Characterisation of alpha3beta1 and alpha(v)beta3 integrin N-oligosaccharides in metastatic melanoma WM9 and WM239 cell lines

It is well documented that glycan synthesis is altered in some pathological processes, including cancer. The most frequently observed alterations during tumourigenesis are extensive expression of beta1,6-branched complex type N-glycans, the presence of poly-N-acetyllactosamine structures, and high sialylation of cell surface glycoproteins. This study investigated two integrins, alpha3beta1 and alpha(v)beta3, whose expression is closely related to cancer progression. Their oligosaccharide structures in two metastatic melanoma cell lines (WM9, WM239) were analysed with the use of matrix-assisted laser desorption ionisation mass spectrometry. Both examined integrins possessed heavily sialylated and fucosylated glycans, with beta1,6-branches and short polylactosamine chains. In WM9 cells, alpha3beta1 integrin was more variously glycosylated than alpha(v)beta3; in WM239 cells the situation was the reverse. Functional studies (wound healing and ELISA integrin binding assays) revealed that the N-oligosaccharide component of the tested integrins influenced melanoma cell migration on vitronectin and alpha3beta1 integrin binding to laminin-5. Additionally, more variously glycosylated integrins exerted a stronger influence on these parameters. To the best of our knowledge, this is the first report concerning structural characterisation of alpha(v)beta3 integrin glycans in melanoma or in any cancer cells.     

Effect of age,size and digestive tract development on weaning effectiveness in crucian carp,Carassius carassius (Linnaeus, 1758)

The study aim was to determine the optimum age, wet body weight (WBW) and total length (TL) of the crucian carp, Carassius carassius (L.), to ensure the effectiveness of weaning directly without a gradual transfer from live food to a compound feed. Moreover, the state of development of the digestive tract was analyzed histologically based on the height of enterocytes. Experimental rearing was conducted between days 5 and 45 post hatch (DPH). Initial WBW of fish was 2.2 ± 0.6 (n = 30) mg and TL 6.1 ± 0.1 (n = 30) mm. Rearing was carried out at 27 ± 0.5°C, with fish divided into six groups: one control (C) fed with Artemia sp. nauplii, and five groups initially fed with Artemia sp. but later replaced by a compound feed. Weaning with the compound diet started at 15, 20, 25, 30 and 35 DPH in groups labeled F15, F20, F25, F30, F35, respectively. Larvae were fed three times per day (08.00 h, 13.00 h, 18.00 h) in equal portions (4% of larvae biomass per day, converted to the dry matter of the feed). Daily biomass growth was adopted as 15%. Each group was triplicated (n = 50 individuals per replicate). Highest values of TL 42.1 ± 0.7 (n = 30) mm and WBW 905.3 ± 50.3 (n = 30) mg were recorded in the control group at 45 DPH; lowest survival rate of 45 DPH was in group F15 (90.7 ± 1.2%, n = 30). The highest value of the enterocyte epithelial length was observed in individuals within groups F30, 34.8 ± 1.2 μm (n = 30) and F35, 35.4 ± 3.6 μm (n = 30), respectively, 30 and 35 DPH; highest percentage of deformations on the final day of the experiment was in group F15 (100 ± 0.0%, n = 30). The results indicate that an effective direct transfer from live food to prepared diets (with no gradual transfer) cannot be performed with crucian carp larvae before 30 DPH at 27°C, when the fish have reached TL = 31.1 ± 0.4 mm (n = 30) and WBW = 436.9 ± 13.7 mg (n = 30).     

Different patterns of genetic structure of relict and isolated populations of endangered peat-bog pine (Pinus uliginosa Neumann)

Recent changes in environmental conditions in populations of peat-bog pine (Pinus uliginosa Neumann) caused rapid decline or even extinction of the species in several stands in Central Europe. Conservation strategies forP. uliginosa require information about the evolutionary history and genetic structure of its populations. Using isozymes we assessed the genetic structure ofP. uliginosa from four isolated stands in Poland and compared the results to genetic structures of other closely related pine species including eight populations ofPinus mugo, ten ofPinus sylvestris and one ofPinus uncinata. The level of genetic variability ofP. uliginosa measured by the mean number of alleles per locus and average heterozygosity was similar to others related toP. uliginosa taxa from the reference group but it differs among populations. High genetic similarity was found between two populations ofP. uliginosa from Low Silesian Pinewood. The populations were genetically distinct as compared to other populations includinglocus classicus of the species from the peat bog at Batorów Reserve. Very low genetic distance (D_n = 0.002) and small genetic differentiation (GST = 0.003) were found betweenP. uliginosa andP. mugo in the sympatric populations of the species from Zieleniec peat bog suggesting the ongoing natural hybridisation and genetic contamination of peat-bog pine from this area. Some evidence for skew in allele frequency distribution potentially due to recent bottleneck was found in population from Low Silesian Pinewood. The analysed open pollinated progeny derived from twoP. uliginosa stands from Low Silesian Pine-wood showed the excess of homozygotes as compared to the maternal trees indicating high level of inbreeding (F = 0.105,F = 0.081). The results are discussed in the context of evolution ofP. uliginosa populations, taxonomic relationships between the analysed species and conservation strategies for active protection of peat-bog pine.     

Conservation genetics of highly isolated populations of the xerothermic beetle Crioceris quatuordecimpunctata (Chrysomelidae)

Xerothermic species are rare and threatened in central and eastern Europe. In light of the continuing loss of steppe‐like habitats due to anthropogenic fragmentation and degradation, the evaluation of genetic variation in populations inhabiting them is of immediate importance if appropriate conservation measures are to be undertaken. Here we report on the genetic diversity of the rare leaf beetle Crioceris quatuordecimpunctata, whose populations in central and eastern Europe inhabit highly geographically isolated areas. All of the studied populations (in Poland, Ukraine, and Slovakia) were differentiated at the mitochondrial marker COI. However, with respect to the nuclear marker ITS1, Polish populations were monomorphic, but distinct from all other populations. The distinctiveness of the studied populations was confirmed by Wolbachia screening, which showed that all populations carried different strains (one or two), which were probably transferred independently from other insects. On the other hand, no diversity was found in any marker within particular populations, which could be caused (at least for mtDNA) by a Wolbachia selective sweep. Crioceris quatuordecimpunctata probably consists of isolated populations, which went through narrow bottlenecks leading to a drastic reduction in their genetic diversity. As these populations are reciprocally monophyletic for mtDNA haplotypes and show a significant divergence of allele frequencies at nuclear loci, they could be classified as evolutionarily significant units (ESUs). In addition, DNA barcodes were used to identify Asparagus officinalis as the host plant for members of all studied populations. These data should be valuable in efforts to conserve populations of C. quatuordecimpunctata (e.g., for guiding reintroductions).     

Reactive oxygen species upregulate expression of PAI-1 in endothelial cells

Second messengers involved in the signal transduction pathway leading to induction of the plasminogen activator inhibitor (PAI-1) have not yet been well characterized. This study focuses on the mechanisms of regulation of PAI-1 expression by reactive oxygen species (ROS) in human endothelial cells. Inhibition of the tumor necrosis factor alpha (TNFalpha?-induced expression of PAI-1 by antioxidant N-acetyl-L-cysteine (NAC) indicated redox-sensitive mechanisms involved in the signaling pathway. Because TNFalpha induces PAI-1 production in endothelial cells, and NAC attenuated this response, we attempted to investigate the possible involvement of ROS in the activation of PAI-1 by TNFalpha. Upregulation of PAI-1 expression in endothelial cells by the stimulation with TNFalpha (50 ng/ml) or H2O2 (10-200 micro M), observed by measurement of the antigen and mRNA levels, was reversed in the presence of NAC (20mM). The stimulatory effect of ROS was detected also at the level of the PAI-1 promoter in endothelial cells transfected with plasmid p800 LUC containing a PAI-1 promoter fragment (+71 to -800). The PAI-1 promoter activity was increased in the presence of ROS, and was suppressed by up to 75% in the presence of antioxidants. On the basis of this study we can conclude that reactive oxygen species play an important role in a cytokine-induced activation of PAI-1 expression, and may act as a signal transduction messenger.     

Characterization of diphosphonucleotide phosphatase/phosphodiesterase from yellow lupin (Lupinus luteus) seeds

A phosphatase cleaving the pyrophosphate bond in diphosphonucleotides and phosphodiester bond in various phosphodiesters (pH optimum at 6.25) was purified from yellow lupin (Lupinus luteus L.) seeds. The enzyme is 75 kDa monomeric glycoprotein (pI=6.4) with 4.4% of carbohydrate (mannose, N-acetylglucosamine, fucose and xylose). Analysis of its partial amino acid sequence (8 peptides, 101 amino acid residues) together with no divalent cation requirements for catalysis points out that the purified enzyme is different from known plant pyrophosphate cleaving enzymes (apyrases and inorganic pyrophosphatases). Its physiological role could be related to a regulation of diphosphonucleotides level in plant metabolism.     

Microbial transformation of neutral fraction and upgraded neutral fraction of Polish tall oil

Summary Microbial transformations of neutral fraction (NF) and upgraded neutral fraction (UNF) of Polish tall oil byMycobacterium sp. MB 3683 were performed. Final metabolites and yields were compared to bioconversion of pure -sitosterol. Additionally, origin of a new metabolite —5-androsta-3,6,17-trione was proved by transformation of UNF in the presence of labeled -sitosterol.     

Female germ unit in Genlisea and Utricularia, with remarks about the evolution of the extra-ovular female gametophyte in members of Lentibulariaceae

Lentibulariaceae is the largest family among carnivorous plants which displays not only an unusual morphology and anatomy but also the special evolution of its embryological characteristics. It has previously been reported by authors that Utricularia species lack a filiform apparatus in the synergids. The main purposes of this study were to determine whether a filiform apparatus occurs in the synergids of Utricularia and its sister genus Genlisea, and to compare the female germ unit in these genera. The present studies clearly show that synergids in both genera possess a filiform apparatus; however, it seems that Utricularia quelchii synergids have a simpler structure compared to Genlisea aurea and other typical angiosperms. The synergids are located at the terminal position in the embryo sacs of Pinguicula, Genlisea and were probably also located in that position in common Utricularia ancestor. This ancestral characteristic still occurs in some species from the Bivalvaria subgenus. An embryo sac, which grows out beyond the limit of the integument and has contact with nutritive tissue, appeared independently in different Utricularia lineages and as a consequence of this, the egg apparatus changes position from apical to lateral.     

Energy metabolism and evaporative water loss in the European free-tailed bat and Hemprich's long-eared bat (Microchiroptera): species sympatric in the Negev Desert

We compared the thermoregulatory abilities of two insectivorous bat species, Tadarida teniotis (mean body mass 32 g) and Otonycteris hemprichii (mean body mass 25 g), that are of different phylogenetic origins and zoogeographic distributions but are sympatric in the Negev Desert. At night, both were normothermic. By day, both were torpid when exposed to ambient temperatures (T(a)) below 25 degrees Celsius, with concomitant adjustments in metabolic rate (MR). Otonycteris hemprichii entered torpor at higher T(a) than T. teniotis, and, when torpid, their body temperatures (T(b)) were 1 degrees -2 degrees Celsius and 5 degrees -8 degrees Celsius above T(a), respectively; MR was correspondingly reduced. At night, the lower critical temperature of T. teniotis was 31.5 degrees Celsius, and that of O. hemprichii was 33 degrees Celsius. Mean nocturnal thermoneutral MR of T. teniotis was 37% greater than that of O. hemprichii. At high T(a), evaporative water loss (EWL) increased markedly in both species, but it was significantly higher in T. teniotis above 38 degrees Celsius. In both species, the dry heat transfer coefficient (thermal conductance) followed the expected pattern for small mammals, by day and by night. Total EWL was notably low in normothermic and torpid animals of both species, much lower than values reported for other bats, indicating efficient water conservation mechanisms in the study species. Comparing thermoregulatory abilities suggests that O. hemprichii is better adapted to hot, arid environments than T. teniotis, which may explain its wider desert distribution. By both standard and phylogenetically informed ANCOVA, we found no differences in basal metabolic rate (BMR) between desert and nondesert species of insectivorous bats, substantiating previous studies suggesting that low BMR is a characteristic common to insectivorous bats in general.