The pathway from arachidonic to docosapentaenoic acid (20:4n-6 to 22:5n-6) and from eicosapentaenoic to docosahexaenoic acid (20:5n-3 to 22:6n-3) studied in testicular cells from immature rats

The concentration-dependent metabolism of 1-¹⁴C-labelled precursors of 22:5n-6 and 22:6n-3 was compared in rat testis cells. The amounts of [¹⁴C]22- and 24-carbon metabolites were measured by HPLC. The conversion of [1-¹⁴C]20:5n-3 to [3-¹⁴C]22:6n-3 was more efficient than that of [1-¹⁴C]20:4n-6 to [3-¹⁴C]22:5n-6. At low substrate concentration (4 μM) it was 3.4 times more efficient, reduced to 2.3 times at high substrate concentration (40 μM). The conversion of [1-¹⁴C]22:5n-3 to [1-¹⁴C]22:6n-3 was 1.7 times more efficient than that of [1-¹⁴C]22:4n-6 to [1-¹⁴C]22:5n-6 using a low, but almost equally efficient using a high substrate concentration. When unlabelled 20:5n-3 was added to a cell suspension incubated with [1-¹⁴C]20:4n-6 or unlabelled 22:5n-3 to a cell suspension incubated with [1-¹⁴C]22:4n-6, the unlabelled n-3 fatty acids strongly inhibited the conversion of [1-¹⁴C]20:4n-6 or [1-¹⁴C]22:4n-6 to [¹⁴C]22:5n-6. In the reciprocal experiment, unlabelled 20:4n-6 and 22:4n-6 only weakly inhibited the conversion of [1-¹⁴C]20:5n-3 and [1-¹⁴C]22:5n-3 to [¹⁴C]22:6n-3. The results indicate that if both n-6 and n-3 fatty acids are present, the n-3 fatty acids are preferred over the n-6 fatty acids in the elongation from 20- to 22- and from 22- to 24-carbon atom fatty acids. In vivo the demand for 22-carbon fatty acids for spermatogenesis in the rat may exceed the supply of n-3 precursors and thus facilitate the formation of 22:5n-6 from the more abundant n-6 precursors.     

The fate of 15NH4 + labeled deposition in a Scots pine forest in the Netherlands under high and lowered NH4 + deposition, 8 years after application

To study the long-term fate of deposited ammonium (NH₄ ⁺) in a Scots pine forest stand under high nitrogen (N) deposition in the Netherlands we re-sampled the plots of a ¹⁵N tracer experiment with high (i.e. ambient) and lowered N deposition in this stand 8 years after application of the tracer. The results were compared with results obtained 7 years earlier. In the 7 years between the samplings the ¹⁵N deltas of needles, twigs and upper organic soil layer had converged to similar values still above the natural ¹⁵N abundance, suggesting equilibration as a result of intensive cycling of N among these pools. Bark and wood had lower deltas than needles and twigs, but if the label found was attributed to tissue synthesized since the start of the labeling only, bark values were similar to needles and twigs, whereas wood values were higher indicating retranslocation of N into older wood. Mineral soil lost all ¹⁵N label it had accumulated after 1 year indicating that this label had not been strongly bound. The first year the low N treatment had retained more of the labeled NH₄ ⁺ deposition than the high N treatment, but in the seven subsequent years relatively more label was retained in the latter. This better retention after 7 years was ascribed to a larger fraction of label taken up by the vegetation in the high N treatment. This shows that the vegetation can affect the label dynamics despite the fact that only a relatively small amount of label was present in the aboveground vegetation.     

N-terminal and core-domain random mutations in human topoisomerase II α conferring bisdioxopiperazine resistance

Random mutagenesis of human topoisomerase II α cDNA followed by functional expression in yeast cells lacking endogenous topoisomerase II activity in the presence of ICRF-187, identified five functional mutations conferring cellular bisdioxopiperazine resistance. The mutations L169F, G551S, P592L, D645N, and T996L confer >37, 37, 18, 14, and 19 fold resistance towards ICRF-187 in a 24 h clonogenic assay, respectively. Purified recombinant L169F protein is highly resistant towards catalytic inhibition by ICRF-187 in vitro while G551S, D645N, and T996L proteins are not. This demonstrates that cellular bisdioxopiperazine resistance can result from at least two classes of mutations in topoisomerase II; one class renders the protein non-responsive to bisdioxopiperazine compounds, while an other class does not appear to affect the catalytic sensitivity towards these drugs. In addition, our results indicate that different protein domains are involved in mediating the effect of bisdioxopiperazine compounds.     

Hydration of human nails investigated by NIR-FT-Raman spectroscopy.

The human nail, although it is usually stable against outer influences, becomes soft and flexible after soaking in water. Frequent washing increases brittleness of nails. Hydration of nails is thought to be the most important factor influencing the physical properties of nails and possibly acts through changes in keratin structure. Here NIR-FT-Raman has been used to examine molecular structural changes of intact moisten nails. Raman spectra were obtained both in vitro from nail samples and in vivo before and after soaking in water. The water uptake of normal nail samples during the first 15 min was reflected in the increasing intensity ratio of the nu(OH)/nu(CH(2)) bands. A saturating effect appeared soon after 10 min which is explained by a defined water holding capacity. R(nu) representation of the low frequency range of the Raman spectra showed that mainly bound water is found both in dry and in wet nails. This implies water-protein interaction. Protein backbone vibration at 932 cm(-1) indicating alpha-helical proteins increased in intensity in the wet nails. The nu(S-S) which is sensitive to changes in conformation of proteins showed a 4% higher intensity. Additional protein-water interactions could lead to a slight change of the dihedral angle of the C-S-S-C bonds and to geometric changes in coiling behavior of the alpha-helical protein. Suggesting a separation between matrix proteins and fiber proteins giving them a greater freedom of flexibility. The in vivo spectra detected from the distal part of the nail resembled spectra in vitro. Raman spectra of the proximal part of the nail showed that it was fully saturated with water. The proximal part of the nail did not show changes in water content and protein structure during nail moisturizing in the Raman spectra. Our results suggest that the softening of the nail following hydration may be due to changed matrix protein molecular structure induced by water.     

In situ autofluorescence detection of a fouling marine diatom on different surfaces

A new technique for quantifying fouling diatoms adhering to different surfaces was developed and tested. The method is based on recording the in vivo chlorophyll autofluorescence of diatom cells in situ. The enhanced signal obtained after addition of DCMU was used as a biomass estimate, and the enhancement itself as an indicator of the photosynthetic capacity. A fluorescence spectrometer equipped with a “well plate reader”; accessory was programmed to scan predetermined positions on the microscope slide based test surfaces. In standard tests, a matrix of 7 × 25 equidistant locations was applied to record the central 17 × 67 mm² area of the test surface. Thus, both spatial distributions and the mean value of chlorophyll associated with the specified area could be obtained directly. Microscopical counting was performed for calibration on transparent glass surfaces as well as PVA‐SbQ based hydrogels. There was a good correlation between counting and fluorescence recordings, with a linear range up to 1100 cells mm^?2. Due to the inherent inaccuracies of background estimates, the detection limit on glass and gel was approximately 200 cells mm^?2. The method was also applied successfully to test non‐transparent surfaces. In addition to standard mass screening of different test surfaces, the method may be found useful in studies of algal physiology related to cell adhesion, photosynthesis, growth, detachment and spatial migration.