Plant tRNA nucleotidyltransferase

A method for purification of tRNA nucleotidyltransferase from Lupinus luteus L. seeds on a preparative scale is presented. This involved ammonium sulfate fractionation and chromatography on DEAE-cellulose, hydroxylapatite, and QAE-Sephadex A-50. The final enzyme prepartion was apparently homogeneous and a 2000-fold purification was achieved. Data presented suggest the existence of only one form of tRNA nucleotidyltransferase in dry lupin seeds.     

Wolf predation and snow cover as mortality factors in the ungulate community of the Bialowieża National Park,Poland

Summary Wolf-ungulate interactions were studied in the pristine deciduous and mixed forests of the Bialowiea National Park in 1985–1989. The study period included two severe and two mild winters. The community of ungulates inhabiting Bialowiea National Park consisted of red deer Cervus elaphus, 55% of all ungulates; wild boar Sus scrofa, 42%; and roe deer Capreolus capreolus, moose Alces alces, and European bison Bison bonasus, about 1% each. The average size of red deer groups increased from 2.7 (SD 2.35) in spring and summer to 6.9 (SD 6.84) in autumn and winter. In winter the group size of red deer was positively correlated with the depth of snow cover and negatively correlated with the mean daily temperature. Average group size of wild boar did not change significantly between seasons; it was 6.8 (SD 5.16) in spring and summer and 5.7 (SD 4.67) in autumn and winter. Analysis of 144 wolf scats showed that wolves preyed selectively on red deer. In October–April, Cervidae (mostly red deer) constituted 91% of biomass consumed by wolves, while wild boar made up only 8%. In May–September deer formed 77% of prey biomass, and the share of wild boar increased to 22%. In all seasons of the year wolves selected juveniles from deer and boar populations: 61% of red deer and 94% of wild boar of determined age recovered from wolves' scats were young <1 year old. Analysis of 117 carcasses of ungulates found in Bialowiea National Park showed that predation was the predominant mortality factor for red deer (40 killed, 10 dead from causes other than predation) and roe deer (4 killed, none dead). Wild boar suffered most from severe winter conditions (8 killed, 56 dead). The percentage of ungulates that had died from undernutrition and starvation in the total mortality was proportional to the severity of winter.     

Equi-replicated balanced block designs generated by a balanced matrix

In this paper it is shown that if N= \documentclass{article}\pagestyle{empty}\begin{document}$ \mathop \sum \limits_{i = 1}^{S_h} $\end{document} c_ihN_ih, where c_ih are some non-negative integer numbers and N_ih are such incidence matrices that A_h = \documentclass{article}\pagestyle{empty}\begin{document}$ \mathop \sum \limits_{i = 1}^{S_h} $\end{document} i N_ih is a balanced matrix defined by SHAH (1959), for h = 1, 2,…, p, then a block design with an incidence matrix Ñ = [N, N,…,N] is an equi-replicated balanced block design. Here the balance of a block design is defined in terms of the matrix M₀ introduced by CALI?SKI (1971).     

Membrane transport of polysialic acid chains: modulation of transmembrane potential

Polysialic acid (polySia) is a long polyanionic polymer (with the degree of polymerization, DP, up to 200) of negatively charged sialic acid monomers. PolySia chains are bound to the external surface of some neuroinvasive bacterial cells and neural cells. PolySia serves as a potent regulator of cell interactions via its unusual biophysical properties. In the present paper, the analysis, based on the Goldman-Hodgkin-Katz equation, of transmembrane potential changes resulting from transmembrane translocation of polySia is performed. The relationships between the transmembrane potential and the polySia DP (up to 200), the temperature, the cation/ anion permeability ratio, and the inner/outer concentration ratio of polySia has been plotted and discussed. The maximal membrane potential changes, up to 118 mV, were found for a permeability ratio greater than one. The increase of the polySia chain length resulted in the diminution of this effect. The temperature-dependent changes in membrane potential were less than 7 mV in the range 0-50 degrees C. The change in the concentration ratio (into its reciprocal) resulted in a mirror reflection of the membrane potential curves. The results show that the expression of polySia chains in bacterial cells can be responsible for the modulation of the transmembrane potential of the bacterial inner membrane. We suggest that the polySia chains can influence the transmembrane potential of neural cell membranes in a similar way. This analysis also describes the effect of the transmembrane translocation of negatively charged polyanionic polynucleotydes on the cell membrane potential.     

Coordination properties of Cu(II) and Ni(II) ions towards the C-terminal peptide fragment -ELAKHA- of histone H2B

The coordination properties of the peptide Ac-GluLeuAlaLysHisAla-amide, the C-terminal 102-107 fragment of histone H2B towards Cu(II) and Ni(II) ions were studied by means of potentiometry and spectroscopic techniques (UV/Vis, CD, EPR and NMR). It was found that the peptide has a unique ability to bind Cu(II) ions at physiological pH values at a Cu(II): peptide molar ratio 1:2, which is really surprising for blocked hexapeptides containing one His residue above position 3. At physiological pH values the studied hexapeptide forms a CuL(2) complex {N(Im),2N(-)}, while in acidic and basic pH values the equimolar mode is preferred. In basic solutions Ac-GluLeuAlaLysHisAla-amide may bound through a {4N(-)} mode forming a square-planar complex, in which the imidazole ring is not any more coordinated or it has been removed in an axial position. On the contrary, Ni(II) ions form only equimolar complexes, starting from a distorted octahedral complex at about neutral pH values to a planar complex, where hexapeptide is bound through a {N(Im),3N(-)} mode in equatorial plane. The results may be of importance in order to reveal more information about the toxicity caused by metals and furthermore their influence to the physiologic metabolism of the cell.     

Pseudomonas aeruginosa LD-carboxypeptidase, a serine peptidase with a Ser-His-Glu triad and a nucleophilic elbow

LD-Carboxypeptidases (EC 3.4.17.13) are named for their ability to cleave amide bonds between l- and d-amino acids, which occur naturally in bacterial peptidoglycan. They are specific for the link between meso-diaminopimelic acid and d-alanine and therefore degrade GlcNAc-MurNAc tetrapeptides to the corresponding tripeptides. As only the tripeptides can be reused as peptidoglycan building blocks, ld-carboxypeptidases are thought to play a role in peptidoglycan recycling. Despite the pharmaceutical interest in peptidoglycan biosynthesis, the fold and catalytic type of ld-carboxypeptidases are unknown. Here, we show that a previously uncharacterized open reading frame in Pseudomonas aeruginosa has ld-carboxypeptidase activity and present the crystal structure of this enzyme. The structure shows that the enzyme consists of an N-terminal beta-sheet and a C-terminal beta-barrel domain. At the interface of the two domains, Ser(115) adopts a highly strained conformation in the context of a strand-turn-helix motif that is similar to the "nucleophilic elbow" in alphabeta-hydrolases. Ser(115) is hydrogen-bonded to a histidine residue, which is oriented by a glutamate residue. All three residues, which occur in the order Ser-Glu-His in the amino acid sequence, are strictly conserved in naturally occurring ld-carboxypeptidases and cannot be mutated to alanines without loss of activity. We conclude that ld-carboxypeptidases are serine peptidases with Ser-His-Glu catalytic triads.     

Chiral discrimination in binding of enantiomers of 2-(aminoalkoxy)-substituted 4-(2-thienyl)pyrimidines and 4,6-bis(2-thienyl)pyrimidines with duplex DNA

Thienylpyrimidines substituted at position 2 of the pyrimidine with a chiral aminoalkoxy group were synthesized. Upon interaction with duplex DNA, the unfused heteroaromatic system of these compounds intercalates with DNA base pairs and the protonated side chain is located in the major groove. The S-enantiomers bind more strongly than their R-counterparts with enantiomeric discrimination, as measured by a ratio of binding constants K(S)/K(R), ranging from 1.2 to 2.4.     

Evaluation of the effect of GHRH(1-44)NH2 on the secretion of interleukin-2 (IL-2) and soluble IL-2 receptor alpha (sIL-2Ralpha) from human peripheral blood mononuclear cells in vitro

OBJECTIVE: The bidirectional communication between the neuroendocrine and the immune systems is now a subject of an intensive investigation. Somatoliberin (GHRH) is a hypothalamic hormone that enhances the synthesis and the release of growth hormone (GH) from the anterior pituitary cells. Few recent reports demonstrate also role of the neuropeptide in the regulation of the immune system function. AIM: The aim of the study was to examine the influence of GHRH on IL -2 as well as sIL -2Ralpha secretion from mitogen-stimulated peripheral blood mononuclear cells. MATERIAL AND METHOD: Mononuclear cells (PBMC) were isolated from the peripheral blood of healthy adults according to the technique described by B?yum. Cells, cultured 24 hours at 37 degrees C in humidified atmosphere of 95% air and 5% CO2, were stimulated with phytohemaglutinin (PHA; 10 microg/ml), and then GHRH(1-44)NH2 at the final concentrations 10(-12), 10(-10), 10(-8) and 10(-6) M was added to appropriate wells. ELISA kits were used to measure IL-2 and sIL-2Ralpha concentrations in the supernatants of cultured cells. Comparisons between tested groups were made by U Mann-Whitney test. The differences were considered significant if p < 0.05. RESULTS: GHRH stimulated the secretion of IL -2 into the supernatants acting significantly at the concentration of 10(-12) M (p < 0.001). Moreover, GHRH at concentrations 10(-10) M and 10(-8) M significantly increased the secretion of sIL-2Ralpha as well (p < 0.001). Strong positive correlation between tested GHRH concentrations and sIL-2Ralpha levels in the supernatants was demonstrated (r = 0.8664; p <0.001). CONCLUSIONS: The results demonstrate the potential involvement of GHRH in the regulation of T lymphocytes secretory function.     

Ascorbate potentiates the cytotoxicity of menadione leading to an oxidative stress that kills cancer cells by a non-apoptotic caspase-3 independent form of cell death

Hepatocarcinoma cells (TLT) were incubated in the presence of ascorbate and menadione, either alone or in combination. Cell death was only observed when such compounds were added simultaneously, most probably due to hydrogen peroxide (H2O2) generated by ascorbate-driven menadione redox cycling. TLT cells were particularly sensitive to such an oxidative stress due to its poor antioxidant status. DNA strand breaks were induced by this association but this process did not correspond to oligosomal DNA fragmentation (a hallmark of cell death by apoptosis). Neither caspase-3-like DEVDase activity, nor processing of procaspase-3 and cleavage of poly(ADP-ribose) polymerase (PARP) were observed in the presence of ascorbate and menadione. Cell death induced by such an association was actively dependent on protein phosphorylation since it was totally prevented by preincubating cells with sodium orthovanadate, a tyrosine phosphatase inhibitor. Finally, while H2O2, when administered as a bolus, strongly enhances a constitutive basal NF-kappaB activity in TLT cells, their incubation in the presence of ascorbate and menadione results in a total abolition of such a constitutive activity.     

Survey of yeast mastitis in dairy herds of small-type farms in the Lublin region,Poland

The purpose of this study was to isolate fungi from the quarter milk of cow udders in 9 dairy farms. The samples were collected between May 1996 and April 2000 in the Lublin region. Six hundred and four milk samples collected from the quarters of 172 cows with clinical and subclinical mastitis were examined. Milk samples were plated as soon as possible on blood agar (BA), Mac Conkey agar, aesculin-tallium acetate-crystal violet blood agar, and Sabouraud agar with chloramphenicol and gentamicin. Fifty eight (9.6%) of the samples were positive for fungi. All of the fungal isolates were yeasts of the genera Candida, Rhodotorula and Trichosporon. We also isolated Streptococcus agalactiae (4.9%), Str. spp.(6.4%), Staphylococcus aureus (10.4%), coagulase-negative staphylococci-CNS (36.6%), Escherichia coli (3.5%), other microorganisms (2.6%) and no growth (25.8%). This revised version was published online in June 2006 with corrections to the Cover Date.