Mario Gabričević

Interaction of α-melanocortin and its pentapeptide antisense LVKAT: effects on hepatoprotection in male CBA mice.

The genetic code defines nucleotide patterns that code for individual amino acids and their complementary, i.e., antisense, pairs. Peptides specified by the complementary mRNAs often bind to each other with a higher specificity and efficacy. Applications of this genetic code property in biomedicine are related to the modulation of peptide and hormone biological function, selective immunomodulation, modeling of discontinuous and linear epitopes, modeling of mimotopes, paratopes and antibody mimetics, peptide vaccine development, peptidomimetic and drug design. We have investigated sense-antisense peptide interactions and related modulation of the peptide function by modulating the effects of α-MSH on hepatoprotection with its antisense peptide LVKAT. First, transcription of complementary mRNA sequence of α-MSH in 3’→5’ direction was used to design antisense peptide to the central motif that serves as α-MSH pharmacophore for melanocortin receptors. Second, tryptophan spectrofluorometric titration was applied to evaluate the binding of α-MSH and its central pharmacophore motif to the antisense peptide, and it was concluded that this procedure represents a simple and efficient method to evaluate sense-antisense peptide interaction in vitro. Third, we showed that antisense peptide LVKAT abolished potent hepatoprotective effects of α-MSH in vivo.

139La NMR Kinetic Study of Lanthanum(III) Complexation with Acetohydroxamic Acid

The application of La-139 NMR spectroscopy to kinetic and equilibrium studies of the substitution of coordinated water by acetohydroxamic acid (HA) is described. The reaction of La3+ with HA can be represented by the following equation (with equilibrium constant K-1): La3+ + HA reversible arrow La(A)(2+) + H+ (1). The following equilibrium parameters were calculated from the La-139 NMR chemical shift of the equimolar reactants measured as a function of acidity and temperature: K-1(298) = 2.36(8)(.)10(-4), Delta(r)H(1) = -28(l) kJ(.)mol(-1), Delta(r)S(1) = -162(4) J(.)K(-1.)mol(-1). The kinetics of the reaction between La3+ and HA can be represented by the following equations: La3+ HA reversible arrow La(A)(2+) + H+ (11) and La3+ + A(-) reversible arrow La(A)(2+) (12). The complexation rate constants were calculated from the line widths of the corresponding signals as a function of acidity and temperature: k(11)(298K) = 1.54(4)(.)10(6) M(-1.)s(-1), DeltaH(11)(double dagger) = 70(14) kJ(.)mol(-1) and DeltaS(11)(dagger) = +108(47) J(.)K(-1.)mol(-1), k(12) (298 K) = 1.6 10(10) M(-1.)s(-1), DeltaH(12)(double dagger) = 35(6) kJ(.)mol(-1) and DeltaS(12)(double dagger) = +68(20) J(.)K(-1.)mol(-1). The spin-spin relaxation times of La-III in the aqua ion and the hydroxamato complex are 4.36(4) ms and 54(l) ps at 298 K, respectively.

Hepatoprotective Effects of Met-enkephalin on Acetaminophen- Induced Liver Lesions in Male CBA Mice

Recent histopathological investigations in patients with hepatitis suggested possible involvement of Met-enkephalin and its receptors in the pathophysiology of hepatitis. Consequently, we evaluated the potential hepatoprotective effects of this endogenous opioid pentapeptide in the experimental model of acetaminophen induced hepatotoxicity in male CBA mice. Met-enkephalin exhibited strong hepatoprotective effects in a dose of 7.5 mg/kg, which corresponds to the protective dose reported for several different animal disease models. In this group plasma alanine aminotransferase and aspartate aminotransferase enzyme activities, as well as liver necrosis score were significantly reduced in comparison to control animals treated with physiological saline (p > 0.01). The specificity of the peptide hepatoprotection was investigated from the standpoint of the receptor and peptide blockade. It was concluded that Met-enkephalin effects on the liver were mediated via δ and ζ opioid receptors. Genotoxic testing of Met-enkephalin confirmed the safety of the peptide.

Modulation of γ2-MSH Hepatoprotection by Antisense Peptides and Melanocortin Subtype 3 and 4 Receptor Antagonists

Melanocortins, i.e., melanocyte stimulating hormones (MSH) are peptides with strong antiinflammatory effects. The most investigated aspects of γ2-MSH are related to cardiovascular effects and natriuresis, with limited research available about its anti-inflammatory and cytoprotective effects. The aims of this study were: 1) to examine the effects of γ2-MSH and its derivative [D-Trp(8)]-γ2-MSH on the acetaminophen model of liver damage in CBA mice; 2) to evaluate the modulation of γ2-MSH hepatoprotection by melanocortin subtypes 3 and 4 receptor antagonists SHU 9119 and HS 024; 3) to define the importance of central MSH pharmacophore region (HFRW) by using antisense peptides LVKAT and VKAT. In this study, specific antagonists and antisense peptides were used to target central pharmacophore region of γ2-MSH and [D-Trp(8)]-γ2-MSH, enabling the evaluation of hepatoprotection from the standpoint of the receptor and pharmacophore blockade. The criteria for monitoring the effects of the hormones on the liver damage were alanine transaminase, aspartate transaminase activities (U/L), and pathohistological scoring of liver necrosis (scale 0-5). γ2-MSH (0.24 mg/kg) indicated hepatoprotective effects in comparison to control (p < 0.001). In contrast, [D-Trp(8)]-γ2-MSH did not show any hepatoprotective effects. Application of antagonists SHU 9119 and HS 024, and antisense peptides LVKAT and VKAT, also did not show any hepatoprotective effects. In fact, when combined with γ2-MSH, it annulled its hepatoprotective effect. The results provide evidence for hepatoprotective and antiinflammatory effects of the γ2-MSH in the liver.

Reactions of the carbonyl group with nitroso compounds. The cases of pyruvic acid and acetaldehyde

Pyruvic acid and acetaldehyde react with substituted nitrosobenzenes to give the corresponding N-phenylacetohydroxamic acids. A mechanism for these reactions involving three sequential steps is proposed. The first step is the nucleophilic attack of the nitroso group on the carbonyl group, which leads to the formation of an unstable dipolar intermediate. This step is followed by proton transfer to the dipolar intermediate to form a more stable cationic intermediate, which, in the subsequent step, undergoes decarboxylation (in the case of pyruvic acid) or elimination of a proton, from the carbon of the nitrosocarbinolic group (in the case of acetaldehyde), leading to the final product, hydroxamic acid.The reaction of pyruvic acid includes an intramolecular reaction pathway, along with an acid-catalysed one. The experimental evidence obtained in support of such a description includes: (a) the order of reactivity of substituted nitrosobenzenes as demonstrated by the plot of log kobsvs. Hammett parameters with slope –1.97 in the case of pyruvic acid and –0.93 in the case of acetaicohyde; (b) the observation of acid-catalysed and ‘uncatalysed’ pathways in the reaction of pyruvic acid; (c)the observation of general acid catalysis in these reactions; (d) the observation of an inverse solvent deuterium isotope effect of 0.41 in the case of acetaldehyde; (e)the observation of a solvent deuterium isotope effect of ca. 1.0 in the acid-catalysed reaction, and solvent isotope effect of ca. 1.2 in the ‘uncatalysed’ reaction of pyruvic acid with nitrosobenzene.

Structural and electronic determinants of flavonoid binding to human serum albumin: An extensive ligand-based study

Flavonoids are ubiquitous plant metabolites that interfere with different biological processes in the human organism. After absorption they bind to human serum albumin (HSA), the most abundant carrier protein in the blood which also binds various hormones and drugs. Binding of flavonoids to HSA may impact their distribution, influencing the active concentration in the blood. To determine the most prominent features responsible for binding of 20 different flavonoid aglycones to the IIA region of HSA, in vitro fluorescence measurements and density functional theory (DFT) calculations were conducted. These results were then integrated to elucidate structure–affinity relationships. The presented results reveal that flavones and flavonoles bind most strongly to the IIA region of HSA. There are several electronic and structural determinants associated with flavonoid binding to this HSA region: high C3 nucleophilicity and partial charge of O4, high HOMO and LUMO energies, and coplanarity of AC and B rings. Both steric and electronic characteristics of flavonoids have a great impact on their binding to HSA, with hydrogen donor and acceptor properties and coplanarity being the most prominent.

Hydrogen Isotope Exchange of Chlorinated Ethylenes in Aqueous Solution: Possibly a Termolecular Liquid Phase Reaction.

This work reports an experimental study of the hydrogen/deuterium exchange in the basic aqueous solutions of trichloroethylene, trans-1,2-dichloroethylene, and cis-1,2-dichloroethylene using (1)H NMR as a monitoring method. 1,1-Dichlorethylene was also investigated but found not to exchange hydrogen isotopes with water. The kinetics of isotope exchange features two different pathways, the first is first order with respect to hydroxide ion, whereas the second is second order. The first pathway is interpreted as a straightforward bimolecular reaction between chloroethylene and hydroxide ion, which leads to the deprotonation of chloroethylene. The second pathway involves a transition state with the association of one molecule of the chloroethylene and two hydroxide ions. It is shown that the second pathway could involve the formation of a precursor complex composed of one chloroethylene molecule and one hydroxide ion, but a direct termolecular elementary reaction is also feasible, which is shown by deriving a theoretical highest limit for the rate constants of termolecular reactions in solution.

Reaction of pyruvic acid with nitrosobenzenes

Pyruvic acid reacts with nitrosobenzes in both acid-catalysed and uncatalysed reactions giving N-phenylacetohydroxamic acids.

Thermodynamic study of inclusion complexes of zaleplon with natural and modified cyclodextrins

The thermodynamics and stoichiometry of zaleplon (ZAL) complexation with different cyclodextrin derivatives [β-CD, hydroxypropyl-β-cyclodextrin (HP-β-CD), randomly methylated-β-cyclodextrin (RAMEB), sulphobutylether-β-cyclodextrin (SBE-β-CD)] in aqueous solution was studied by spectrofluorimetry and 1H NMR spectroscopy in order to obtain a more general understanding of the driving forces behind the inclusion phenomena. Job’s plot derived from the NMR spectral data and statistical analysis of spectrofluorimetric titration data confirmed the formation of equimolar complexes in all systems tested, excluding the possibility of higher order complex formation. Furthermore, thermodynamic parameters obtained by both techniques gave similar and negative values of ΔG° for all complexes, indicating spontaneous inclusion of drug into CDs. From a thermodynamic point of view, two types of inclusions were determined. One is enthalpy driven ZAL complexation with β-CD, HP-β-CD and RAMEB, while the other is entropy driven complexation observed in the case of SBE-β-CD. The mechanisms behind each type of inclusion were discussed in detail.

Betainohydroxamic acid chloride.

The title compound, N-hydroxy-2-(trimethylammonio)acetamide chloride, C(5)H(13)N(2)O(2)(+).Cl(-), has been synthesized and structurally characterized. The structure consists of betainohydroxamic acid cations and Cl(-) anions linked by N-H.Cl and O-H.Cl hydrogen bonds into chains along [001]. It was found that the positive inductive effect of the charged N atom in close proximity to the hydroxamate carbonyl O atom has a negligible effect on the hydroxamic C-N bond length.