The pyrimidinones mhcpe, 2-methyl-3
H
-5-hydroxy-6-carboxy-4-pyrimidinone ethyl ester (mhcpe,
1
), 2,3-dimethyl-5-benzyloxy-6-carboxy-4-pyrimidinone ethyl ester (dbcpe,
2
) and
N
...-methyl-2,3-dimethyl-5-hydroxy-6-carboxyamido-4-pyrimidinone (
N
-MeHOPY,
3
), are synthesized and their structures determined by single crystal X-ray diffraction. The acid-base properties of
1
are studied by potentiometric and spectrophotometric methods, the p
K
a
values being 1.14 and 6.35. DFT calculations were carried out to determine the most stable structure for each of the H
2
L
+
, HL and L
−
forms (HL = mhcpe) and assign the groups involved in the protonation-deprotonation processes. The mhcpe
−
ligand forms stable complexes with V
IV
O
2+
in the pH range 2 to 10, and potentiometry, EPR and UV-Vis techniques are used to identify and characterize the V
IV
O-mhcpe species formed. The results are consistent with the formation of V
IV
O, (V
IV
O)L, (V
IV
O)L
2
, (V
IV
O)
2
L
2
H
−2
, (V
IV
O)L
2
H
−1
, (V
IV
O)
2
L
2
H
−3
, (V
IV
O)LH
−2
species and V
IV
O-hydrolysis products. Calculations indicate that the global binding ability of mhcpe towards V
IV
O
2+
is similar to that of maltol (Hmaltol = 3-hydroxy-2-methyl-4
H
-pyran-4-one) and lower than that of 1,2-dimethyl-3-hydroxy-4-pyridinone (Hdhp). The interaction of V
IV
O-complexes with human plasma proteins (transferrin and albumin) is studied by circular dichroism (CD), EPR and
51
V NMR spectroscopy. V
IV
O-mhcpe-protein ternary complexes are formed in both cases. The binding of V
IV
O
2+
to transferrin (hTF) in the presence of mhcpe involves mainly (V
IV
O)
1
(hTF)(mhcpe)
1
, (V
IV
O)
2
(hTF)(mhcpe)
1
and (V
IV
O)
2
(hTF)(mhcpe)
2
species, bound at the Fe
III
binding sites, and the corresponding conditional formation constants are determined. Under the conditions expected to prevail in human blood serum, CD data indicate that the V
IV
O-mhcpe complexes mainly bind to hTF; the formation of V
IV
O-hTF-mhcpe complexes occurs in the presence of Fe
III
as well, distinct EPR signals being clearly obtained for Fe
III
-hTF and to V
IV
O-hTF-mhcpe species. Thus this study indicates that transferrin plays the major role in the transport of V
IV
O-mhcpe complexes under blood plasma conditions in the form of ternary V
IV
-ligand-protein complexes.
A new pyrimidinone is synthesized and the binding of its V
IV
O
2+
complexes with serum proteins is reported.
Cell-penetrating peptides (CPPs) have been extensively exploited in gene therapy approaches as vectors for intracellular delivery of bioactive molecules. The ability of CPPs to be internalized into ...cells and their capacity to complex nucleic acids depend on their molecular structure, both primary and secondary, namely regarding hydrophobicity/hydrophilicity. CPP acylation has been used as a strategy to improve this structural feature.
Acyl groups (from 6 to 18 carbon atoms) were attached to the S4
-PV peptide and their effects on the peptide competence to complex siRNAs and to mediate gene silencing in glioblastoma (GBM) cells were studied. A systematic characterization of membrane interactions with S4
-PV acyl-derivatives was also conducted, using different biophysical techniques (surface pressure-area isotherms in Langmuir monolayers, DSC and
P NMR) to unravel a relationship between CPP biological activity and CPP effects on membrane stability and lipid organization.
A remarkable concordance was noticed between acylated-S4
-PV peptide competence to promote gene silencing in GBM cells and disturbance induced in membrane models, the lauroyl- and myristoyl-S4
-PV peptides being the most effective. A cut-off effect was described for the first time regarding the influence of acyl-chain length on CPP bioactivity.
C12-S4
-PV showed high capacity to destabilize lipid bilayers, to escape from lysosomal degradation and to mediate gene silencing without promoting cytotoxicity.
Besides unraveling a new CPP with high potential to be employed as a gene delivery vector, this work emphasizes the benefit from allying biophysical and biological studies towards a proper CPP structural refinement for successful pre-clinical/clinical application.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Double quantum and triple quantum filtered ^sup 23^Na nuclear magnetic resonance techniques were used to characterise in detail the isotropic and anisotropic binding and dynamics of intra- and ...extracellular Na^sup +^ in different cellular systems, in the absence and presence of Li^sup +^. The kinetics of Li^sup +^ influx by different cell types was evaluated. At steady state, astrocytes accumulated more Li^sup +^ than red blood cells (RBCs), while a higher intracellular Li^sup +^ concentration was found in chromaffin than in SH-SY5Y cells. Anisotropic and isotropic motions were detected for extracellular Na^sup +^ in all cellular systems studied. Isotropic intracellular Na^sup +^ motions were observed in all types of cells, while anisotropic Na^sup +^ motions in the intracellular compartment were only detected in RBCs. ^sup 23^Na triple quantum signal efficiency for intracellular Na^sup +^ was SH-SY5Y > chromaffin > RBCs, while the reverse order was observed for the extracellular ions. ^sup 23^Na double quantum signal efficiency for intracellular Na^sup +^ was non-zero only in RBCs, and for extracellular Na^sup +^ the order RBCs > chromaffin > SH-SY5Y cells was observed. Li^sup +^ loading generally decreased intracellular Na^sup +^ isotropic movements in the cells, except for astrocytes incubated with a low Li^sup +^ concentration and increased anisotropic intracellular Na^sup +^ movements in RBCs. Li^sup +^ effects on the extracellular signals were more complex, reflecting Li^sup +^/Na^sup +^ competition for isotropic and anisotropic binding sites at the extracellular surface of cell membranes and also at the surface of the gel used for cell immobilisation. These results are relevant and contribute to the interpretation of the in vivo pharmacokinetics and sites of Li^sup +^ action.PUBLICATION ABSTRACT
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DOBA, EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, IZUM, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, SIK, UILJ, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
The uptake of the oxidation products of two oxovanadium(IV) compounds,
N,
N′-ethylenebis(pyridoxylaminato)oxovanadium(IV), V
IVO(Rpyr
2en), and ...bis-3-hydroxy-1,2-dimethyl-4-pyridinonatooxovanadium(IV), V
IVO(dmpp)
2, by human erythrocytes was studied using
51V and
1H NMR and EPR spectroscopy. V
IVO(Rpyr
2en) in aerobic aqueous solution is oxidized to its V
V counterpart and the neutral form slowly enters the cells by passive diffusion. In aerobic conditions, V
IVO(dmpp)
2 originates V
V complexes of 1:1 and 1:2 stoichiometry. The neutral 1:1 species is taken up by erythrocytes through passive diffusion in a temperature-dependent process; its depletion from the extracellular medium promotes the dissociation of the negatively charged 1:2 species, and the protonation of the negatively charged 1:1 species. The identity of these complexes is not maintained inside the cells, and the intracellular EPR spectra suggest N
2O
2 or NO
3 intracellular coordinating environments. The oxidative stress induced by the oxovanadium compounds in erythrocytes was not significant at 1
mM concentration, but was increased by both vanadate and oxidized V
IVO(dmpp)
2 at 5
mM. Only 1
mM oxidized V
IVO(dmpp)
2 significantly stimulated erythrocytes glucose intake (0.75
±
0.13 against 0.37
±
0.17
mM/h found for the control,
p
<
0.05).
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Abstract
Several studies have suggested the involvement of biogenic monoaminergic neurotransmission in bipolar disorder and in the therapy for this disease. In this study, the effects of the ...mood‐stabilizing drugs lithium, carbamazepine or valproate on the dopaminergic and adrenergic systems, particularly on D
2
‐like and β‐adrenergic receptors, were studied both in cultured rat cortical neurones and in rat prefrontal cortex.
In vitro
and
in vivo
data showed that stimulation of β‐adrenergic receptors with isoproterenol increased cyclic adenosine monophosphate (cAMP) levels and this effect was significantly inhibited by lithium, carbamazepine or valproate. The activation of dopamine D
2
‐like receptors with quinpirole decreased the isoproterenol‐induced rise in cAMP in control conditions. This inhibition was observed
in vivo
after chronic treatment of the rats with carbamazepine or valproate, but not after treatment with lithium or in cultured rat cortical neurones after 48 h exposure to the three mood stabilizers. Dopamine D
2
and β
1
‐adrenergic receptors were found to be co‐localized in prefrontal cortical cells, as determined by immunohistochemistry, but western blot experiments revealed that receptor levels were differentially affected by treatment with the three mood stabilizers. These data show that mood stabilizers affect D
2
receptor‐mediated regulation of β‐adrenergic signalling and that each drug acts by a unique mechanism.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
The basic catalysis of melamine with epichlorohydrin gives prepolymers that can be used in the preparation of energetic materials. In this work, sodium hydroxide and triethylamine were used as ...catalysts and ethyleneglycol as initiator. Different reaction conditions were tested and the characterization of the products was carried out by IR and NMR spectroscopy and MS spectrometry, hydroxyl groups content, vapour pressure osmometry and elemental and thermal analysis. Epichlorohydrin reacts with the amine groups of melamine and forms lateral chains with hydroxyl and epoxide end groups, which can be used for curing purposes. The two catalysts lead to similar products, confirmed both by the structure and number of the lateral chains. The melamine/epichlorohydrin ratio was found important for the structure of the final compounds. Chlorine atoms leave the molecules during reaction due to basic catalysis. In the light of the use of the prepolymers in energetic materials, the presence of the 1,3,5-
s-triazine ring and the lateral chains with end groups curable by e.g. isocyanates was accomplished with success. However, the loss of chlorine atoms limits to a certain extent their possible substitution by energetic groups.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
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