Cloning and expression analysis of an ADP-ribosylation factor from Solanum tuberosum L.

Summary A cDNA clone encoding an ADP-ribosylation factor from potato (Solanum tuberosum L.) was isolated. The nucleotide and deduced amino acid sequences show high homology to known ADP-ribosylation factor sequences from Arabidopsis, yeast, cow and man. In northern blot experiments, all tissues analysed showed expression of the corresponding mRNA. Strongest expression was found, however, in potato tubers.Abbreviations ARF, ADP ribosylation factor - BSA bovine serum albumin - EDTA ethylenediaminetetraacetic acid - SDS sodium dodecyl sulfate - SSC sodium saline citrate     

Design of a bubble-swawm bioreactor for animal cell culture

A stationary bubble-swarm has been used to aerate a mammalian cell culture bioreactor with an extremely low gas flow rate. Prolonging the residence time of the gas bubbles within the medium improved the efficiency of the gas transfer into the liquid phase and suppressed foam formation. An appropriate field of speed gradients prevented the bubbles from rising to the surface. This aeration method achieves an almost 90% transfer of oxygen supplied by the bubbles. Consequently, it is able to supply cells with oxygen even at high cell densities, while sparging with a gas flow of only 0.22·10^–3–1.45·10^–3 vvm (30–200 ml/h).The reactor design, the oxygen transfer rates and the high efficiency of the system are presented. Two repeated batch cultures of a rat-mouse hybridoma cell line are compared with a surface-aerated spinner culture. The used cell culture medium was serum-free, either with or without BSA and did not contain surfactants or other cell protecting agents. One batch is discussed in detail for oxygen supply, amino acid consumption and specific antibody production.     

A spectral correlation function for efficient sequential NMR assignments of uniformly 15N-labeled proteins

Summary A new computer-based approach is described for efficient sequence-specific assignment of uniformly ¹⁵N-labeled proteins. For this purpose three-dimensional ¹⁵N-correlated [¹H, ¹H]-NOESY spectra are divided up into two-dimensional ¹H-¹H strips which extend over the entire spectral width along one dimension and have a width of ca. 100 Hz, centered about the amide proton chemical shifts along the other dimension. A spectral correlation function enables sorting of these strips according to proximity of the corresponding residues in the amino acid sequence. Thereby, starting from a given strip in the spectrum, the probability of its corresponding to the C-terminal neighboring residue is calculated for all other strips from the similarity of their peak patterns with a pattern predicted for the sequentially adjoining residue, as manifested in the scalar product of the vectors representing the predicted and measured peak patterns. Tests with five different proteins containing both -helices and -sheets, and ranging in size from 58 to 165 amino acid residues show that the discrimination achieved between the sequentially neighboring residue and all other residues compares well with that obtained with an unguided interactive search of pairs of sequentially neighboring strips, with important savings in the time needed for complete analysis of 3D ¹⁵N-correlated [¹H, ¹H]-NOESY spectra. The integration of this routine into the program package XEASY ensures that remaining ambiguities can be resolved by visual inspection of the strips, combined with reference to the amino acid sequence and information on spin-system types obtained from additional NMR spectra.Abbreviations 1D, 2D, 3D, 4D one-, two-, three-, four-dimensional - NOE nuclear Overhauser enhancement - NOESY nuclear Overhauser enhancement spectroscopy - COSY correlation spectroscopy - TOCSY total correlation spectroscopy     

Solution structure by 1H and dynamics by natural abundance 13C NMR of a receptor recognising peptide derived from a C-terminal fragment of neuropeptide Y

Summary A peptide consisting of 20 amino acid residues, derived from a C-terminal fragment of neuropeptide Y (NPY) and showing high affinity to NPY receptors, was synthesised. Its sequence is PAADLARYRHYIN-LITRQRY-NH₂, and the solution structure was calculated from NMR-derived distance and torsion angle restraints, obtained at 15°C in a solvent mixture of water and 30% (v/v) 1,1,1,3,3,3-hexafluoro-2-propanol, by using DIANA and restrained energy minimisation. The structure was found to consist of a well-defined -helix in the centre, with a few residues at the termini having less well defined conformations. The spinlattice and spin-spin relaxation rates of -carbons have been determined on ¹³C at natural abundance. From 1D experiments the global rotational correlation time was determined and from 2D experiments the dynamics of each individual residue was obtained. The results demonstrate that the C-H vectors in the -helix essentially follow the global motion. Towards the termini, contributions from local dynamics increase. This tendency is correlated to the increasing uncertainty of the structure towards the peptide ends. An effective molecular volume was calculated from the temperature dependence of the global rotational correlation time. This is well compatible with a monomeric peptide, solvated by water and 1,1,1,3,3,3-hexafluoro-2-propanol. The presence of peptide dimers was ruled out as being inconsistent with the relaxation data.Supplementary material available from the authors: Two data tables and 10 PDB coordinate files of the calculated NMR structures of P7. One data table contains all detected and integrated NOE intensities; the other connects each proton and pseudoatom to an atom number used in the NOE table. The table contents served as input data files for CALIBA.Currently on leave from the Institute of Chemical Physics and Biophysics, Tallinn, Estonia.     

HNCCH-TOCSY,a triple resonance experiment for the correlation of backbone 13Cα and 15N resonances with aliphatic side-chain proton resonances and for measuring vicinal 13CO, 1Hβ coupling constants in proteins

Summary A 3D triple resonance experiment has been designed to provide intraresidual and sequential correlations between amide nitrogens and -carbons in uniformly ¹³C¹⁵N-labeled proteins. In-phase ¹³C magnetization is transferred to the aliphatic side-chain protons via the side-chain carbons using a CC-TOCSY mixing sequence. Thus, the experiment alleviates the resonance assignment process by providing information about the amino acid type as well as establishing sequential connectivities. Leaving the carbonyl spins untouched throughout the transfer from ¹³C to ¹H leads to E.COSY-type cross peaks, from which the ³J_{H co} coupling constants can be evaluated. The pulse sequence is applied to oxidized Desulfovibrio vulgaris flavodoxin.     

Cell-substrate adhesion and metastatic potential of cultured mesothelioma cells induced by asbestos

Cell-substrate adhesion was quantified for two cultured mesothelioma cell lines (epitheliomatus and sarcomatous) on glass, fibronectin and laminin substrates. Interference reflection microscopy (IRM) was used to image the adhesion patterns of cells and a grey level analysis was employed to quantify adhesion. Sarcomatous cells demonstrated marked adhesion to glass and fibronectin-coated substrates but not to laminin-coated substrate, with the greatest adhesion occurring on the fibronectin-coated surface. This adhesion was accompanied by cytoplasmic spreading. By contrast, epitheliomatous cells showed little tendency to adhere to any of the substrates and only showed significant spreading when in contact with the laminin substrate (P < 0.01). A bioassay was used to determine the metastatic potential of each of the cell lines. Via the intravenous route, the sarcomatous cells killed the host rats in 24.7 ± 1.5 (S.D.) days compared to 27.3 ± 0.9 (S.D.) days for the epitheliomatous cells (P < 0.01). After subcutaneous inoculation of tumour cells, the sarcomatous cells killed the host rats in 54.7 ± 0.7 (S.D.) days compared to 48.5 ± 0.5 (S.D.) days for the epitheliomatous cells (P < 0.01). We conclude that the results of the metastasis bioassays were consistent with the predicted behavior of these cell lines based on their ability to adhere to substrates in the in vitro adhesion assays.     

Temperate rainforest lichens in New Zealand: high thallus water content can severely limit photosynthetic CO2 exchange

CO₂ exchange rate in relation to thallus water content (WC, % of dry weight) was determined for 22 species of lichens, mainly members of the genera Pseudocyphellaria and Sticta, from a temperate rainforest, Urewere National Park, New Zealand. All data were obtained in the field, either using a standard technique in which the lichens were initially wetted (soaked or sprayed, then shaken) and allowed to slowly dry, or from periodic measurements on samples that were continuously exposed in their natural habitat. A wide range of WC was found, with species varying from 357 to 3360% for maximal WC in the field, and from 86 to 1300% for optimal WC for photosynthesis. Maximal WC for lichens, wetted by the standard technique, were almost always much less than the field maxima, due to the presence of water on the thalli. The relationships between CO₂ exchange rate and WC could be divided into four response types based on the presence, and degree, of depression of photosynthesis at high WC. Type A lichens showed no depression, and Type B only a little at maximal WC. Type C had a very large depression and, at the highest WC, CO₂ release could occur even in the light. Photosynthetic depression commenced soon after optimal WC was reached. Type D lichens showed a similar depression but the response curve had an inflection so that net photosynthesis was low but almost constant, and never negative, at higher WC. There was little apparent relationship between lichen genus or photobiont type and the response type. It was shown that high WC does limit photosynthetic CO₂ uptake under natural conditions. Lichens, taken directly from the field and allowed to dry under controlled conditions, had net photosynthesis rates that were initially strongly inhibited but rose to an optimum, before declining at low WC. The limiting effects of high WC were clearly shown when, under similar light conditions, severe photosynthetic depression followed a brief, midday, rain storm. Over the whole measuring period the lichens were rarely at their optimal WC for photosynthesis, being mostly too wet or, occasionally, too dry. Photosynthetic performance by the lichens exposed in the field was similar to that expected from the relationship between the photosynthetic rate and WC established by the standard procedure.     

Behavioural and morphological changes in ciliates induced by the predator Amoeba proteus

The predator Amoeba proteus induced behavioural and morphological changes in ciliates of the genus Euplotes. The frequency of avoidance behaviour in E. octocarinatus increased from 16±5% to 84±5% (SD) after 14 h of coexistence with the predator. The ciliate's width increased from 59±3 μm to 77±4 μm (SDM) within 48 h. Similar behavioural, but not morphological, change was induced in E. daidaleos, but neither morphological nor behavioural responses occurred in E. aediculatus. E. octocarinatus and E. daidaleos populations survived in the presence of A. proteus, whereas E. aediculatus populations became extinct by predation. Induced behavioural response seemed to be the reason for the low predation risk of E. octocarinatus and E. daidaleos. The results suggest that Euplotes ciliates have evolved specific defence mechanisms to various predators. Defensive changes are induced by a chemical substance released from A. proteus. This “kairomone” has a molecular weight between 5000 and 10000 Da. Proteolytic digestion of its activity indicated that the avoidance-inducing substance is a peptide. After the turbellarian Stenostomum sphagnetorum had induced a defensive morphology in E. octocarinatus or E. aediculatus, neither of these ciliates immediately avoided Amoeba proteus. Thus, Euplotes ciliates with a defensive morphology do not have behavioural defences in reaction to all predators.     

Effects of short-term salinity on leaf gas exchange of the fig (Ficus carica L.)

The influence of short-term salinity (day 1–day 2: 50 mol m^–3 NaCl, day 3–day 7: 100 mol m^–3 NaCl in the nutrient solution) on leaf gas exchange characteristics were studied in two fig clones (Ficus carica L.), whose root mass had been varied in relation to the leaf area. The stomatal conductance was diminished by NaCl in the first week of treatment. NaCl slightly reduced the calculated intercellular partial pressure of CO₂. The net photosynthetic rate of plants with many roots was stimulated by NaCl on some days of the first week of treatment, whereas the net assimilation rate of the plants with few roots remained unaltered or decreased by NaCl. Only the assimilation of the salt-treated plants of one clone for some days during the first week of treatment seemed to be influenced by stomatal conductance. Nonstomatal factors were primarily responsible for the changes in CO₂ uptake in response to salt and/or root treatment. The water use efficiency increased during several days of the first week of NaCl treatment. Decreased stomatal conductance, increased water use efficiency and stimualtion of the net CO₂ assimilation rate appear to enhance salt tolerance during the first few days of salinity. ei]H Lambers