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.     

Strategies for the identification and purification of ligands for orphan biomolecules

The isolation of related genes with evolutionary conserved motifs by the application ofpolymerase chain reaction-based molecular biology techniques, or from database searchingstrategies, has facilitated the identification of new members of protein families. Many of theseprotein molecules will be involved in protein–protein interactions (e.g. growth factors,receptors, adhesion molecules), since such interactions are intrinsic to virtually every cellularprocess. However, the precise biological function and specific binding partners of these novelproteins are frequently unknown, hence they are known as orphan molecules.Complementary technologies are required for the identification of the specific ligands orreceptors for these and other orphan proteins (e.g., antibodies raised against crude biologicalextracts or whole cells). We describe herein several alternative strategies for the identification,purification and characterisation of orphan peptide and protein molecules, specifically thesynergistic use of micropreparative HPLC and biosensor techniques.     

A spatial model of interspecific competition and selective predation: The case of the two Hippolais

Mutual exclusion between congeneric species has been observed such as the case of the grey and red squirrels in Great Britain and the case of the twoHippolais warbler speciesHippolais icterina andH. polyglotta in Europe. This process can lead to the formation of an extinction wave which propagates. Two main assumptions are tested, competition and selective predation. The aim of this work is to present spatial models of these two processes. The animals of two species are assumed to move on a two dimensional array of spatial patches with local interactions of competition or of selective predation between them. We focus on the case of mutual exclusion. Initially, the two competing species occupy complementary areas in an array of spatial patches with a small common zone. Numerical simulations show that under particular conditions, one species gets extinct and the other invades the whole set of spatial patches. These simulations show that with time, the length of the overlapping zone stabilizes and moves at a constant velocity. The limit length of the overlapping band and the velocity of the extinction wave are found to be functions of the parameters of the models. We relate this general model to the case of two sibling species of birds:H. icterina andH. polyglotta.     

Functional Morphology of the Introvert and Digestive System of Myzostoma cirriferum (Myzostomida)

Myzostoma cirriferum feeds by diverting food particles carried by the ambulacral grooves of its comatulid host Antedon bifida. When searching for food, the myzostome uses its protrusible introvert to fulfil two major functions: sensory perception and the capture of food particles. The digestive system is composed of four parts, viz. a pharynx, that is contained within the introvert, a stomach, a series of paired caeca and an intestine that lie in the myzostome's trunk. The pharynx is supplied with a thick muscle which, thanks to peristaltic movements, carries food particles from the mouth to the stomach. Both stomach and caecal cells are able to absorb dissolved nutriments and to store lipids, whereas intestinal cells are only capable of absorption. Due to the beating of their cilia, stomach cells also carry food particles into the caecal lumen, where they are subjected to endocytosis and intracellular digestion by caecal cells. Undigested food fragments eventually gather in a very large, apical vacuole, and the cell apices containing vacuoles are eliminated into the caecal lumen by an apocrinal process. Detached cell apices reach the stomach, where they are embedded in a matrix, together forming a spindle-shaped faecal mass that is expelled through the postero-ventral anus. The observed digestive process—entailing the regular elimination of the apical part of the caecal digestive cells—appears to be unique among the Spiralia.     

Stabilization of the ternary complex EF-Tu.GTP.valyl-tRNA by ammonium salts

In a search for crystallizing conditions for the ternary complex EF-Tu.GTP.valyl-tRNA^val, the influence of various salts on its stability has been examined by measuring the rate of deacylation of the aminoacyl-tRNA in the complex. The most striking result is the general higher stability in solutions of ammonium salts and, in particular, the enhancement of this effect by sulphate and citrate. Thus sodium sulphate and citrate lead to destabilization of the complex, as expected from conventional considerations of adding salt, whereas the corresponding ammonium salts stabilize the complex as shown, for example, by an increase in the half-life of the valyl-tRNA^val in the complex from about 20 hours to at least 300 hours in the presence of 1.2 M ammonium sulphate. These results suggest that ammonium sulphate and ammonium citrate might be very suitable precipitants for crystallization studies of the ternary complex.     

Differential effects of manganese ions on Blastocladiella emersonii adenylate cyclase.

Adenylate cyclase (ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1) activity in Blastocladiella emersonii is associated with particulate subcellar fractions. Solubilization after treatment with detergent suggests its localization in a membrane fraction of the zoospore homogenate. The enzyme specifically requires Mn2+ for activity and is not stimulated by NaF. The kinetic characteristics of substrate utilization by B. emersonii adenylate cyclase were investigated with various concentrations of ATP and Mn2+, and in the presence of inhibitors. Plots of enzyme activity versus the actual concentration of the MnATP2- complex give sigmoid curves. An excess of Mn2+ activates the enzyme at low concentrations of substrate and leads to a modification of the enzyme kinetics. The nucleotides 5'-AMP and GTP were shown to be competitive inhibitors of the enzyme. In addition, kinetic data, obtained under conditions in which an inhibitor (ATP) is added in constant proportion to the variable substrate (MnATP2-) concentration, produced reciprocal plots that were linear and intersecting to the right of the ordinate, and secondary replots that were hyperbolic. These kinetic patterns support a model in which: MnATP2- is the substrate; free Mn2+ is an activator at low substrate concentrations, but an inhibitor at high substrate concentrations; and free ATP is not an efficient inhibiyor (Ki greater than 1.10(-4) M).     

Conformational properties of the N-terminal residues of S-peptide. I. The ribonuclease S′ system

The contribution of the 1–6 N-terminal sequence to the conformational properties of the S-peptide (the 1–20 sequence of ribonuclease A) was assessed by determining in the ribonuclease S′ system the helical content and the binding capability of synthetic [Orn¹⁰]-S-peptide analogs, in which lysine¹, glutamic² and threonine³ were progressively deleted, alanine⁴ and alanine⁵were alternatively replaced by serine, and alanine⁶ was substituted by serine or proline. Both the deletion of the three N-terminal residues and the alanine⁶/proline replacement produces the loss of the helical structure up to lysine⁷. No or minor effects are found in all other cases. From the comparison of the binding data, the energy for the conformational stabilization of the N-terminal region was calculated to amount to 1.4 kcal/mol. The results are discussed in comparison with the known x-ray data of the enzyme, with some predictive rules of secondary structure which were applied to this region and with the known phylogenetic variance of the residues in this region.