Unusual Structures and Cytotoxicities of Chitonoidosides A,A1, B,C, D,and E,Six Triterpene Glycosides from the Far Eastern Sea Cucumber Psolus chitonoides

Six new triterpene tetra-, penta- and hexaosides, chitonoidosides A (1), A₁ (2), B (3), C (4), D (5), and E (6), containing one or two sulfate groups, have been isolated from the Far-Eastern sea cucumber Psolus chitonoides, collected near Bering Island (Commander Islands) from the depth of 100–150 m. Three of the isolated compounds (1, 3 and 6) are characterized by the unusual aglycone of new type having 18(20)-ether bond and lacking a lactone in contrast with wide spread holostane derivatives. Another unexpected finding is 3-O-methylxylose residue as a terminal unit in the carbohydrate chains of chitonoidosides B (3), C (4), and E (6), which has never been found before in the glycosides from holothurians belonging to the Psolidae family. Moreover, this monosaccharide is sulfated in the compound 4 into unprecedented 3-O-methylxylose 4-O-sulfate residue. Chitonoidoside C (4) is characterized by tetrasaccharide moiety lacking a part of the bottom semi-chain, but having disaccharide fragment attached to C-4 of Xyl1. Such architecture is not common in sea cucumber glycosides. Cytotoxic activities of the compounds 1–5 against mouse and human erythrocytes and human cancer cell lines: adenocarcinoma HeLa, colorectal adenocarcinoma DLD-1, and leukemia promyeloblast HL-60 cells were studied. The cytotoxic effect of chitonoidoside d (5) was the most significant in this series due to the presence of pentasaccharide disulfated sugar chain in combination with holostane aglycone. Surprisingly, the glycosides 1 and 3, comprising the new aglycone without γ-lactone, demonstrated similar activity to the known compounds with holostane aglycones. Chitonoidoside C (4) was less cytotoxic due to the different architecture of the carbohydrate chain compared to the other glycosides and probably due to the presence of a sulfate group at C-4 in 3-O-MeXyl4.     

Development of a HPLC/tandem-MS method for the analysis of the larvicides methoprene, hydroprene, and kinoprene at trace levels using Diels-Alder derivatization

The invasion and subsequent spread of the mosquito-borne West Nile virus in the United States has resulted in increased use of methoprene. With the increased need for sensitive detection and monitoring of methoprene in the environment, an analytical LC/ESI-MS/MS method has been developed for the analysis of methoprene and two analogues, kinoprene and hydroprene, in water. To improve the ionization efficiency of the nonpolar analytes, a derivatization step with the Cookson-type reagent 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD) was used. Derivatization improved the limit of detection 100-fold. For tandem MS analyses, limits of detection in environmental water samples (S/N = 3) are about 6 pg/mL for methoprene and 20 pg/mL for kinoprene and hydroprene, resulting in limits of quantification (S/N = 10) of 20 pg/mL for methoprene and 60 pg/mL for hydroprene and kinoprene extracted from 10 mL of water. This method was applied to measure methoprene concentrations in water samples from a treated site.     

Similarity of fine specificity of IgA anti-gliadin antibodies between patients with celiac disease and humanized α1KI mice

Gliadins, and primarily α-gliadins containing several sequences such as aa 31-49, aa 56-88 (33-mer), aa 57-68, and aa 69-82, are critical in the induction of immune response or toxic reaction leading to the development of celiac disease (CLD). The role of IgA anti-gliadin antibodies (IgA AGA) is unknown. To this end, we prepared several humanized monoclonal IgA AGA using transgenic α1KI mice. Employing Pepscan with overlapping decapeptides of α-gliadin we observed a robust similarity between the specificity of humanized mouse monoclonal IgA AGA and IgA AGA from patients with florid CLD. The common immunodominant region included several sequential epitopes localized in the N-terminal part of α-gliadin (QFQGQQQPFPPQQPYPQPQPFP, aa 29-50, and QPFPSQQPYLQL, aa 47-58). Notably, IgA AGA produced by clones 8D12, 15B9, 9D12, and 18E2 had significant reactivity against sequences localized in the 33-mer, LQLQPFPQPQ (aa 56-65) and PQLPYPQPQPFL (aa 69-80). Humanized mouse monoclonal IgA AGA that have a known specificity are suitable as standard in ELISAs to detect serum IgA AGA of CLD patients and for studying the AGA pathogenic role in CLD, especially for analyzing the translocation of complex of specific IgA antibodies and individual gliadin peptides through enterocyte barrier.     

Molecular evidence of genetic diversity changes in pea (<Emphasis Type="Italic">Pisum sativum</Emphasis> L.) germplasm after long-term maintenance

Maintaining germplasm genetic integrity is a key objective of long-term ex situ conservation. Periodic regeneration, performed on limited plots with small number of individuals, increases the risk of genetic drift and genetic diversity changes. In this study, six accessions of white flowered, dry seed pea varieties (Pisum sativum L. subsp. sativum var. sativum): Bohatyr, Klatovsky zeleny, Hanák, Moravsky hrotovicky krajovy, Raman and Viktoria-75 and four accessions of colour flowered, fodder pea (P. sativum subsp. sativum var. arvense (L.) Poiret: Arvika, Česky banán, Moravská krajová and Niké, representing Czechoslovak varieties and landraces, bred over the last 40–80 years, were analyzed using ten microsatellite locus specific markers. Each accession was represented by 20 individual seeds of two temporally different samples, spanning the period of 20 or 40 years. Together with intra-accession variation (except of cv. Hanák), evidence of genetic changes, e.g. differences in allele frequencies as well as genetic composition of sample, was detected in six out of ten accessions (Arvika, Bohatyr, Česky banán, Moravsky hrotovicky krajovy, Moravská krajová and Raman). Evidence of genetic erosion was found in three accessions (Česky banán, Moravsky hrotovicky krajovy and Raman), while in another three (Arvika, Bohatyr and Moravská krajová) the level of diversity was found to have increased. Moreover in three samples of Bohatyr (2004) and Klatovsky zeleny (1963 and 2004), low levels of heterozygosity was detected. These results demonstrate that in pea, a self-pollinating and highly homozygous plant, the danger of the loss of genetic integrity exists. These findings are significant for long-term ex situ germplasm management.     

A new genus and two new species of Anteroporidae (Cestoda: Lecanicephalidea) from the darkspotted numbfish, Narcine maculata (Torpediniformes: Narcinidae), off Malaysian Borneo

The cestode fauna of the darkspotted numbfish, Narcine maculata (Shaw) (Torpediniformes: Narcinidae), from Malaysian Bomrneo was examined for the first time. This work resulted in the discovery of a new genus and two new species of Anteroporidae (Lecanicephalidea). Sesquipedalapex comicus gen. n., sp. n. was erected on the basis of the peculiarities of its scolex, in particular its possession of an extremely long apical modification of the scolex proper, which readily distinguishes it from the other genus in the family. The genus is also distinct in its possession of acetabula that are in the form of suckers, rather than bothridiate in form. This species was found to deeply embed its elongate apical structure for much of its length within the intestinal mucosa, provoking a papilliform expansion of the outer wall of the spiral intestine at the site of attachment. The second new species, Anteropora klosmamorphis sp. n., is readily distinguished from its congeners on the basis of testis number and bothridial shape. Both new species are hyperapolytic. The diagnosis of Anteroporidae is amended to accommodate both new taxa. This increases the total number of genera in the family to two, and the total number of species to five.     

The distribution and spreading pattern of Dermacentor reticulatus over its threshold area in the Czech Republic--how much is range of this vector expanding?

Host-seeking Dermacentor reticulatus ticks were detected by flagging method at 46 localities at south-east part of the Czech Republic, in the basins of rivers Morava and Dyje. Exact north-west distribution limits of D. reticulatus were defined in this area for the first time. Detailed prediction map of probabilities of D. reticulatus occurrence was obtained using GIS analysis. Spatial model delimited a south-north gradient in probability across the studied area, with highest probabilities above 0.8 in its southernmost part. Abundance of D. reticulatus varied markedly between localities in interval 0.33-222 of ticks per flag per hour. The highest abundances were in flooded areas at lower streams, towards upper streams abundance and density of these ticks decreased. Females prevailed in samples with population sex ratio of 0.413, significantly deviating from parity. Larvae and nymphs of this species were not detected by flagging. Although D. reticulatus range expansion probably did not reach such a degree as reported in other countries, these ticks became very abundant in some parts of studied area. Since spreading of vector-borne diseases became a problem in Europe, the knowledge of their exact recent geographic ranges is important for future modelling of their shift predictability.     

West-to-east differences of Babesia canis canis prevalence in Dermacentor reticulatus ticks in Slovakia

Babesia canis canis is the most frequent causative agent of canine babesiosis in Central Europe, frequently causing severe disease. Recently, many new endemic foci of this disease have been reported from European countries. Growing incidence of canine babesiosis was recorded also in Slovakia during the last decade, from first cases in eastern Slovakia ten years ago to recent cases all over the south of the country. We have used nested PCR-RFLP method to study prevalence of B. c. canis in its natural tick vector Dermacentor reticulatus, collected at three geographically isolated lowland areas of southern Slovakia situated in the southeast, southwest, and west of Slovakia, respectively. The highest prevalence of B. c. canis was observed in D. reticulatus from eastern Slovakia (14.7%; n=327), whereas the prevalence in southwest was significantly lower (2.3%; n=1205). Notably, all 874 D. reticulatus ticks collected at Záhorská ní?ina lowland (W Slovakia) were B. c. canis-negative. Recorded differences in Babesia prevalence concurs well with the shift in incidence of clinical cases of canine babesiosis as observed by vet practitioners. Presented results revealed that eastern Slovakia represents an area of high risk of B. c. canis infection, whereas western areas of the country still remain Babesia canis-free.     

Spatial and volume patterns of an unmanaged submontane mixed forest in Central Europe: 160 years of spontaneous dynamics

The long-term development of stand characteristics and tree spatial patterns (TSP), their mutual relation, and linkage to site and tree species were studied in the Boubín primeval forest (protected since 1858). Surveys were carried out in 1851, 1961/1964, 1972, 1984/1989, 1996 and 2010 on one to six research plots sized 0.58-1.00 ha. To see how results from these surveys could be generalized, results were also compared with whole-area data sets (46 ha) from 1972 to 2010.The proportion of Abies alba continually decreased, from 20% in 1851 to 2-3% in 2010. This decrease started no later than at the beginning of the 20th century. In contrast, the proportion of Fagus sylvatica slightly increased. The sum of dead and living wood volume was stable during the 159 year period, with deviations of only up to 5%. From 1961/64 to 2010, the number of living trees continually decreased, but the mean-tree volume and volume of dead wood increased. The distribution of dead wood always differed from the distribution of living trees.A random TSP was always most common, which seems to be typical for this type of forest. This was true even when A. alba was gradually forced out from the stand structure. Nevertheless, the pronounced decrease of A. alba found during the 1961/64 survey compared with 1851 was reflected in a tendency towards clustering, as gaps from A. alba dieback gradually closed. In these gaps, competitive pressure was lower, resulting in more clustered distributions. Regular distribution was recorded only rarely. F. sylvatica maintained a random or clustered TSP over long periods, depending on site conditions. On water-affected plots, it had a consistently higher tendency towards clustering. The only step change in TSP occurred due to Hurricane Emma in 2008. Otherwise, the most marked TSP changes over time were found for Picea abies, in which there was a gradual trend from clustered to random, connected with a decreasing number of individuals.The main reason for the decline of A. alba and the decreased number of living trees from 1961/64-2010 was the on-going effect of a high stock of red deer at the turn of the 19th and 20th centuries. The decreasing trend in the number of living trees showed no marked change, even 60 years after the game pressure was strongly reduced. However, the vertical structure became increasingly homogenized due to the long-term absence of severe abiotic and/or biotic disturbances.     

The influence of flesh colour and growing locality on polyphenolic content and antioxidant activity in potatoes

Compounds contained in potato tubers that may act as antioxidants have been attracting increased attention. The main antioxidants in potatoes are polyphenolic compounds. The total polyphenolic (TP) content and antioxidant activity (AA) in potato tubers of four yellow-fleshed cultivars and two purple-fleshed cultivars from the 2004 harvest were tested. The total polyphenolic content was determined using the Folin-Ciocalteau reagent (FC) and the antioxidant activity, with the DPPH method using stable free radical 1,1-diphenyl-2-picrylhydrazyl (DPPH·). Significant differences were found in total polyphenolic content between the yellow-fleshed cultivars Karin, Impala, Ditta, and Saturna. Significant differences between yellow and purple-fleshed potatoes were found: the yellow-fleshed cultivars had an average of 2.96 g gallic acid equivalents (GAE) per kg DM, the purple-fleshed cultivars 4.68 g GAE per kg DM. Significant differences in AA between the yellow and purple-fleshed cultivars were determined (yellow-fleshed in an average of 139.3 mg equivalents of ascorbic acid [EAA] per kg DM, purple-fleshed 332.3 mg EAA per kg DM) as well as between the purple-fleshed cultivars Valfi (298 mg EAA per kg DM) and Violette (366 mg EAA per kg DM). The major antioxidants in purple-fleshed cultivars were anthocyanidins petunidin (Valfi 92%_rel) and malvidin (Violette 85%_rel). Cv. Violette contained three times higher content of anthocyanidins than cv. Valfi. Among four experimental localities, the higher situated potato-growing region of Stachy (locality with the lowest average annual and daily minimum temperatures and the highest sum of precipitation in the vegetative period) differed significantly from other localities by a higher TP content, AA and yield in the analyzed cultivars. A high positive correlation between antioxidant activity and total polyphenolic content with R = 0.8648, R² = 0.7478 (P = 0.0001) was found.     

Land use effects on carbon quality and soil biological properties in Eutric Cambisol

Abstract

The choice of prospective type of farming requires knowledge about the specific relationships that exist between farm management practices and base environmental conditions. Nowadays the protection of soil organic carbon is one of the main tasks, because organic carbon in addition to soil fertility can act in elimination of soil contamination and carbon sequestration. Field experiments were focused on the effect of intensive farming without organic inputs versus grassland on organic carbon content. Organic carbon content (C_ox) and humic substance fractions (C-humic acids and fulvic acid fractions), hot water extractable carbon and selected microbial characteristics in Eutric Cambisol were monitored during the period 1999–2010. A priming effect of soil cultivation was detected immediately after tillage. Arable soil with ‘intensive’ crop sequences (exclusively cash crops, cereals, oil plants) and with an optimal level of chemical inputs (mineral fertilizers, pesticides), but without organic farmyard manure had lower content of all carbon forms compared with grassland. ¹³C NMR spectroscopy and thermal analysis (TGA) were applied to characterize humic acid (HA) structure and stability. More carbon, less oxygen and more aromatic compounds were detected in grassland HA. Slight differences were found in HA thermo-oxidative stability and degradability, which was probably caused by changes in elemental composition and structure. Even the land use had no significant effect on basic microbiological characteristics (basal respiration, microbial biomass and qCO₂); the physiology of the microbial community of grassland was altered by a higher ability to utilize L- and D-glutamic acid. The L/D ratio of glutamic acid mineralization indicated no occurrence of stress in soil for both types of farming. It has been demonstrated that although losses of carbon as a result of land-use conversions are generally more rapid, gains of carbon in grassland followed by changes in management practices can also occur.