Comparative substrate-inhibitor analysis of liver monoamine oxidases of minks

Comparative substrate-inhibitor analysis of catalytic properties of liver monoamine oxidases (MAO) was performed in the mature males of the American mink Mustela vison and the European mink Mustela lutreola. The action on the MAO activity was studied of alkaloids of the benzo[c]phenanthridine group: sanguinarine and chelidonine, diisoquinoline alkaloid berberine, medication agents Ukrain and Sanguirythrin as well as derivatives of 2-propylamine: deprenyl and clorgylin. The latter turned out to be irreversible inhibitor of the MAO A form, whereas deprehyl--irreversible inhibitor of the MAO B form in both studied mink species. The selectivity of action of each inhibitor on the corresponding liver MAO form for the species M. vison was one order of magnitude stronger than for the species M. lutreola. All studied alkaloids as well medication agents on their basis have been shown to be specific irreversible inhibitors of the intermediate strength of the liver MAO A form of both mink species. They inhibit the enzymatic deamination of serotonin, tyramine, and tryptamine without affecting the deamination reaction of benzylamine and beta-phenylethylamine (at concentrations of 10 mM and lower). Out of the studied five isoquinoline agents, the medication Ukrain and alkaloid chelidonine have the highest inhibitory action; the agent Sanguirythrin and alkaloids berberine and sanguinarine produce the weaker monoamine oxidase effect. The revealed specificity of action of the studied inhibitors is an indirect evidence for the presence in the liver enzymes of both mink species, like in the rat liver enzyme, of several molecular forms.     

Comparative study of catalytic properties of mink and rat liver monoamine oxidase

Comparative substrate-inhibitor analysis of catalytic properties of mitochondrial monoamine oxidase (MAO) of liver of the American mink Mustela vison Schreber and of liver of Wistar rat has been performed. It has been found that MAO of mink, like MAO of rat, has properties of classic mammalian MAO: it deaminates tyramine, tryptamine, serotonin, benzylamine, β-phenylethylamine and does not deaminate histamine as well as does not have sensitivity to semicarbazide. Study of kinetics of the monoamine oxidase deamination revealed both qualitative and quantitative differences between these enzymes. Specificity of action on MAO-A form of four irreversible inhibitors—acridine derivatives—has been shown; this specificity was several times higher for the mink liver MAO than for the rat liver MAO. It is suggested that the liver MAO of both species of the studied animals has several isoenzyme forms or several centers of the substrate binding.     

Inhibition of liver mitochondrial monoamine oxidase activity by alkaloids isolated from Chelidonium and Macleaya and by their derivative drugs

It has been shown that the major alkaloids from plants Chelidonium majus L. and Macleaya (Bocconia) cordata and microcarpa, namely, berberine, sanguinarine, chelidonine, and drugs "Ukrain" (thiophosphoric acid derivative of a sum of the alkaloids isolated from Ch. majus L.) and "Sanguirythrine" (a mixture of the alkaloids sanguinarine and chelerythrine, w/w 3:7, isolated from Macleaya), are irreversible inhibitors of oxidative deamination reaction of serotonin and tyramine as substrates, catalyzed by rat liver mitochondrial monoamine oxidase (MAO). At the same time these substances do not influence the oxidative deamination reaction of benzylamine as substrate (in concentration 1 mM or less). The substrate specificity of this inhibition manifests that mainly the oxidative deamination reactions catalyzed by MAO form A are inhibited by the agents studied. Among the examined agents, alkaloid chelidonine and drug "Ukrain" are the strongest inhibitors of the reaction. Alkaloids berberine and sanguinarine and drug "Sanguirythrine" exhibit a weaker action. Judging from the data obtained, sanguinarine and chelerythrine appear to exert similar inhibitory effects in this reaction, since sanguinarine and "Sanguirythrine" have similar values of bimolecular rate constants of their interaction with mitochondrial MAO. As it is well known, the MAO inhibitors appear to be, as a rule, pronounced antidepressants. The combination of malignotoxicity and antidepressive activity in drug "Ukrain" seems to be favourable for its clinical applications.     

Effect of some isoquinoline alkaloids on enzymatic activity of acetylcholinesterase and monoamine oxidase

It has been shown, that some benzo[c]-phenanthridine and diisoquinoline alkaloids isolated from Chelidonium majus L. and Macleaya (Bocconia) cordata and M. microcarpa (berberine, sanguinarine, chelidonine) and of drugs ("Ukrain" and "Sanguirythrine") inhibited the enzyme activity of acetylcholinesterase from human erythrocyte and monoamine oxidase from the rat liver. All agents under study have been shown to be reversible inhibitors of the enzymatic hydrolysis of acetylthiocholine. It has been determined that chelidonine belonged to reversible inhibitors of a competitive type, all other examined agents have been demonstrated to be inhibitors of a mixed competitive-noncompetitive type, and a greater contribution to the inhibition was made by the competitive constituent. Among all examined agents berberine, sanguinarine and "Sanguirythrine" were the strongest inhibitors of this reaction and chelidonine and "Ukrain" were much weaker. All agents under study have been shown to be irreversible inhibitors of the oxidative deamination reaction of serotonine and tyramine and not to influence the oxidative deamination reaction of benzylamine as a substrate. Among the examined agents, alkaloid sanguinarine and drug "Ukrain" are the strongest inhibitors of the reaction, alkaloids berberine, sanguinarine and "Sanguirythrine" exhibit a weaker action.     

Reactivity of liver monoamine oxidase in the seal Phoca hispida ladogensis

The goal of this work consisted in substrate and inhibitor specificity of liver monoamine oxidase (MAO) of the freshwater Ladoga subspecies of the ringed seal Phoca hispida ladogensis. The studied enzyme has been found to have the large substrate specificity by deaminating, apart from eight classic substrates of the terrestrial mammalian MAO, also histamine, the substrate of diamino oxidase. It has been revealed that the studied enzyme realizes wide substrate specificity by deaminating, apart from eight classic MAO substrates of terrestrial mammals, also histamine, the substrate of diamino oxidase. The deamination rates of benzylamine, β-phenylethylamine, and N-methylhistamine are found to be almost by one order higher than the deamination rates of serotonin and noradrenaline. The seal liver MAO did not deaminate putrescine and cadaverine and was insensitive to 10^?2 M semicarbaside. There were calculated bimolecular rate constants of interaction of inhibitors: chlorgyline, deprenyl, berberine, sanguinarine, chelidonine, and four derivatives of acridine with the enzyme at deamination of nine substrates. By the method of substrate-inhibitor analysis we have shown heterogeneity of the enzyme, i.e., the presence in the seal liver of at least of two different MAO.     

Population aspects of sexual dimorphism in Mustelidae from the example of four species (Mustela lutreola, Neovison vison, Mustela putorius, and Martes martes)

Size sexual dimorphism was studied on 695 crania of four species of the Mustelidae. According to the degree of increasing differences between sexes, the species were arranged in the following order: common marten (Martes martes), European mink (Mustela lutreola), American mink (Neovison vison), and black polecat (Mustela putorius). The value of dimorphism characterizes the ecological flexibility of species and is an integrated population characteristic. It was demonstrated that M. martes occupies a specific and relatively narrow ecological niche of the forest ecosystem and joins weak competitive relations with smaller species of Mustelidae. The level of sexual dimorphism in M. lutreola and N. vison, as well as M. putorius, reflects the tension of their interspecific relationships in a given territory. A high level of sexual dimorphism in M. putorius is caused by further divergence of the ecological niches of males and females, being a compensatory mechanism, which mitigates the consequences of toughened environmental requirements.     

Inhibition of Human Blood Acetylcholinesterase and Butyrylcholinesterase by Some Alkaloids

A comparative study has been carried out on effects of berberine (diisoquinoline alkaloid) and sanguinarine and chelidonine (benzophenanthridine alkaloids( on erythrocyte acetylcholinesterase and serum butyrylcholinesterase from human blood. The studied alkaloids have been shown to be strong reversible inhibitors of the cholinesterase activity. Acetylcholinesterase is more sensitive to their action, than butyrylcholinesterase. The type of reversible inhibition was determined, and inhibitor constants were calculated. It is revealed that the character of inhibition is identical for the both cholinesterases. Berberine and sanguinarine are competitive-noncompetitive inhibitors, whereas chelidonine, a competitive inhibitor.     

Catalytical properties of liver monoamine oxidases of the Commander squid <Emphasis Type="Italic">Berryteuthis magister</Emphasis> from various habitation zones

There has been performed kinetic analysis of enzymatic reactions of deamination of tyramine, tryptamine, serotonin, benzylamine, β-phenylethylamine, and histamine under action of liver monoamine oxidase (MAO) of the Commander squid Berryteuthis magister from various habitation zones in the Bering and Japan Seas. A substrate inhibition by high concentrations of all studied substrates has been revealed, which seems to indicate mutual effect of various MAO forms present in liver of the studied squids. Analysis of kinetic parameters of enzymatic reactions of deamination of six studied substrates and the substrate-inhibitory analysis with use of two derivatives of acridine and deprenyl indicate the enzyme heterogeneity, the presence of at least two MAO-A forms, and the absence of intraspecies differences in MAO of the Commander squids from various habitation zones. The most active was the MAO form responsible for serotonin deamination. There were obtained quantitative difference in substrate specificity and reaction ability with respect to inhibitor of proflavin for the liver MAO of the Commander and Pacific squids.     

Population genetic structure and distribution of introduced American mink (<Emphasis Type="Italic">Mustela vison</Emphasis>) in Spain,based on microsatellite variation

The population genetic structure of an invasive species in Spain, the American mink (Mustela vison), was investigated using microsatellite DNA markers. This semi-aquatic carnivore, originating from North America, was imported into Europe for fur farming since the beginning of the 20th century. Due to massive escapes, farm damages, deliberate releases and/or accidents, feral mink populations were established in the aquatic ecosystems of many European countries, including Spain. We genotyped 155 American mink originating from the Spanish regions Basque Country, Catalonia, Castilla-Leon, Aragon, Valencia and Galicia using 10 polymorphic microsatellite loci to highlight population genetic structure, distribution and dispersal. M. vison populations in Spain appear differentiated and not yet connected by gene flow. Bayesian clustering analyses and spatial analyses of molecular variance detected four inferred clusters, overall coinciding with the sampled geographical localities. Preliminary testing shows moderate to large estimated effective population sizes. Molecular analyses result useful to provide baseline data for further research on the evolution of invasive mink populations, as well as support local management strategies and indirectly benefit the conservation of threatened species in Spain, such as the endangered European mink (Mustela lutreola), and the polecat (Mustela putorius), which share the habitat with the American mink. This paper is dedicated to the memory of Xavier Domingo-Roura.     

Sexual dimorphism in the skull of the European mink<Emphasis Type="Italic">Mustela lutreola</Emphasis> from NW part of Russia

Morphometric variation in 22 characters of 86 skulls of the European minkMustela lutreola (Linnaeus, 1761), from the NW part of Russia, has been analysed. Stepwise discriminant analysis was used to estimate craniometric variables for sex determinations. Two characters (zygomatic breadth and interorbital width) are enough for the 96.5% correct classification. The male skull ofM. lutreola is characterised by a relatively high neurocranium, widely arranged zygomatic arches, a wide rostrum, and by wider auditory bullae and higher mandibles. Sexual size dimorphism ofM. lutreola is less than that of other similar-sized mustelids —Mustela putorius, M. eversmanii, M. sibirica, Neovison vison. The results are discussed in relation to the existing theories on sexual dimorphism in mustelids.     

Isozyme Patterns of Lactate Dehydrogenase from Tissues of Mink and Arctic Fox during Postnatal Development

Isozymes of lactate dehydrogenase extracted from heart, kidney, and liver of mink (Mustela vison Briss.) and Arctic fox (Alopex lagopus L.) during postnatal development were separated by agarose gel electrophoresis. Tissue-specific isozyme pattern of lactate dehydrogenase can be revealed at the age of one month, while the definitive pattern is formed at the age of four months. The isozyme patterns of lactate dehydrogenase in the studied tissues of mink and Arctic fox share the properties specific for animal species of various ecogenesis.     

Comparative enzymological study of catalytic properties of liver monoamine oxidases in frogs

Comparative enzymological study of catalytical properties of monoamine oxidase (MAO) of liver of the marsh frog Rana ridibunda and common frog Rana temporaria has revealed certain features of similarity and differences between these enzymes. The MAOs from both studied biological sources show catalytic properties resembling those of the classical MAO of terrestrial vertebrates: they deaminate tyramine, tryptamine, serotonin, and benzylamine and do not deaminate histamine, have sensitivity to clorgyline, the specific inhibitor of the MAO A form, and deprenyl, the specific inhibitor of the MAO B form, and are not inhibited by 10⁻² M semicarbazide. Based on data of substrate-inhibitor analysis, a suggestion is put forward about the existence of two molecular forms of the enzyme in liver of the studied frog species. Quantitative interspecies differences have been revealed between liver MAO of Rana ridibunda and Rana temporaria in values of kinetic parameters of reactions of deamination of several substrates and in sensitivity to the inhibitors, deprenyl and clorgyline. In the species Rana temporaria the MAO activity in reaction of deamination of serotonin and benzylamine were virtually identical, whereas in the species Rana ridibunda these parameters for serotonin were almost one order higher than for benzylamine. In the species Rana ridibunda, selectivity of action of deprenyl was expressed many times weaker, while selectivity of the clorgyline—one order of magnitude stronger than in the species Rana temporaria. The catalytic activities towards all studied substrates of liver MAO of both studied amphibian species were several times lower as compared with the enzyme of rat liver.     

The inhibition enzymatic hydrolysis of acetylthiocholine by acetylcholinesterase using principal alkaloids isolated from celandine and macleya and their derivatives

A study was made of a possible inhibitory action on the enzymatic hydrolysis of acetylthiocholine by human erythrocyte acetylcholinesterase of principal alkaloids isolated from Chelidonium majus L. and Macleaya (Bocconia) cordata and microcarpa (namely sanguinarine, chelidonine, berberine), and of drugs "Ukrain" (thiophosphoric acid derivative of a sum of the alkaloids isolated from Chelidonium majus L.) and "Sanguirythrine" (a mixture of unseparated closely related to benzo[c]phenanthridine alkaloids sanguinarine and chelerythrine, isolated from Chelidonium majus L. and other plants of Papaveraceae family). All agents under study have been shown to be reversible inhibitors of the enzymatic hydrolysis of acetylthiocholine. On the basis of the kinetic data it has been determined that chelidonine belonged to reversible inhibitors of a competitive type. All other examined agents have been demonstrated to be inhibitors of a mixed competitive-noncompetitive type, and a greater contribution to the inhibition was made by the competitive constituent. Among all examined agents berberine, sanguinarine and "Sanguirythrine" were the strongest inhibitors of this reaction (the values of generalized inhibitory constants being 0.23, 0.23 and 0.29 microM, respectively) and cheliodonine and "Ukrain" were much weaker (2.0 and 2.5 microM, respectively). Judging from the data obtained, sanguinarine and chelerythrine exert similar inhibitory effects on the reaction of enzymatic hydrolysis of acetylthiocholine, since sanguinarine and "Sanguirythrine" have nearly equal generalized inhibitory constants.     

Microsatellite markers for the endangered European mink (Mustela lutreola) and closely related mustelids

The European mink (Mustela lutreola L., 1761) is an endangered carnivore species whose populations suffered a severe decline during the last century. The genotyping of eight polymorphic microsatellite loci revealed a relatively low number of alleles per locus (two to eight), as well as low levels of polymorphism (observed and expected heterozygosity values per locus were 0.49 and 0.54, respectively). Cross‐specific polymerase chain reaction amplifications were successful in seven closely related mustelid species suggesting that these loci may be useful not only for assessing genetic variability in European mink populations but also for determining potential hybridization events between M. lutreola and other mustelid species.     

Comparative sensitivity of liver monoamine oxidases of frog and whitefish to some tricyclic compounds

There is performed a comparative analysis of action of four acridine derivatives and of one xanthene derivative (pyronin G) on activity of liver monoamine oxidase (MAO) of two species of poikilothermal freshwater animals: a representative of amphibians—the common frog Rana temporaria and a representative of the Salmonidae order—the European whitefish Coregonus lavaretus. The studied synthetic hexamember tricyclic compounds show the irreversible character of inhibition of intermediate potency towards the enzyme from the both biological sources. There are obtained qualitative and quantitative differences in the reaction ability and selectivity of action of the studied inhibitors for liver MAO of frog and whitefish. The obtained data of the inhibitory analysis with use of specific substrates are an indirect proof for the existence in liver of the studies frog species of two molecular forms, whereas in the whitefish liver—the single molecular MAO form.     

Molecular characterization of the Himalayan mink

A rare color variant of the American mink (Neovison vison), discovered on a ranch in Nova Scotia and referred to as the “marbled” variety, carries a distinctive pigment distribution pattern resembling that found in some other species, e.g., the Siamese cat and the Himalayan mouse. We tested the hypothesis that the color pattern in question—light-colored body with dark-colored points (ears, face, tail, and feet)—is due to a mutation in the melanin-producing enzyme tyrosinase (TYR) that results in temperature-sensitive pigment production. Our study shows that marbled mink carry a mutation in exon 4 of the TYR gene (c.1835C > G) which results in an amino acid substitution (p.H420Q). The location of this substitution corresponds to the amino acid position that is also mutated in the TYR protein of the Himalayan mouse. Thus, the marbled variant is more aptly referred to as the Himalayan mink.     

Effect of alkaloids from celandine on calcium accumulation and oxidative phosphorylation in mitochondria depending on their DNA intercalating properties

The effect of principal alkaloids (sanguinarine, chelerythrine, coptisine, chelidonine) of greater celandine Chelidonium majus L., as well as the alkaloids from Colchicum autumnale L. (colchicine and colchamine) on calcium accumulation and oxidative phosphorylation in rat liver mitochondria has been studied. The obtained data were compared with DNA intercalating properties of alkaloids detected by the method of thermodenaturation (DNA melting curve plots). It was found that chelerythrine and sanguinarine blocked absorption and accumulation of calcium cations and inhibited oxidative phosphorylation, while the coptisine significantly diminished those indices. Chelidonine, colchicines and colchamine had no influence on the studied characteristics. The effect of alkaloids upon mitochondria functional state correlated tightly with their DNA intercalating properties: chelerythrine and sanguinarine were strong intercalators, while coptisine was a weak one, and chelidonine, colchicine and colchamine did not interact with DNA and caused no changes in its melting point. Correlation coefficient between the intercalating properties of alkaloids and their inhibition of calcium accumulation was 0.89, and with their oxidative phosphorylation inhibition - 0.93. It is suggested that the effect of studied alkaloids upon functional properties of mitochondria can be mediated by mtDNA.     

Interaction of benzo[c]phenanthridine and protoberberine alkaloids with animal and yeast cells

We compared the effects of four quaternary benzo[c]phenanthridine alkaloids – chelerythrine, chelilutine, sanguinarine, and sanguilutine – and two quaternary protoberberine alkaloids – berberine and coptisine – on the human cell line HeLa (cervix carcinoma cells) and the yeastsSaccharomyces cerevisiae andSchizosaccharomyces japonicus var. versatilis. The ability of alkaloids to display primary fluorescence, allowed us to record their dynamics and localization in cells. Cytotoxic, anti-microtubular, and anti-actin effects in living cells were studied. In the yeasts, neither microtubules nor cell growth was seriously affected even at the alkaloid concentration of 100 μg/ml. The HeLa cells, however, responded to the toxic effect of alkaloids at concentrations ranging from 1 to 50 μg/ml. IC₅₀ values for individual alkaloids were: sanguinarine IC₅₀ = 0.8 μg/ml, sanguilutine IC₅₀ = 8.3 μg/ml, chelerythrine IC₅₀ = 6.2 μg/ml, chelilutine IC₅₀ = 5.2 μg/ml, coptisine IC₅₀ = 2.6 μg/ml and berberine IC₅₀ >10.0 μg/ml. In living cells, sanguinarine produced a decrease in microtubule numbers, particularly at the cell periphery, at a concentration of 0.1 μg/ml. The other alkaloids showed a similar effect but at higher concentrations (5–50 μg/ml). The strongest effects of sanguinarine were explained as a consequence of its easy penetration through the cell membrane owing to nonpolar pseudobase formation and to a high degree of molecular planarity. This revised version was published online in July 2006 with corrections to the Cover Date.     

Experimental evaluation of the reliability of placental scar counts in American mink (Mustela vison)

Counting of placental scars in the uterus is a valuable method to estimate female fecundity in wild-ranging mammalian populations, but the reliability of placental scar counts (PSC) has only been thoroughly assessed for a few species. Here we evaluate the reliability of PSC in American mink (Mustela vison) through examination of farm animals with known reproductive history. PSC estimated pregnancy rate and litter size accurately in individuals up to 3 months postpartum. Placental scars were detectable in most female American mink sampled 7–8 months postpartum, but the proportion of minks misclassified as barren was overestimated, and mean litter size was underestimated. We found a large variation in the intensity of pigmentation in placental scars with similar age. Differentiation of scars according to shading as indicating either successful full-term pregnancies or post-implantation loss of embryos was not possible. We propose that these results are applicable for fecundity analysis in American mink and closely related mustelids.     

Mitodepressive effects ofChelidonium alkaloids on root tip cells ofAllium cepa L

A comparison of the effects of a water extract of the fresh vegetation apex ofChelidonium majus L. var.majus, with those of berberine sulphate and of chelidonine hydrochloride is reported. The water extract of fresh stems and leaves ofChelidonium majus and the berberine sulphate solution had marked mitodepressive effects on onion root tip cells. Chelidonine hydrochloride had similar but less marked effects. On the basis of the results obtained it can be assumed that the most active group of substances with cytostatic effects, hindering the cells from entering mitosis, are protoberberine bases contained in the latex ofChelidonium majus. Within the range of the investigated concentrations the extract of fresh stems and leaves was less toxic for the cells than berberine sulphate. The data ascertained provide evidence that the mechanism of the cytostatic action of chelidonine, of berberine, as well as of the extract of fresh stems and leaves ofChelidonium, majus differs from the mechanism of the action of colchicine. Of the testedChelidonium alkaloids only chelidonine produced a partial inactivation of the mitotic spindle.