA) and venous blood samples from a wedged PA catheter were used to calculate transpulmonary B-type natriuretic peptide (BNP) uptake and cGMP release. The H-PVR patients were re-examined 1 h after a 40-mg oral dose of sildenafil. RESULTS: Although transpulmonary BNP uptake was similar (p = 0.2), cGMP release was diminished in the H-PVR patients (-1.9 vs. 27.8 nmol.min(-1); p = 0.005). Transpulmonary BNP uptake and cGMP release correlated in the L-PVR patients (R = 0.6, p = 0.004) but not in the H-PVR. The H-PVR patients also had lower PA compliance, systemic arterial compliance (by 47% and 20%, p < 0.001 and p < 0.03), and cardiac index. Sildenafil reduced PVR (-47%), systemic resistance (-24%) and heart rate (-8%), increased cardiac index (+24%), and PA compliance (+87%, all p < 0.001), with a parallel increase of cGMP release (from -5.6 to 16.5 nmol.min(-1), p = 0.047), without affecting BNP uptake or norepinephrine(PA). The PVR response was not dependent on PA wedge pressure or pulmonary hypertension reversibility with prostaglandin E(1). CONCLUSIONS: The H-PVR patients have stiffening of both pulmonary and systemic arteries, preserved transpulmonary BNP uptake, but diminished cGMP release, which is reversible by the administration of sildenafil. This study provides in vivo evidence that phosphodiesterase 5A inhibition restores sensitivity of pulmonary vasculature to endogenous cGMP-dependent vasodilators.
- MeSH
- 3',5'-Cyclic-GMP Phosphodiesterases antagonists & inhibitors MeSH
- Pulmonary Artery drug effects MeSH
- Vascular Resistance physiology drug effects MeSH
- Adult MeSH
- Hemodynamics drug effects MeSH
- Guanosine Monophosphate metabolism MeSH
- Middle Aged MeSH
- Humans MeSH
- Natriuretic Peptide, Brain metabolism MeSH
- Piperazines pharmacology MeSH
- Purines pharmacology MeSH
- Heart Failure drug therapy physiopathology MeSH
- Sulfones pharmacology MeSH
- Vasodilation physiology MeSH
- Vasodilator Agents therapeutic use MeSH
- Check Tag
- Adult MeSH
- Middle Aged MeSH
- Humans MeSH
- Male MeSH
- Female MeSH
- Publication type
- Research Support, Non-U.S. Gov't MeSH
