Analogues of cyclolinopeptide A containing alpha-hydroxymethyl amino acid residues

Linear and cyclic cyclolinopeptide A (CLA) analogues containing alpha-hydroxymethylleucine (HmL) in positions 1, 4, and 1&4, and alpha-hydroxymethylvaline (HmV) in position 5, were synthesized by the solid-phase peptide strategy and cyclized with the 1-Ethyl-3-(3-dimethylaminopropyl)-carbodiimide/1-hydroxy-7-azabenzotriazole (EDC/HOAt) reagent. The peptides were examined for their immunosuppressive activity in the lymphocyte proliferation assays (LPA). Only HmL-containing peptides demonstrated at about 25% lower immunosuppressive activity, but they are four times more soluble in water solutions than the native CLA. It seems from the LPA results that peptide [(HmL4)CLA] is the most promising for further studies. This peptide was characterized in solution, at room temperature in CDCl3, and the conformation compared with that observed for CLA in the solid state.     

Effects of tamoxifen dose and nutrition scheme during growth on stimulation of the reproductive system in cornish breed cocks

The effects of tamoxifen (TAM) antiestrogen dose as well as protein and energy content in feed on stimulation of the reproductive system development in meat type cocks were studied. The experiment was conducted on males of the Dominant White Cornish chicken breed. The optimum TAM dose for stimulation of reproductive system development was at the level of 5 mg per kg body weight. The effects of TAM administration on the following traits were investigated: body weight, breast muscle thickness, age at sexual maturity, egg fertilization and hatchability. No effect of TAM on breast muscle thickness was found. It was also noted that semen obtained from 11 to 12 week old males of the Dominant White Cornish breed could be used successfully in the insemination of females.     

Genetic structure is influenced by environmental barriers: empirical evidence from the common voleMicrotus arvalis populations

One of the major challenges in population biology is the identification of barriers to gene flow and/or secondary contacts between differentiated entities. The level of genetic differentiation among eight populations of the common voleMicrotus arvalis (Pallas, 1779) around the Biebrza Wetlands, NE Poland was examined by analyzing seven microsatellite loci for 140 voles and testing for the presence of barriers to gene flow. Overall population differentiation was moderate and significant (F _ST = 0.081,p < 0.001) and there was no correlation between geographical and genetic distances among populations. We found a relatively high level of genetic variability within the populations studied. This could be explained by male bias in dispersal, a phenomenon recently found inM. arvalis. Patterns of genetic structure visualized in synthetic genetic maps showed clear gradients along a southeast-northwest axis across the study area, as well as the presence of a potential barrier to dispersal. The position of a barrier to gene flow identified using Monmonier’s maximum difference algorithm likely corresponds to humid habitats of the Biebrza Wetlands. These results suggest that the presence of environmental barriers to gene flow and drift may be responsible for the observed spatial genetic structure ofM. arvalis in the Biebrza Valley. Institute of Biology, University of Białystok, OEwierkowa 20 B, 15-950 Białystok, Poland,     

Midgut glycosidases activities in monophagous larvae of Apollo butterfly, Parnassius apollo ssp. frankenbergeri

Parnassius apollo (Lepidoptera, Papilionidae) declines on numerous localities all over Europe. Its local subspecies frankenbergeri, inhabiting the Pieniny Mts (southern Poland) and successfully recovered from extinction, is monophagous in larval stage. In natural conditions, it completes development on the orpine Sedum telephium ssp. maximum. Since proper quality and quantity of necessary nutritional compounds of the food plant ensure developmental success, the digestive processes in the insect midgut should reflect adaptation to a specific food source. The paper presents, for the first time, the activity of detected glycolytic enzymes in midgut tissue and liquid gut contents of the L4 and L5 instars of P. apollo larvae. alpha-Amylase plays the main role in utilization of carbohydrates, contrary to cellulase activity. Saccharase seems to be the main disaccharidase, and high activity of beta-glycosidase enables hydrolysis of the plant glycosides. Trehalase activity was unexpectedly low and comparable to those of cellobiase and lactase. alpha-Amylolytic and other glycolytic activities indicate that larvae utilize starch and other carbohydrate compounds as energy sources. Possible use of some plant allelochemicals as energy sources by Apollo larvae is discussed.     

Generation of cloned and chimeric embryos/offspring using the new methods of animal biotechnology

The article summarizes results of studies concerning: 1/ qualitative evaluation of pig nuclear donor cells to somatic cell cloning, 2/ developmental potency of sheep somatic cells to create chimera, 3/ efficient production of chicken chimera. The quality of nuclear donor cells is one of the most important factors to determine the efficiency of somatic cell cloning. Morphological criteria commonly used for qualitative evaluation of somatic cells may be insufficient for practical application in the cloning. Therefore, different types of somatic cells being the source of genomic DNA in the cloning procedure were analyzed on apoptosis with the use of live-DNA or plasma membrane fluorescent markers. It has been found that morphological criteria are a sufficient selection factor for qualitative evaluation of nuclear donor cells to somatic cell cloning. Developmental potencies of sheep somatic cells in embryos and chimeric animals were studied using blastocyst complementation test. Fetal fibroblasts stained with vital fluorescent dye and microsurgically placed in morulae or blastocysts were later identified in embryos cultured in vitro. Transfer of Polish merino blastocysts harbouring Heatherhead fibroblasts to recipient ewes brought about normal births at term. Newly-born animals were of merino appearance with dark patches on their noses, near the mouth and on their clovens. This overt chimerism shows that fetal fibroblasts introduced to sheep morulae/blastocysts revealed full developmental plasticity. To achieve the efficient production of chicken chimeras, the blastodermal cells from embryos of the donor breeds, (Green-legged Partridgelike breed or GPxAraucana) were transferred into the embryos of the recipient breed (White Leghorn), and the effect of chimerism on the selected reproductive and physiological traits of recipients was examined. Using the model which allowed identification of the chimerism at many loci, it has been found that 93.9% of the examined birds were chimeras. The effect of donor cells on the reproduction and physiology of the recipients was evident.     

Spontaneous apoptosis of melanotic and amelanotic melanoma cells in different phases of cell cycle: relation to tumor growth

Since the spontaneous alteration of native melanotic (Ma) into amelanotic (Ab) transplantable melanoma line it has been observed that this alteration is accompanied by the acceleration of growth of Ab line. The aim of the present study was to check and estimate spontaneous apoptosis of cells from cell cycle phases. Cytometric cell cycle analysis was performed by staining cells with propidium iodide (PI). Apoptosis estimated by the TUNEL method, alterations in the plasma membrane structure (annexin V staining), changes in the mitochondrial transmembrane potential--delta psi m (JC-1 staining) showed that amelanotic melanoma cells have decreased ability to undergo spontaneous apoptosis. The obtained results showing that in the native melanotic line about 30% of cells are in S+G2/M phases and that 33% of these cells undergo apoptosis could lead to the conclusion that the slower growth of this melanoma line is the result of lower proliferation activity and higher rate of apoptosis of these tumor cells. The number of cells in S+G2/M phases in amelanotic melanoma line increases up to 40% and only 7% of them undergo apoptosis. This observation seems to suggest that the expansive growth of this melanoma line depends mainly on the decreased ability to undergo spontaneous apoptosis, especially in case of cells from S+G2/M phases. Moreover, the obtained results indicate that alteration of melanotic line into amelanotic one, accompanied by differences in many biological features also concerns basic cell processes such as cell cycle and cell death.     

Inhibition of jack bean urease by p-benzoquinone: elucidation of the role of thiols and reversibility of the process

p-Benzoquinone (pBQ) was studied as an inhibitor of jack bean urease in 20 mM phosphate buffer, pH 7.0, 1 mM EDTA, 25 degrees C. The inhibition was carried out by the use of a preincubation procedure in the absence of substrate. The influence of the inhibitor concentration and the preincubation time on the enzyme activity was elucidated. It was found that increase in pBQ concentration resulted in a linear decrease of urease activity. The dependence of the enzyme activity on the preincubation time showed that the rate of inhibition rapidly decreased at the beginning of the process in order to achieve the constant value. The inhibition became time independent in the studied time range. This observation is characteristic of a slow binding mechanism of inhibition. The protective experiment proved that the urease active site is involved in the binding of pBQ. High effectiveness of thiol protectors against pBQ inhibition indicates the strategic role of the active site sulfhydryl group in the blocking process. There were two methods used for reactivation of pBQ-inhibited urease. The dilution of the urease-pBQ complex in urea solution did not result in a regain of enzyme activity. Alternatively, the addition of dithiothreitol into the urease-pBQ mixture caused the instant and efficient reactivation of the enzyme. The experiments showed that the nature of the urease-pBQ complex is irreversible but the application of a specific thiol reagent can release the active enzyme from the complex.     

Inhibition of jack bean urease by tetrachloro-o-benzoquinone and tetrachloro-p-benzoquinone

Tetrachloro-o-benzoquinone (TCoBQ) and tetrachloro-p-benzoquinone (TCpBQ) were studied as inhibitors of jack bean urease in 20 mM phosphate buffer, pH 7.0, 1 mM EDTA, 25 degrees C. The mechanisms of inhibition were evaluated by analysis of the progress curves obtained with two procedures: the reaction initiated by addition of the enzyme and the reaction initiated by addition of the substrate after preincubation of the enzyme with the inhibitor. The obtained results were characteristic of slow-binding inhibition. The effects of different inhibitor concentrations on the initial and steady-state velocities obeyed the relationships of two-step enzyme-inhibitor interaction, qualified as mechanism B. It was found that TCoBQ and TCpBQ are strong urease inhibitors. TCpBQ is more effective than TCoBQ with the overall inhibition constant of K(i)* = 4.5 x 10(-7) mM. The respective inhibition constant of TCoBQ was equal to: K(i)* = 2.4 x 10(-6) mM. The protective experiment proved that the urease active site is involved in the tetrachlorobenzoquinone inhibition process. High effectiveness of thiol protectors against inhibition by TCoBQ and TCpBQ indicates the strategic role of the active site sulfhydryl group in the blocking process. The stability of the complexes: urease-TCoBQ and urease-TCpBQ was tested in two ways: by dilution or addition of dithiothreitol. No recovery of urease activity bound in the urease-inhibitor complexes proves that the complexes are stable and strong.     

Ectomycorrhizal fungal communities of native and non-native Pinus and Quercus species in a common garden of 35-year-old trees

Non-native tree species have been widely planted or have become naturalized in most forested landscapes. It is not clear if native trees species collectively differ in ectomycorrhizal fungal (EMF) diversity and communities from that of non-native tree species. Alternatively, EMF species community similarity may be more determined by host plant phylogeny than by whether the plant is native or non-native. We examined these unknowns by comparing two genera, native and non-native Quercus robur and Quercus rubra and native and non-native Pinus sylvestris and Pinus nigra in a 35-year-old common garden in Poland. Using molecular and morphological approaches, we identified EMF species from ectomycorrhizal root tips and sporocarps collected in the monoculture tree plots. A total of 69 EMF species were found, with 38 species collected only as sporocarps, 18 only as ectomycorrhizas, and 13 both as ectomycorrhizas and sporocarps. The EMF species observed were all native and commonly associated with a Holarctic range in distribution. We found that native Q. robur had ca. 120% higher total EMF species richness than the non-native Q. rubra, while native P. sylvestris had ca. 25% lower total EMF species richness than non-native P. nigra. Thus, across genera, there was no evidence that native species have higher EMF species diversity than exotic species. In addition, we found a higher similarity in EMF communities between the two Pinus species than between the two Quercus species. These results support the naturalization of non-native trees by means of mutualistic associations with cosmopolitan and novel fungi.