Characterization of calcium, phosphate and peroxide interactions in activation of mitochondrial swelling using derivative of the swelling curves

We describe a new method for the analysis of mitochondrial swelling curves. Using classical swelling curves, only the maximum extent of the swelling can be calculated in a numerical form. However, taking the derivative of the classical swelling curves enables the evaluation of two additional parameters of the swelling process in a numerical form, namely, the maximum swelling rate after the addition of the swelling inducer (as dA???/10 s) and the time (in sec) at which the maximum swelling rate after the addition of the swelling inducer is obtained. The use of these three parameters enables the better characterization of the swelling process as demonstrated by the evaluation of calcium and phosphate interactions in the opening of the mitochondrial permeability transition pore and by the characterization of the peroxide potentiating action.     

Sequencing, cloning and high-yield expression of a fungal β-N-acetylhexosaminidase in Pichia pastoris

The β-N-acetylhexosaminidase from Talaromyces flavus has a remarkable synthetic ability, processing even carbohydrates with various functionalities. Its broader use is partially hampered by low-yield production in the native fungus. Here, we present an optimized 3-day production of this enzyme in the eukaryotic host of Pichia pastoris, in ca 10-fold higher volume activity (10 U/ml) and close-to-perfect purity (one chromatographic step needed). Importantly, the recombinant enzyme features the same biochemical and catalytic properties, including the syntheses with derivatized carbohydrate substrates. This is the first example of the overexpression of a fungal β-N-acetylhexosaminidase by a single-cell producer in liquid medium. It represents a promising solution for wider biotechnological applications of this outstanding enzyme.     

Prostate-cancer-targeted N-(2-hydroxypropyl)methacrylamide copolymer/docetaxel conjugates

Biodistribution, pharmacokinetics, and efficacy of prostate-cancer-targeted HPMA copolymer/DTX conjugates are evaluated in nude mice bearing prostate cancer C4-2 xenografts. PSMA-specific monoclonal antibodies 3F/11 are used as the targeting moiety. Control conjugates tumor accumulation to total background organs (heart, lung, kidney, liver, spleen and blood) accumulation increase substantially with time for the targeted conjugate, and the ratio at 48 h is 7-fold higher than that at 6 h. Preliminary evaluation of the efficacy of the conjugates in vivo show tumor growth inhibition for all HPMA copolymer/DTX conjugates.     

Sward structure and diet selection after sheep introduction on abandoned grassland in the Giant Mts, Czech Republic

The effect of rotational grazing on the sward structure of long-term abandoned grassland and the sheep diet selection were investigated in the Giant Mts (Krkono?e/Karkonosze). The aim of the study was to answer the following questions: (1) Does sheep grazing affect the sward structure of previously abandoned mountain grassland? (2) How does sheep diet selection develop within and among grazing seasons? (3) Which section of a pasture do sheep prefer to graze? Data was collected three times during each grazing season in spring, summer and in autumn in the years 2001, 2002 and 2003 Fifty 1 × 1 m plots were used where the plant species occurrence and damage of plants by grazing for all species were repeatedly recorded. 1) Nonsignificant changes in plant species occurrences were recorded in spite of obvious visual changes in sward structure due to grazing e.g. the retreat of tall dominant species characteristic of long-term unmanaged grasslands. 2) Seasonal as well as inter-annual changes in sheep diet selection were detected. In spring 2001, sheep grazed over a wide variety of plant species in low quantities. In subsequent springs, they preferred species favoured during previous autumns (Veratrum album subsp. lobelianum; Ranunculus platanifolius; Senecio ovatus) and changed the diet only as a result of the elimination of favoured plants. 3) Sheep preferred to graze at the highest elevated part of the pasture probably as a consequence of anti-predator behaviour not due to higher occurrence of favoured plant species or the presence of a drinking place or salt licks. Changes in the sward structure were mostly of a quantitative not qualitative character, thus the presence-absence data collection is not a suitable method for monitoring the effects of management restoration. The diet selection changed probably due to the animals’ experience; sheep had no experience with montane species in spring 2001. The sheep were able to recognize favoured plant species after 6 months of wintering in lowland.     

On the mechanism of sequence-specific DNA-dependent acetylation of p53: the acetylation motif is exposed upon DNA binding

P53 acetylation requires p300-docking to two contiguous sites in the activation domain that in turn mediates DNA-dependent acetylation of the tetramer. In an attempt to further define the mechanism of DNA-dependent acetylation of p53, an in vitro system has been reconstituted with distinct p53 isoforms and has been used to reveal conformational constraints on p53 acetylation. Two native p53 tetrameric isoforms purified from Sf9 cells differing by the extent of phosphorylation within the C-terminal acetylation site are both acetylated in a sequence-specific DNA-dependent manner. By contrast, p53 purified from an Escherichia coli expression system is in a largely denatured conformation and its acetylation is DNA-independent. Heating native p53 to destroy the folded structure restores DNA-independent acetylation similar to that seen with bacterially expressed p53. There are at least two sites of conformational flexibility in the p53 tetramer: the first in the flexible S10 beta-sheet within the MDM2 ubiquitination sequence and the second in the C-terminal regulatory domain. We analysed therefore whether DNA-dependent acetylation correlated with conformational changes in either of these two regions. DNA-dependent acetylation of p53 is maintained in a dose-dependent manner by low concentrations of consensus site DNA under conditions where flexibility in the S10 beta-sheet region is maintained. Oligonucleotide DNAs that promote acetylation stimulate the binding of monoclonal antibodies PAb421 and ICA-9; two antibodies whose contiguous epitopes overlap the C-terminal acetylation motif. By contrast, bent oligonucleotide DNAs that conceal both the S10 beta-sheet from binding of the monoclonal antibody DO-12 and attenuate binding of the monoclonal antibody PAb421 can preclude acetylation. These data suggest that, in the absence of DNA, the acetylation motif of p53 is in a cryptic state, but after DNA binding, allosteric effects mediate an exposure of the acetylation motif to allow DNA-dependent acetylation of the tetramer.     

The use of styrene-maleic acid copolymer (SMA) for studies on T cell membrane rafts

An emerging alternative to the use of detergents in biochemical studies on membrane proteins is apparently the use styrene-maleic acid (SMA) amphipathic copolymers. These cut the membrane into nanodiscs (SMA-lipid particles, SMALPs), which contain membrane proteins possibly surrounded by their native lipid environment. We examined this approach for studies on several types of T cell membrane proteins, previously defined as raft or non-raft associated, to see whether the properties of the raft derived SMALPs differ from non-raft SMALPs. Our results indicate that two types of raft proteins, GPI-anchored proteins and two Src family kinases, are markedly present in membrane fragments much larger (>250?nm) than those containing non-raft proteins (<20?nm). Lipid probes sensitive to membrane fluidity (membrane order) indicate that the lipid environment in the large SMALPs is less fluid (more ordered) than in the small ones which may indicate the presence of a more ordered lipid L_o phase which is characteristic of membrane rafts. Also the lipid composition of the small vs. large SMALPs is markedly different – the large ones are enriched in cholesterol and lipids containing saturated fatty acids. In addition, we confirm that T cell membrane proteins present in SMALPs can be readily immunoisolated. Our results support the use of SMA as a potentially better (less artifact prone) alternative to detergents for studies on membrane proteins and their complexes, including membrane rafts.     

Seasonal shifts in Fe-acquisition strategies in Southern Ocean microbial communities revealed by metagenomics and autonomous sampling

Iron (Fe) governs the cycling of organic carbon in large parts of the Southern Ocean. The strategies of diverse microbes to acquire the different chemical forms of Fe under seasonally changing organic carbon regimes remain, however, poorly understood. Here, we report high-resolution seasonal metagenomic observations from the region off Kerguelen Island (Indian Sector of the Southern Ocean) where natural Fe-fertilization induces consecutive spring and summer phytoplankton blooms. Our data illustrate pronounced, but distinct seasonal patterns in the abundance of genes implicated in the transport of different forms of Fe and organic substrates, of siderophore biosynthesis and carbohydrate-active enzymes. The seasonal dynamics suggest a temporal decoupling in the prokaryotic requirements of Fe and organic carbon during the spring phytoplankton bloom and a concerted access to these resources after the summer bloom. Taxonomic assignments revealed differences in the prokaryotic groups harbouring genes of a given Fe-related category and pronounced seasonal successions were observed. Using MAGs we could decipher the respective Fe- and organic substrate-related genes of individual taxa assigned to abundant groups. The ecological strategies related to Fe-acquisition provide insights on how this element could shape microbial community composition with potential implications on organic matter transformations in the Southern Ocean.     

Host-guest Interactions in Intercalates Zr(HPO4)2·2C2H5OH and VOPO4·2C2H5OH

Molecular mechanics simulations using Cerius² modelling environment combined with vibrational spectroscopy (IR and Raman) have been used to study the host-guest interactions in zirconium and vanadyl phosphate intercalated with ethanole. The strategy of investigation is based on the comparison of vibrational spectra for the host compound, intercalate and guest species. This comparison confirmed the rigidity of VOPO₄- and Zr(HPO₄)₂-layers during the intercalation and provided us with the basis for the strategy of modelling. Molecular mechanics simulations revealed the structure of intercalates and enabled to analyse the host-guest interaction energy and bonding geometry. The bilayer arrangement of ethanole molecules in the interlayer space with two differently bonded ethanole molecules has been found in both intercalates. The average interaction energy ethanole-layer for two differently bonded ethanole molecules is : 127.5 and 135.7 kcal·mol^-1 in Zr(HPO₄)₂·2C₂H₅OH, respectively 94.0 and 104.4 kcal·mol^-1 in VOPO₄·2C₂H₅OH. The Coulombic contribution to the ethanole-layer interaction energy is predominant in all cases, but the hydrogen bonding contribution is much higher in Zr(HPO₄)₂·2C₂H₅OH than in VOPO₄·2C₂H₅OH. Present results of modelling enabled the interpretation of vibrational spectra and explanation of small changes in positions and shapes of spectral bands, in infrared and Raman spectra, proceeding from the host structure to intercalates.     

The human leucocyte surface antigen CD53 is a protein structurally similar to the CD37 and MRC OX-44 antigens

CD53 is an N-glycosylated pan-leucocyte antigen of 35–42 000 M _r. The sequence of the CD53 polypeptide deduced from a cDNA clone is 219 amino acids in length. It appears to lack a conventional leader sequence because the deduced NH₂-terminal amino acid sequence is very similar to the rat MRC OX-44 and human CD37 antigens. The CD53 molecule is likely to consist of four transmembrane regions and a major extracellular hydrophilic loop containing two potential N-glycosylation sites. It is suggested that the CD53 glycoprotein is the true human homologue of the rat OX-44 antigen, rather than the CD37 antigen of more restricted expression and lower NH₂-terminal sequence similarity to OX-44.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession number M37033. Offprint requests to: P. Angelisová.