Volume 67, Issue 2

PP1, PP2A and PP2B all belong to PPP family of serine/threonine protein phosphatases. Their primary structures are highly conserved, particularly in the catalytic domain. In order to obtain correlative information about this conserved region, we constructed N-, C-deletion and N/C double-deletion mutants. We found that the N- and Csingle-deletion mutants exhibited higher enzymatic activities, while specific activity of N/C double-deletion mutant PP1 (9-306) did not notably change. The results of kinetics analysis showed that kcat and kcat/Km increased about 16-fold in the single-deletion mutants; while the two parameters of the double-deletion were lower than the single-deletions. We further explored stability of all mutants in existing denaturant guanidine hydrochloride (GdnHCl). It was noticeable that stability of PP1-(9-306) in all mutants was the highest. We speculated that PP1-(9-306) maybe retains a compact spherical structure, thus accordingly affected molecular catalysis. On the other hand the structures of single-deletion mutants were relatively relaxed, which were able to bind substrate easily, so activities of single-deletion mutants were higher than that of double-deletion mutant. We therefore deduced that PP1-(9-306) may be close to core region of PP1 molecule. In order to further solidify this idea, we used fluorescence spectra method to explore changes of space conformation. We found that emission peaks of all single-deletions were blue shifted in different degree in the absence of denaturant, while emission peak of N/C double-deletion mutant did not change obviously compared with that of the wild-type PP1. Conformation change of N/C double-deletion mutant was significantly less than those of single-deletion mutants in different GdnHCl concentration.

The first record of Coniochaeta prunicola Damm & Crous (Coniochaetales, Sordariomycetes, Ascomycota) as a pathogen of Persica vulgaris Mill. described and illustrated from Nitra district, Slovakia. C. prunicola was isolated from symptomatic leaves of host tree and based on morphological attributes identified for the first time as a causative agent of peach tree damage. C. prunicola is characterized by dark brown ascomata clothed with setae, the fasciculate, unitunicate, cylindrical asci and broadly almond-shaped, ellipsoidal ascospores with a longitudinal germ slit.

Hieracium nigrescens subsp. brachytrichellum Zahn, known to date only from the Southern Carpathians, was discovered in the Vranica Mountains in Bosnia. This is the first station of H. nigrescens s.l., an arctic-alpine species of H. sect. Alpina (Griseb.) Gremli, on the Balkan Peninsula. A map of general distribution of H. nigrescens s.l. and illustration of the specimens collected in Bosnia are given. The likely origin of the Balkan population is briefly discussed.

Morphological, reproductive and karyological variability were investigated in fifteen populations of Allium oleraceum transferred from natural populations in Lithuania to the field collection of medicinal and aromatic plants of the Institute of Botany of the Nature Research Centre in Vilnius. Nine populations were tetraploid (2n = 4x = 32), four pentaploid (2n = 5x = 40) and two consisted of both cytotypes. The greatest differences among the populations and between the ploidy levels were observed in stem height and mass of aerial bulbils per plant. The seed production was very low and did not differ significantly between the ploidy levels, while neither tetraploids nor pentaploids did produce any seed if insects were prevented from visiting their flowers. The means of the morphological characters were higher in pentaploids than in tetraploids. However, none of them provided the possibility of determining ploidy level without chromosome counting.

Cytological analyses were performed in nineteen accessions belonging to seven Achillea species. The results indicate that two species, A. filipendulina and A. tenuifolia, are diploid (2n = 2x = 18) and three species, A. bieberstinii, A. pachycephala, and A. aucheri are tetraploid (2n = 4x = 36). For two latter species, the chromosome numbers are new. Two ploidy levels of 2n = 6x = 54 and 2n = 8x = 72 in A. millefolium and two ploidy levels of 2n = 2x = 18 and 2n = 4x = 36 in A. santolina species were found. Cluster analysis based on chromosomal characteristics and karyotype asymmetry indices clustered the studied genotypes into three major groups. The first group included the diploid species (A. filipendulina, A. santolina and A. tenufolia), while the second group comprised tetraploid species (A. santolina, A. aucheri and A. pachycephala). A. millefolium (hexaploid, octaploid) and A. bieberstinii (tetraploid) were classified in the third group. Total form percentage (TF%) in groups 1, 2 and 3 were 42.03, 42.15 and 41.08, respectively. Group 1 possessed the highest average of symmetry index (S% = 70.8). Stebbins classification method grouped all accessions in class A. Moreover, group 1 (diploid level species), had the most symmetric karyotype from point of view of centromere and chromosome length. Therefore, it can be concluded that the genotypes belonging to group 1 are the earliest evolutionary form.

The karyotype of fifteen Leucocoryne taxa was studied, assessing characteristics such as chromosome morphology and size, secondary constriction location, and asymmetry level. Two groups of Leucocoryne taxa were described based on chromosome number (2n = 10 and 2n = 18) and karyotype asymmetry. The haploid karyotype formula for the group 2n = 10 was 3m + 2st (or 2t), whereas for the group 2n = 18 was 7m + 2st (or 2t). Such results corroborate the karyotype descriptions previously carried out for some taxa of the genus. Leucocoryne taxa showed a high resemblance in chromosome morphology, but inter-specific differences were found in mean chromosome size. These data and previous studies based on gross chromosome morphology support Crosa’s hypothesis, which suggests that the cytotype 2n = 10 is diploid and perhaps ancestral, whereas that the cytotype 2n = 18 is tetraploid but with an additional chromosome fusion being probably a derived status.

The paper presents results of morphometric analysis of Anthyllis vulneraria L. in Poland based on 828 herbarium specimens. This study investigates five taxa which have been recognized in Poland: Anthyllis vulneraria L. ssp. vulneraria, Anthyllis vulneraria ssp. polyphylla (DC.) Nyman, A. vulneraria ssp. maritima (Schweigg.) Corb., A. vulneraria ssp. alpestris Asch. et Graebn. and A. vulneraria ssp. carpatica (Pant.) Nyman as well as three intermediate taxa of presumably hybrid origin. Multivariate statistical analyses of 12 quantitative and 10 qualitative characteristics revealed conspicuous variation within A. vulneraria. Analysis of variance (ANOVA), principal coordinate analysis (PCoA), correspondence analysis (CA) and discriminant analysis (DA) proved the taxonomic usefulness of quantitative characteristics such as number of rosette leaves, calyx length, inflorescence length, the ratio of leaf length/width, bract length as well as qualitative characteristics such as stem hairiness, calyx colour, calyx indumentum, distances between stem leaves and form of rosette leaves. The issues concerning intermediate morphotypes occurring in Poland and their status are discussed. Finally, a key for determination of taxa within A. vulneraria in Poland and distribution maps based on the material examined are provided.

The deciduous forests represent dominant natural vegetation of Central European landscape and an important functional component for maintenance of biological diversity. However, their syntaxonomy and ecological gradients still remain unclear. The numerical classification was conducted to determine the main units of forest vegetation, while ordination techniques were used to explain the structure of vegetation-environmental data matrix consisting of 110 forest stands in the Štiavnické vrchy Mts (central Slovakia). Ten vegetation types within the phytosociological classes of deciduous forests Quercetea robori-petraeae and Querco-Fagetea were distinguished. The major environmental driver responsible for variation in forest species composition was interpreted as a response to soil moisture which also accounted for a large part of species variability (3.74%). Soil nutrient/acidity complex expressed by pH, Ca and Al concentration was also an important source of vegetation variability. Relevance of soil conditions in relation to plant survival and community distribution was discussed. Along the soil moisture gradient, vegetation types were arranged from the subxerophilous oak forests through the mesophilous beech and ravine forests to the hygrophilous alder ash vegetation.

Ecological models have often been used in order to answer questions that are in the limelight of recent researches such as the possible effects of climate change. The methodology of tactical models is a very useful tool comparison to those complex models requiring relatively large set of input parameters. In this study, a theoretical strategic model (TEGM) was adapted to the field data on the basis of a 24-year long monitoring database of phytoplankton in the Danube River at the station of Göd, Hungary (at 1669 river kilometer - hereafter referred to as “rkm”). The Danubian Phytoplankton Growth Model (DPGM) is able to describe the seasonal dynamics of phytoplankton biomass (mg L−1) based on daily temperature, but takes the availability of light into consideration as well. In order to improve fitting, the 24-year long database was split in two parts in accordance with environmental sustainability. The period of 1979–1990 has a higher level of nutrient excess compared with that of the 1991–2002. The authors assume that, in the above-mentioned periods, phytoplankton responded to temperature in two different ways, thus two submodels were developed, DPGM-sA and DPGM-sB. Observed and simulated data correlated quite well. Findings suggest that linear temperature rise brings drastic change to phytoplankton only in case of high nutrient load and it is mostly realized through the increase of yearly total biomass.

By generating stress conditions soil flooding can induce alterations in the cell metabolism and thus is detrimental to plant growth. This study was done under the greenhouse conditions to determine the effect of soil flooding on the chlorophyll fluorescence of some hybrids of canola (Brassica napus L.). Fifty five days old plants were subjected to flooding for six days. There was no difference in the parameters modulate chlorophyll fluorescence, in contrast, some the parameters related to the energy flux in photosystem II varied due to flooding stress. At the end of the six days, the performance indexes (PItotal and PIABS) decreased, in all hybrids except in ‘Hyola 420’. The difference kinetics of the chlorophyll a fluorescence transient showed different effects on different sites of the photosynthetic machinery. It could be concluded that compared to the other hybrids, ‘Hyola 420’ was less sensitive to flooding.

The self-incompatibility of tea plant (Camellia sinensis (L.) O. Kuntze) was studied with the methods of aniline blue fluorescence assay and paraffin sections. The characteristics of pollen tube elongation after hand pollination was analyzed in 4 tea cultivars, including ‘Keemenzhong’, ‘Longjing-changye’, ‘Fuding-dabaicha’ and ‘Yabukita’, under self-pollination and cross-pollination, respectively. Although there were some difference among cultivars, pollen tubes elongated through the style and reach the ovary successfully at 48 h after pollination for both cross- and self-pollen tubes in all the four cultivars of tea. Pollen tubes entered into the ovule micropyles, however, only for cross-pollination, but not for self-pollination. Pollen tubes of selfing plants, failed in fertilizing, seemed have some difficulties to enter the ovule. All of which indicated that the self-incompatibility of tea plant is a late-acting self-incompatibility system (LSI) or an ovarian sterility (OS), in which the self incompatibility was due to none self pollen tube penetrating into the ovule and no fertilization.

Tropane alkaloids (TAs) including hyoscyamine, anisodamine, anisodine and scopolamine are widely used as anticholinergic agents, which act on the parasympathetic nervous system, and their clinical demands are increasing rapidly. Anisodus acutangulus is a solanaceous perennial plant that is endemic to China with good potential for TA production and classified as an endangered species in China. Therefore there is a need to improve the TA production in this resource plant by biology methods. In our research, three different important elicitors including methyl jasmonate (MJ), yeast elicitor (YE) and abscisic acid (ABA) were used to study their effects on TA production, and genes expressions in A. acutangulus plant under each treatment were examined. The results revealed that TA production was induced by all the three treatments. However, its quantity varies with inducers used. The effect induced by MJ and YE were significant, while the effect induced by ABA was not obviously. To our knowledge, this is the first report on the relationship between TA production and genes expression profile induced by different elicitors in cultivated A. acutangulus plant, which should provide some useful information on how to improve TA production with the cultivated A. acutangulus plant in the near future.

Cyclin-dependent kinases (CDKs) play an essential role in cell cycle regulation during the embryonic and postembryonic development of organisms. To better understand the molecular mechanisms of CDKs involved in embryogenesis regulation in the endangered medicinal plant Dendrobium candidum Wall. ex Lindl., a 1229-bp full-length cDNA of an A-type CDK gene, Denca;CDKA;1, was identified using 3′ rapid amplification of cDNA end (RACE) PCR. Denca;CDKA;1 was predicted to encode a 294 amino acid residue-long protein of 33.76 kDa with an isoelectric point of 7.72. The deduced Denca;CDKA;1 protein contained a conserved serine/threonine-protein kinase domain (S-TKc) and a canonical cyclinbinding “PSTAIRE” motif. Multiple sequence alignment indicated that members of CDKA family from various plants exhibited a high degree of sequence identity ranging from 82% to 93%. A neighbor-joining phylogenetic tree showed that Denca;CDKA;1 was clustered into the plant group and was distant from the animal and fungal groups. The modeled three-dimensional structure of Denca;CDKA;1 exhibited the similar functional structure of a fold consisting of β-sheets and α-helices joined by discontinuous random coils forming two relatively independent lobes. Quantitative real-time PCR analysis revealed that Denca;CDKA;1 transcripts were the most abundant in protocorm-like bodies with 4.76 fold, followed by that in roots (4.19 fold), seeds (2.57 fold), and stems (1.57 fold). This study characterized the novel Denca;CDKA;1 gene from D. candidum for the first time and the results will be useful for further functional determination of the gene.

This article provides an ultrastructural atlas of microsporogenesis in the tobacco model line SR1. The stages of cell-wall remodeling and reorganization of the intercellular channels, accompanying this process, are reported for the microspore mother cells. The meiotic changes in the cell nucleus and cytoplasm are traced. The appearance of single-, double-, or multi-membrane nuclear vacuoles in microspore mother cells and their further elimination from the nucleus are for the first time described for the genus Nicotiana as well as deviations from a normal course for this process. Intercellular chromatin migration (cytomixis) was observed in the microsporogenesis of the line SR1 and behavior of the nuclear vacuoles within the cytomictic nucleus was described for the first time. The enzymatic activity of spherosome-like vesicles in the tobacco microsporogenesis is discussed. The features of microsporogenesis in the tobacco line SR1 are compared with those of other plant species and its association with the transition from a diploid to a haploid phase of the life cycle is discussed.

A microcosm experiment was used to examine the response of nematode in terms of density and diversity at different levels of permethrin contamination. The sediments were contaminated with three permethrin concentrations [P1: low (5 mg kg−1), P2: medium (25 mg kg−1) and P3: high (250 mg kg−1)] and the effects were evaluated after 30 days. The results from univariate and multivariate analyses showed significant differences between nematode assemblages from uncontaminated control and those from permethrin treatments. All univariate indices changed significantly at all the levels of permethrin contamination. In fact, the total nematode abundance (I), Shannon-Weaner index (H′), species richness (d), evenness (J′) and number of species (S) decreased significantly in all the contaminated microcosms. In addition, the results from multivariate analyses of the species abundance data demonstrated that permethrin affects the responses of nematode species. These significant modifications in nematode community structures with response to permethrin contamination were the consequences of a different specific tolerance to this pesticide. Thus, Araeolaimus bioculatus, Calomicrolaimus honestus, Oncholaimus campylocercoides and Theristus pertenuis characterized by increased abundances in all treated replicates, appeared to be “permethrin-resistant” species. Daptonema trabeculosum was eliminated in all the doses tested and seemed to be a very sensitive species to permethrin contamination.

The aim of our study is to describe and visualise the spread of two non-indigenous land snail species Cepaea nemoralis and Monacha cartusiana in the Czech Republic during more than 100 years period. Several factors play an important role in changes of the distribution of these species: ecological (climate change), ethological (passive dispersal potencial) and economic (increasing traffic as a vector of spreading). The spreading of M. cartusiana has a rapidly increasing trend. More than half sites in the Czech Republic were colonised by this species in 2000–2010. While the spread of C. nemoralis has been continuous during the last century, the rapid range extension was recorded in the last two decades.

Ecological research of gamasid mites was carried out in three natural forest ecosystems with Picea abies (1350 m a.s.l.), Abies alba (950–1000 m a.s.l.) and Fagus sylvatica (1200 m a.s.l.) situated in the Bucegi Massif, Southern Carpathians, Romania during 2001–2003. In the studied forest ecosystems, the taxonomic structure of gamasids was represented by 11 families (Epicriidae, Parasitidae, Veigaiidae, Ameroseiidae, Aceosejidae, Rhodacaridae, Macrochelidae, Pachylaelapidae, Laelapidae, Eviphididae and Zerconidae), with 39 genera and 97 species. The maximum number of species was recorded in the ecosystem with Abies alba (80), followed by the ecosystem with Fagus sylvatica (73), while the minimum in the ecosystem with Picea abies (68). 2,016 samples, 97 species with 23,441 individuals were analysed. In order to show similarities between predator mite populations from these areas, the vegetation and some abiotic parameters of the soil (temperature, humidity, pH) were analysed and described. Similarities between gamasid mite populations from the investigated ecosystems were analysed using the Jaccard index (q). The highest similarity index (q = 0.706) was recorded between gamasids identified in Picea abies and Abies alba forests, at soil level, in comparison with those from Abies alba and Fagus sylvatica forests (q = 0.656). Based on the similarity index, it is discussed that the gamasid population structure was influenced by the type of soil, the specific composition of the herbaceous, shrub and tree layers, the microclimatic factors, all characteristic for each studied ecosystem.

Toxoptera aurantii (Homoptera: Aphididae) is one of the most important pests of many agricultural plants such as camellia, cocoa and coffee worldwide. The culturable bacterial flora of the pest was determined to find new microbes that can be used as biocontrol agent against T. aurantii. A total of six bacteria were isolated and identified by a variety of morphological, physiological, biochemical and molecular tests. In addition, an approximately 1,150 bp fragment of the 16S rRNA gene region was sequenced to verify isolate identification. According to the identification studies, the isolates were identified as Bacillus tequilensis Cb1, Chryseobacterium stagni Cb2, Pseudomonas flourescens Cb3, Rahnella aquatilis Cb4, Staphylococcus sp. Cb5 and Cb6. Pathogenicity of the bacterial isolates were carried out against the last instar nymphs of T. aurantii. The highest activity was obtained from Pseudomonas flourescens Cb3 with 50% mortality within 10 days after application (p<0.05). Mortalities of other treatments ranged from 6.6 to 20%. The results presented here show that Pseudomonas flourescens Cb3 appears to be a significant candidate as a possible biocontrol agent against T. aurantii and should be beneficial in the future biocontrol programs of the pest.

Herpes simplex virus type 2 (HSV-2) is the major cause of genital herpes in humans. The glycoprotein D of HSV-2 (gD2) is a promising subunit vaccine candidate for the treatment of genital herpes. The aim of the present study was to express a biologically active recombinant gD2 in eukaryotic baculovirus system in quantities sufficient for further studies. Human cDNA encoding a gD2 protein with 393 amino acids was subcloned into the pFastBac HTb vector and the recombinant protein was expressed in Spodoptera frugiperda (Sf9) cells by high-density cell culture. In a stirred bioreactor, the key limiting factors including glucose concentration, glutamine concentration and dissolved oxygen (DO) were optimized for high-density cell growth. The Sf9 cell density could reach 9.6×106 cells/mL and the yield of recombinant gD2 protein was up to 192 mg/L in cell culture under the optimal conditions of 15 mM glucose, 0.4 g/L glutamine and 40% DO. Production of significant amounts of pure, full-length gD2 opened up the possibility to investigate novel functions of gD2. Moreover, the purified recombinant gD2 protein revealed a partial prophylactic immune function in genital herpes of guinea pigs infected with HSV-2.

Distal parts of the fish intestine were analyzed for presence of bifidobacteria and lactobacilli using selective agars. Seventy seven samples from Cyprinus carpio, Oncorhynchus mykiss, Carassius auratus, Tinca tinca, Perca fluviatilis, Rutilus rutilus, Scardinius erythrophthalmus, Oreochromis niloticus, and Squalius cephalus were collected randomly throughout years 2008 and 2009. Bifidobacteria were detected in 5 samples from 4 fish species at counts 2.18–4.29 log CFU/g, lactobacilli were present in 6 fish species at counts 1.21–3.65 log CFU/g. Seven bifidobacterial isolates were identified to the species level using biochemical tests and by sequencing of 16S rRNA gene. Three strains belonged to species B. longum, two isolates were identified as B. dentium, one strain as B. asteroides and one isolate was not determined to the species level by employed methods. As identified bifidobacterial species are considered to be of human, animal or honeybee origin, they probably derived as contamination from sewage or other sources. After further more detail testing, the possible use of isolated bifidobacteria as probiotics is promising since they were able to pass through the digestive tract successfully.

The study reported in this manuscript is a part of extended investigations on transmission of fungi potentially pathogenic to man as well as parasitic flatworms and nematodes by wild fowl. The objective of this study was to identify the presence of fungi and parasites in the gastrointestinal tract of cormorants. Mycological and parasitological analyses were carried out on swabs taken from ontocenoses of beak, stomach, duodenum, jejunum, ileum and cloaca of 21 cormorants. The presence of fungi in the gastrointestinal tract was detected in 60% of the birds examined. They were isolated most frequently from beaks and cloacae of birds, duodenum was free of fungi. The most frequently isolated fungi were: Candida krusei and Debaryomyces hansenii. All isolates of fungi selected for enzymatic analyses were characterized by a high activity of esterases. Stomachs of all cormorants analyzed were found to contain Contracaecum rudolphii s.l. nematode. In turn, intestinal digesta of most of the birds were shown to contain Paradilepis scolecina tapeworm. Apart from tapeworms, duodenal digesta were also found to contain Paryphostomum radiatum digenea. The highest prevalence and intensity of infection with digenea was determined in the case of the birds shot in July.

Data have demonstrated that whey protein (WP) enhances the immune system. The aim of this study was to investigate and compare the effects of WP from three camel breeds on oxidative stress, blood lipid profile and the cytokine levels. Seventy five male mice were randomly split into five groups. The first served as a control group. The second, the third and the fourth groups were orally administrated the WP from Majaheim, Maghateer and Soffer camel breeds, respectively, at a dose of 100 mg/kg mouse body weight. The fifth group was supplemented with bovine serum albumin (BSA). Results showed similar electrophoretic patterns of the three whey proteins. WP was found to significantly inhibit the hydroperoxide and the Reactive Oxygen Species (ROS) in leukocytes, liver and skin as well as the blood cholesterol level in a time dependent manner. A significant enhancement of glutathione was revealed in WP groups. Furthermore, WP was found to significantly elevate the IL-2 with a significant time dependent enhance of IL-8. On contrast, a significant lowering effect of whey proteins on the pro-inflammatory cytokines, IL-1α, IL-1β, IL-6 and IL-10 was detected. Moreover, a mitogenic activity of WP was observed on the lymphocytes. Non-significant changes were observed in AST, ALT, creatinine and glucose level. These findings suggest that WP significantly improved the levels of the oxidative markers and the immune functions without any difference in the bioactivities of the three studied whey proteins.