Flow cytometric detection of mitotic cells using the bromodeoxyuridine/DNA technique in combination with 90 degrees and forward scatter measurements

Mitotic cells could be well discriminated from the cells in the G1-, S- and G2-phases of the cell cycle using pulse labeling of S-phase cells with bromodeoxy-uridine (BrdUrd) and staining of the cells for incorporated BrdUrd and total DNA content. Unlabeled G2- and M-phase cells could be measured as two separate peaks according to propidium iodide fluorescence. M-phase cells showed lower propidium iodide fluorescence emission compared to G2-phase cells. The fluorescence difference of M- and G2-phase cells was caused by the different thermal denaturation of their DNA. Best separation of M- and G2-phase cells was obtained after 30-50 min heat treatment at 95 degrees C. Mitotic index could be measured if no unlabeled S-phase cells were present in the cell culture. With additional measurements of 90 degree scatter and/or forward scatter signals, mitotic cells could be clearly discriminated from both unlabeled G2- and S-phase cells. The correct discrimination (about 99%) of mitotic cells from interphase cells was verified by visual analysis of the nuclear morphology after selective sorting. Unlabeled and labeled mitotic cells could be observed as pulse-labeled cells progressed through the cell cycle. We conclude that this modified BrdUrd/DNA technique using prolonged thermal denaturation and the simultaneous measurement of scatter signals may offer additional information especially in the presence of BrdUrd-unlabeled S-phase cells.     

Automatic control of the extra-corporal bypass: system analysis, modelling and evaluation of different control modes.

Automatic control of the blood gas parameters during extracorporeal circulation has the potential to improve the quality of this procedure and to relieve the personnel from a time consuming task. This paper describes a model of the underlying system for a standard clinical set-up and pinpoints the major difficulties which are the variations of the process gains and the blood- and gas-flow dependent dead times and time constants. Scheduled PI-controllers both for the arterial oxygen as well as for the carbon dioxide partial pressure were designed. Scheduling was based on the blood flow rate. These controllers were tested in a simulation environment. The control systems remained stable under all tested operating condition, but if the blood flow rate was changed abruptly rather large load errors occurred. The performance was improved markedly by adding a feed-forward control path which directly influences the actuating signals based on the actual blood flow rate and the hemoglobin contents, variables which are measured anyway. The major conclusion of this study is to use such direct feed-forward compensation even if more sophisticated control algorithms are used.     

A new Cephalaria species (Dipsacaceae) from the European part of Turkey

A new species, Cephalaria tuteliana, (Dipsacaceae) from the European part of Turkey is described and illustrated. The characteristics of the new species, and its relationships with Cephalaria tenuiloba is discussed.     

Crystal structure and conformational analysis of ampullosporin A.

Ampullosporin A is a 15-mer peptaibol type polypeptide that induces pigment formation by the fungus Phoma destructiva, forms voltage-dependent ion channels in membranes and exhibits hypothermic effects in mice. The structure of ampullosporin A has been determined by x-ray crystallography. This is the first three-dimensional (3D) structure of the peptaibol subfamily SF6. From the N-terminus to residue 13 the molecule adopts an approximate right-handed alpha-helical geometry, whereas a less regular structure pattern with beta-turn characteristics is found in the C-terminus. Even though ampullosporin A does not contain a single proline or hydroxyproline it is significantly bent. It belongs to both the shortest and the most strongly bent peptaibol 3D structures. The straight structure part encompasses residues Ac-Trp(1)-Aib(10) and is thus less extended than the alpha-helical subunit. The 3D structure of ampullosporin A is discussed in relation to other experimentally determined peptaibol structures and in the context of its channel-forming properties. As a part of this comparison a novel bending analysis based on a 3D curvilinear axis describing the global structural characteristics has been proposed and applied to all 3D peptaibol structures. A sampling of 2500 conformations using different molecular dynamics protocols yields, for the complete ampullosporin A structure, an alpha-helix as the preferred conformation in vacuo with almost no bend. This indicates that solvent or crystal effects may be important for the experimentally observed peptide backbone bending characteristics of ampullosporin A.     

Conjugative plasmid transfer in gram-positive bacteria.

Conjugative transfer of bacterial plasmids is the most efficient way of horizontal gene spread, and it is therefore considered one of the major reasons for the increase in the number of bacteria exhibiting multiple-antibiotic resistance. Thus, conjugation and spread of antibiotic resistance represents a severe problem in antibiotic treatment, especially of immunosuppressed patients and in intensive care units. While conjugation in gram-negative bacteria has been studied in great detail over the last decades, the transfer mechanisms of antibiotic resistance plasmids in gram-positive bacteria remained obscure. In the last few years, the entire nucleotide sequences of several large conjugative plasmids from gram-positive bacteria have been determined. Sequence analyses and data bank comparisons of their putative transfer (tra) regions have revealed significant similarities to tra regions of plasmids from gram-negative bacteria with regard to the respective DNA relaxases and their targets, the origins of transfer (oriT), and putative nucleoside triphosphatases NTP-ases with homologies to type IV secretion systems. In contrast, a single gene encoding a septal DNA translocator protein is involved in plasmid transfer between micelle-forming streptomycetes. Based on these clues, we propose the existence of two fundamentally different plasmid-mediated conjugative mechanisms in gram-positive microorganisms, namely, the mechanism taking place in unicellular gram-positive bacteria, which is functionally similar to that in gram-negative bacteria, and a second type that occurs in multicellular gram-positive bacteria, which seems to be characterized by double-stranded DNA transfer.     

Mechanical versus drug prevention of thrombosis after total hip endoprosthesis implantation. A randomized, controlled clinical study]

Pharmacological prophylaxis is routinely applied after total hip replacement. Although it effectively reduces deep-vein thrombosis, side effects (bleeding, haematoma, swelling, thrombocytopenia) are not infrequent. Since in Germany use of foot pumps as only means of prophylaxis is unpopular, we investigated their efficacy and safety in a randomized study. 106 patients used either low molecular weight heparin (Fraxiparin, Sanofi-Synthelabo, Germany) or the foot-pump (A-V Impulse System, Orthofix, Mühltal, Germany), and were monitored for deep-vein thrombosis using serial duplex sonography on postoperative days 4, 12 and 45. Clinical observations included daily measurements of thigh circumference, recording of postoperative drainage amounts, and monitoring of wound healing. None of the 50 patients treated with the foot-pump developed deep-vein thrombosis, while 4 of the 50 patients (8 per cent) on pharmacological prophylaxis did so. Six patients stopped using the foot-pump during the study. One patient developed heparin-induced thrombocytopenia. Patients on mechanical prophylaxis had smaller amounts of drainage (mean 247 ml vs. 272 ml, p = 0.485) and significantly less swelling of the thigh (10 mm compared with 15 mm, p or = < 0.001), The good results in terms of prevention of thromboembolic complications and soft tissue swelling favour the general use of foot pumps as mechanical prophylaxis.     

Coastal plain halophytes and their relation to soil ionic composition

Halophytic coastal communities in Bahia Blanca, Argentina, were examined in relation to soil salinity along a transect from inland to the sea. The relationships between soil electrical conductivity and dominance-abundance values for different species were determined. The Chañaral community with its most conspicuous species Geoffroea decorticans was present in soils with the lowest salinity levels in the area. Halophytic underbush develops at soil salinity values of intermediate soil electrical conductivity, the characteristic species being Cyclolepis genistoides, Atriplex undulata and Salicornia ambigua. Halophytic shrub-like steppe vegetation, dominated by Allenrolfea patagonica or Heterostachys ritteriana, is present in soils with high salinity. It is concluded that the ionic composition of plants was a stable characteristic for different species and did not vary with changes in soil salinity.     

Mercury translocation in and evaporation from soil. III. quantification of evaporation of mercury from podzolized soil profiles treated with Hg Cl

Mercury evaporation from undisturbed iron‐humus podzol lysimeters was measured over 3 months after treatment with HgCl₂ spiked with radioactive ²⁰³Hg. The relative evaporation rate from HgCl₂ treated soils followed the sum of two exponential functions. Because evaporation asymptotically approaches zero with time, the integral of the fit curve represents the evaporative loss in percent of atmospheric deposition. For the soil investigated, about 5% of atmospheric Hg deposition was reemitted into the atmosphere. It is hypothesized that mercury evaporation can decrease the leaching of mercury in and from soil significantly; this effect is probably increasing with decreasing rain acidity or soil acidity. Mercury deposited as soluble salt remains susceptible to reemission to air for 300 d after incorporation into the soil matrix. Indications are found that Hg evaporation from soils in geological background areas predominantly derives from recent atmospheric Hg deposition and not from geological sources.     

Do NOE distances contain enough information to assess the relative populations of multi-conformer structures?

Summary The feasibility of determining the relative populations of multi-conformer structures from NOE-derived distances alone is assessed. Without cross-validation of the NOE restraints, any population ratio can be refined to a similar quality of the fit. Complete cross-validation provides a less biased measure of fit and allows the estimation of the correct population ratio when used in conjunction with very tight distance restraints. With the qualitative distance restraints most commonly used in NMR structure determination, cross-validation is unsuccessful in providing the correct answer. Other experimental sources are therefore needed to determine relative populations of multi-conformer structures.To whom correspondence should be addressed.     

The new program OPAL for molecular dynamics simulations and energy refinements of biological macromolecules

Summary A new program for molecular dynamics (MD) simulation and energy refinement of biological macromolecules, OPAL, is introduced. Combined with the supporting program TRAJEC for the analysis of MD trajectories, OPAL affords high efficiency and flexibility for work with diferent force fields, and offers a user-friendly interface and extensive trajectory analysis capabilities. Salient features are computational speeds of up to 1.5 GFlops on vector supercomputers such as the NEC SX-3, ellipsoidal boundaries to reduce the system size for studies in explicit solvents, and natural treatment of the hydrostatic pressure. Practical applications of OPAL are illustrated with MD simulations of pure water, energy minimization of the NMR structure of the mixed disulfide of a mutant E. coli glutaredoxin with glutathione in different solvent models, and MD simulations of a small protein, pheromone Er-2, using either instantaneous or time-averaged NMR restraints, or no restraints.Abbreviations D diffusion constant in cm²/s - Er-2 pheromone 2 from Euplotes raikovi - GFlop one billion floating point operations per second - Grx(C14S)-SG mixed disulfide between a mutant E. coli glutaredoxin, with Cys¹⁴ replaced by Ser, and glutathione - MD molecular dynamics - NOE nuclear Overhauser enhancement - rmsd root-mean-square deviation - density in g/cm³