The cholesterol lowering properties of the complex compound simvastatin with glycyrrhizic acid (simvaglyzin) in experimental models

A molecular complex of simvastatin (SV) and glycyrrhizic acid (GA) (at their ratio of 1 : 4) has been synthesized. The complex named “simvaglyzin” (SVG) was stable in aqueous and aqueous-alcohol solutions at GA concentrations exceeding 0.2 mM. In vitro SVG acted as an uncompetitive inhibitor of 3-hydroxy-3-methyl-glutaryl-CoA (3-HMG-CoA) reductase (Ki of 94 nM). Appearance of this inhibitory activity is associated with cytochrome P450-dependent conversion of SVG, because the addition of 1 mM metyrapone to the incubation medium fully prevented the inhibition of 3-HMG-CoA reductase. SV and SVG (used at 300 nM concentration) inhibited mevalonate synthesis rate by 39.15±8,27 and 38.85±3,04%, respectively. In vivo SVG showed a dose-dependent cholesterol lowering effect. In rats the cholesterol lowering effect of SVG used at daily doses equivalent to 66 and 100 mg/kg of SV was the same as the effect of SV administered at the daily dose of 200 mg/kg. The decrease in total cholesterol of blood serum was 7% and 9% (p < 0.05) and 8%, respectively. Myotoxicity of these SVG doses estimated by blood serum creatine phosphokinase (CPK) activity was lower than that of SV. In rats treated with SV the activity of CPK increased by 79% (p < 0.01), while in SVG treated rats it decreased by 30% and 36% (p < 0.05). Any increase of the hepatotoxicity markers alanine aminotransferase or aspartate aminotransferase in blood serum was not observed. The data suggest pharmacological synergism attributed to the SV-GA complex formation and increased safety of the resultant complex compared with a parent compound.     

Carotenoids as antioxidants: spin trapping EPR and optical study.

The role of several natural and synthetic carotenoids as scavengers of free radicals was studied in homogeneous solutions. A set of free radicals: *OH, *OOH, and *CH(3) were generated by using the Fenton reaction in dimethyl sulfoxide. It was shown that the spin trapping technique is more informative than optical methods for the experimental conditions under study. 5,5-Dimethyl-pyrroline-N-oxide (DMPO) and N-tert-butyl-alpha-phenylnitrone (PBN) were used as spin traps for the EPR studies. The results show that the scavenging ability of the carotenoids towards radical *OOH correlates with their redox properties.     

EPR spin trapping detection of carbon-centered carotenoid and beta-ionone radicals

Free radical intermediates were detected by the electron paramagnetic resonance spin trapping technique upon protonation/deprotonation reactions of carotenoid and beta-ionone radical ions. The hyperfine coupling constants of their spin adducts obtained by spectral simulation indicate that carbon-centered radicals were trapped. The formation of these species was shown to be a result of chemical oxidation of neutral compounds by Fe(3+) or I(2) followed by deprotonation of the corresponding radical cations or addition of nucleophilic agents to them. Bulk electrolysis reduction of beta-ionone and carotenoids also leads to the formation of free radicals via protonation of the radical anions. Two different spin adducts were detected in the reaction of carotenoid polyenes with piperidine in the presence of 2-methyl-2-nitroso-propane (MNP). One is attributable to piperidine radicals (C(5)H(10)N*) trapped by MNP and the other was identified as trapped neutral carotenoid (beta-ionone) radical produced via protonation of the radical anion. Formation of these radical anions was confirmed by ultraviolet-visible spectroscopy. It was found that the ability of carotenoid radical anions/cations to produce neutral radicals via protonation/deprotonation is more pronounced for unsymmetrical carotenoids with terminal electron-withdrawing groups. This effect was confirmed by the radical cation deprotonation energy (H(D)) estimated by semiempirical calculations. The results indicate that the ability of carotenoid radical cations to deprotonate decreases in the sequence: beta-ionone > unsymmetrical carotenoids > symmetrical carotenoids. The minimum H(D) values were obtained for proton abstraction from the C(4) atom and the C(5)-methyl group of the cyclohexene ring. It was assumed that deprotonation reaction occurs preferentially at these positions.     

Antioxidant and redox properties of supramolecular complexes of carotenoids with beta-glycyrrhizic acid

Supramolecular complexes between carotenoids and a triterpene glycoside, beta-glycyrrhizic acid (GA), were found to exhibit unusual antioxidant activity. Complexation with GA increases a scavenging rate of canthaxanthin and 7',7'-dicyano-7'-apo-beta-carotene toward OOH radicals more than 10 times, but has no effect on the scavenging rate of zeaxanthin. Scavenging rate constants were measured in DMSO solution of carotenoids using the EPR spin-trapping technique. EPR parameters of spin adducts were determined as a(H) = 2.3 G, a(N) = 13.9 G for PBN (N-tert-butyl-alpha-phenylnitrone)-OOH, and a(H) = 3.4 G, a(N) = 14.9 G for the PBN-CH3 adduct. Taking into account the previously measured dependence of the scavenging rate constants toward OOH radicals on the oxidation potential of carotenoids, this result can be explained by the hypothesis that the complexation with GA affects the value of oxidation potentials. This hypothesis was confirmed by CV measurements.     

Larvae of related Diptera species from thermally contrasting habitats exhibit continuous up-regulation of heat shock proteins and high thermotolerance

A population of Stratiomys japonica, a species belonging to the family Stratiomyidae (Diptera), common name ‘soldier flies’, occurs in a hot volcanic spring, which is apparently among the most inhospitable environments for animals because of chemical and thermal conditions. Larvae of this species, which naturally often experience temperatures more than 40 °C, have constitutively high concentrations of the normally inducible heat-shock protein Hsp70, but very low level of corresponding mRNA. Larvae of three other species of the same family, Stratiomys singularior, Nemotelus bipunctatus and Oxycera pardalina, are confined to different type semi-aquatic habitats with contrasting thermal regime. However, all of them shared the same pattern of Hsp70 expression. Interestingly, heat-shock treatment of S. japonica larvae activates heat-shock factor and significantly induces Hsp70 synthesis, whereas larvae of O. pardalina, a species from constant cold environment, produce significantly less Hsp70 in response to heat shock. Adults of the four species also exhibit lower, but detectable levels of Hsp70 without heat shock. Larvae of all species studied have very high tolerance to temperature stress in comparison with other Diptera species investigated, probably representing an inherent adaptive feature of all Stratiomyidae enabling successful colonization of highly variable and extreme habitats.     

Molecular phylogeny of fungus gnats (Diptera: Mycetophilidae) revisited: position of Manotinae,Metanepsiini, and other enigmatic taxa as inferred from multigene analysis

The phylogeny of selected genera from four subfamilies of fungus gnats (Diptera: Mycetophilidae) – Manotinae, Leiinae, Sciophilinae and Gnoristinae (including Metanepsiini) – is reconstructed based on the combined analysis of five mitochondrial (12S, 16S, COI, COII, cytB) and two nuclear (28S, ITS2) gene markers. Results of the different analyses all support Manotinae as a monophyletic group, with Leiinae as the sister group. Allactoneura DeMeijere is nested in the monophyletic and strongly supported clade of Leiinae. The tribe Metanepsiini is revealed as paraphyletic and the genera Metanepsia Edwards and Chalastonepsia Søli do not appear to be closely related. The genera Docosia Winnertz, Ectrepesthoneura Enderlein, Novakia Strobl and Syntemna Winnertz were placed with a group of genera included traditionally in the Gnoristinae. The monophyly of Dziedzickia Johannsen and Phthinia Winnertz is not supported. The genera of Sciophilinae (excluding Paratinia Mik but including Eudicrana Loew) form a monophyletic group in the Bayesian model.     

A genomic walking method for screening sequence length polymorphism

We adapted a recently developed nonrestrictional, nonligational genome walking method, Universal Fast Walking (UFW), for detection of length polymorphism in the proximal promoter region of genes. We demonstrate its efficacy at discovering naturally occurring transposition into heat‐shock genes of wild Drosophila and show that it surmounts limitations of simple polymerase chain reaction (PCR) approaches. We further present modifications to the standard UFW protocol and provide some guidelines to improve specificity. Although the resultant banding pattern of a standard UFW can be regarded as a DNA fingerprint, many amplicons result from false priming and not real polymorphisms. We describe ways to distinguish between UFW amplicons and false priming products in a high‐throughput assay.