Phylogeographic patterns of steppe species in Eastern Central Europe: a review and the implications for conservation

The phylogeography of species associated with European steppes and extrazonal xeric grasslands is poorly understood. This paper summarizes the results of recent studies on the phylogeography and conservation genetics of animals (20 taxa of beetles, butterflies, reptiles and rodents) and flowering plants (18 taxa) of such, "steppic" habitats in Eastern Central Europe. Most species show a similar phylogeographic pattern: relatively high genetic similarity within regional groups of populations and moderate-to-high genetic distinctiveness of populations from currently isolated regions located in the studied area. This distinctiveness of populations suggests a survival here during glacial maxima, including areas north of the Bohemian Massif-Carpathians arc. Steppic species generally do not follow the paradigmatic patterns known for temperate biota (south-north “contraction–expansion”), but to some extent are similar to those of arctic-alpine taxa. There are three main groups of taxa within Eastern Central Europe that differ in their contemporary distribution pattern, which may reflect historical origin and expansion routes. Present diversity patterns of the studied steppic species suggest that they share a unique genetic signature and distinct assemblages exist in each of the now isolated areas rich in steppic habitats. At least some of these areas probably act as present “interglacial refugia” for steppic species. This study strongly supports the need to protect steppic species throughout their entire ranges in the region, as the continuous destruction of steppic habitats in some areas may lead not only to the disappearance of local populations, but also to the extinction of unique evolutionary units.     

Effect of amyloid beta peptide on poly(ADP-ribose) polymerase activity in adult and aged rat hippocampus

It is suggested that the fibrillar amyloid beta peptide (A beta) in brain plays a direct role in neurodegeneration in Alzheimer's disease, probably through activation of reactive oxygen species formation. Free radicals and numerous neurotoxins elicit DNA damage that subsequently activates poly(ADP-ribose) polymerase (PARP, EC 2.4.2.30). In this study the effect of neurotoxic fragment (25-35) of full length A beta peptide on PARP activity in adult and aged rat hippocampus was investigated. In adult (4 month old) rat hippocampus the A beta 25-35 peptide significantly enhanced PARP activity by about 80% but had no effect on PARP activity in cerebral cortex and in hippocampus from aged (24-27 month old) rats. The effect of A beta peptide was reduced by half by the nitric oxide synthase inhibitor N-nitro-L-arginine. Stimulation of glutamate receptor(s) itself enhanced PARP activity by about 80% in adult hippocampus. However, A beta 25-35 did not exert any additional stimulatory effect. These results indicate that A beta, through NO and probably other free radicals, induces activation of DNA bound PARP activity exclusively in adult but not in aged hippocampus.     

Phytochemical variability during vegetation of Chamerion angustifolium (L.) Holub genotypes derived from in vitro cultures

The purpose of the study was to evaluate Chamerion angustifolium (L.) Holub genotypes for preliminary selection and further breeding programs aimed at obtaining a suitable industrial form for the pharmaceutical applications. Clonally propagated plants representing 10 genotypes of Ch. angustifolium were regenerated under in vitro conditions, hardened and planted in the field. Studies included an evaluation of shoot proliferation, phytochemical assessment of in vitro and ex vitro plants as well as investigations of intraspecies variability regarding four phenological stages: vegetative, beginning of blooming, full blooming, and green fruit phases. Quantitative and qualitative analyses of bioactive compounds were performed using high-performance liquid chromatography coupled with diode array detector and tandem mass spectrometer (HPLC–DAD–MS/MS) and high-performance liquid chromatography (HPLC) methods. The efficiency of shoot multiplication varied between genotypes from 8.12 to 21.48 shoots per explant. A high reproduction rate (>?20 shoots per explant) was recorded for four lines (PL_45, PL_44, PL_58, DE_2). Plants grown in vitro synthesized oenothein B (11.2–22.3 mg g^?1 DW) and caffeic acid derivatives. Plants harvested from field contained the full spectrum of polyphenols characteristic for this species, and oenothein B and quercetin 3-O-glucuronide were the most abundant. The maximal content of oenothein B was determined in the vegetative phase of fireweed, while some flavonoids were found in the highest amount in full blooming phase. The results of analysis of variance indicated significant differences among genotypes in oenothein B, 3-O-caffeoylquinic acid and flavonoids accumulation in four phenological phases. PL_44 plants were characterized by high content of oenothein B and quercetin 3-O-glucuronide as well as a relatively high level of other flavonoids. Based on our phytochemical and micropropagation studies, PL_44 genotype was the best candidate for early selection and further breeding programs.

     

The effect of polyphenols and vitamin E on the antioxidant status and meat quality of broiler chickens exposed to high temperature

The aim of this study was to determine the effect of a polyphenol product (PP) (Proviox) and vitamin E on the antioxidant status and meat quality of broiler chickens exposed to high temperature. The experimental materials comprised 120 ROSS 308 broilers (6 treatments, 10 replications, 2 birds per replication). Dietary supplementation with vitamin E and PP was applied in the following experimental design: group I (negative control) – without supplementation; group II (positive control) – without supplementation; group III – supplementation with 100 mg vitamin E/kg; group IV – 200 mg vitamin E/kg; group V – 100 mg vitamin E/kg and 100 mg PP/kg; group VI – 200 mg PP/kg. In groups II–VI, broiler chickens aged 21–35 d were exposed to increased temperature (34°C for 10 h daily). In chickens exposed to high temperature, dietary supplementation with antioxidants, mostly PP, improved growth performance parameters, including body weight, body weight gain and feed intake until 28 d of age. Vitamin E added to broiler chicken diets at 200 mg/kg and vitamin E combined with PP was most effective in improving the total antioxidant status of birds, enhancing blood antioxidant enzyme activities and increasing vitamin E concentrations in the liver and breast muscles. Broilers fed diets supplemented with 200 mg/kg of vitamin E alone and vitamin E in combination with PP were characterised by a higher percentage content of breast muscles in the carcass. Dietary supplementation with antioxidants improved the water-holding capacity of meat, reduced natural drip loss and increased the crude ash content of meat. The breast muscles of chickens fed diets supplemented with PP had a lower contribution of yellowness. The breast muscles of chickens receiving diets with 100 mg vitamin E/kg(group III) and diets supplemented with PP (groups V and VI) were characterised by the highest concentrations of polyunsaturated fatty acids. The PP can be a valuable component of diets for broiler chickens exposed to high temperature.     

Selection of low-molecular-mass trypsin and chymotrypsin inhibitors based on the binding loop of CMTI-III using combinatorial chemistry methods

Using a combinatorial chemistry approach, a decapaptide library containing the N-terminal fragment of trypsin inhibitor CMTI-III was synthesized by the solid-phase method. The peptide library was screened for trypsin and chymotrypsin inhibitory activity applying the iterative method in solution. Two decapeptides were selected and resynthesized for each enzyme. The association equilibrium constants ((1.1+/-0.2)x10(8) and (7.3+/-1.6)x10(7)) determined for peptides with trypsin inhibitory activity indicate that they are 3-4-fold less active than the CMTI inhibitors. On the other hand, they are significantly more effective as compared with the starting sequence. Two peptides selected as chymotrypsin inhibitors displayed about 10 times higher activity (1.7+/-0.4)x10(7) and (1.1+/-0.2)x10(7), respectively) than those monosubstituted in position P(1) of the CMTI-III analogue. Considering low molecular weight of peptides selected and the lack of conformational constraints in their structures, the results are promising. They are good templates as starting sequences for further selection of small, peptidomimetic proteinase inhibitors.     

Interaction Between Polyamine and Nitric Oxide Signaling in Adaptive Responses to Drought in Cucumber

The effect of polyamines (PAs) on nitric oxide (NO) generation was investigated in cucumber (Cucumis sativus cv. Dar) primary leaves using bio-imaging with an NO-selective fluorophor, DAF-2DA. Seedlings pretreated with PAs and subjected to water deficit showed early (after 5 h) and transient NO production. The amplitude of the response depended on the form of the applied polyamine. Spermine (1.0 mM) and spermidine (1.0 mM) induced higher NO-dependent fluorescence compared with putrescine (1.0 mM) and the control. The NO production was blocked by tungstate, an inhibitor of nitrate reductase, and partially by an inhibitor of nitric oxide synthase (NOS-like) enzymes. NO donor administration preceding drought had no effect on endogenous PA levels but was positively correlated with an alleviation of water deficit-induced membrane permeability and lipid peroxidation. Application of 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO), a membrane-permeable NO scavenger, markedly reversed the NO donor effects. Similarly, pretreating seedlings with PAs resulted in lower ion leakage from the membrane and modified lipid peroxidation. The results indicate that NO may act downstream of PAs in cucumber seedlings under water stress.     

Perturbation of the lipid phase of a membrane is not involved in the modulation of MRP1 transport activity by flavonoids

The expression of transmembrane transporter multidrug resistance-associated protein 1 (MRP1) confers the multidrug-resistant phenotype (MDR) on cancer cells. Since the activity of the other MDR transporter, P-glycoprotein, is sensitive to membrane perturbation, we aimed to check whether the changes in lipid bilayer properties induced by flavones (apigenin, acacetin) and flavonols (morin, myricetin) were related to their MRP1 inhibitory activity. All the flavonoids inhibited the efflux of MRP1 fluorescent substrate from human erythrocytes and breast cancer cells. Morin was also found to stimulate the ATPase activity of erythrocyte ghosts. All flavonoids intercalated into phosphatidylcholine bilayers as judged by differential scanning calorimetry and fluorescence spectroscopy with the use of two carbocyanine dyes. The model of an intramembrane localization for flavones and flavonols was proposed. No clear relationship was found between the membrane-perturbing activity of flavonoids and their potency to inhibit MRP1. We concluded that mechanisms other than perturbation of the lipid phase of membranes were responsible for inhibition of MRP1 by the flavonoids.     

Arene- and quinoline-sulfonamides as novel 5-HT7 receptor ligands

Novel arene- and quinolinesulfonamides were synthesized using different solutions and a solid-support methodology, and were evaluated for their affinity for 5-HT(1A), 5-HT(2A), 5-HT(6), and 5-HT(7) receptors. Compound 54 (N-Ethyl-N-[4-(1,2,3,4,4a,5,6,7,8,8a-decahydroisoquinolin-2-yl)butyl]-8-quinolinesulfonamide) was identified as potent 5-HT(7) antagonist (K(i)=13 nM, K(B)=140 nM) with good selectivity over 5-HT(1A), 5-HT(2A), 5-HT(6) receptors. In the FST in mice, it reduced immobility in a manner similar to the selective 5-HT(7) antagonist SB-269970.