Mesodermal fate decisions of a stem cell: the Wnt switch

Stem cells are a powerful resource for cell-based transplantation therapies in osteodegenerative disorders, but before some kinds of stem cells can be applied clinically, several aspects of their expansion and differentiation need to be better controlled. Wnt molecules and members of the Wnt signaling cascade have been ascribed a role in both these processes in vitro as well as normal development in vivo. However some results are controversial. In this review we will present the hypothesis that both canonical and non-canonical signaling are involved in mesenchymal cell fate regulation, such as adipogenesis, chondrogenesis and osteogenesis, and that in vitro it is a timely switch between the two that specifies the identity of the differentiating cell. We will specifically focus on the in vitro differentiation of adipocytes, chondrocytes and osteoblasts contrasting embryonic and mesenchymal stem cells as well as the role of Wnts in mesenchymal fate specification during embryogenesis

Factors affecting the energy-range relation of fast electrons in aluminium.

Factors which can influence measurements of the practical range of fast electrons are stated and discussed. These factors can be divided into geometrical parameters, like beam divergence, diameter of the beam and diameter of the detector, and into factors which influence particle energy. It is concluded that the energy-range relations of Katz and Penfold in aluminium and of Markus in water can be used in the energy range from 10 to 30 Mev with a maximum uncertainty of about 2%

Synchronization of mammalian cells by selection and additional chemical block studied by DNA distribution analysis and BrdUrd-Hoechst 33258-technique.

Ehrlich ascites tumour cells growing in vitro in suspension culture were separated according to volume by the technique of velocity sedimentation in a zonal rotor with a reorienting gradient. Using DNA distribution analysis the sedimentation pattern of the cells could be analysed in detail. With appropriate conditions it was possible to separate pure G1 cells. Samples could also be obtained which were enriched in S or G2 + M cells. The main limitation of the selection in this type of rotor was the reorientation of the gradient which caused disturbances during deceleration of the rotor. The synchronous growth of selected G1 cells has been studied in detail to investigate the reasons for the rather poor synchrony of these cells. The poor synchrony was found to be caused mainly by the small volume of the selected G1 cells compared with the normal volume of G1 cells in an asynchronous population. The synchronization of these cells could be essentially improved by a short treatment with excess thymidine causing a metabolic block at the G1/S border. The duration of this treatment could be minimized using DNA distribution analysis of growing cells after releasing of the block. The durations of the cell cycle phases in synchronized cells agreed with the values calculated in asynchronous cells by DNA distribution analysis and the BrdUrd-Hoechst 33258-technique

Analysis of micronuclei induced by 2-chlorobenzylidene malonitrile (CS) using fluorescence in situ hybridization with telomeric and centromeric DNA probes, and flow cytometry.

Micronuclei (MN) induced in NIH 3T3 cells by the tear gas 2-chlorobenzylidene malonitrile (CS) were studied in detail using fluorescence in situ hybridization (FISH). The chromosomal composition of CS-induced MN was analysed by simultaneous use of DNA probes for the telomeric hexamer repeat (TTAGGG) and for mouse major satellite DNA. The majority of CS-induced MN, 63-73% of all CS-induced MN at doses from 10 to 30 μM CS, revealed centromeric signals and several telomeric signals suggesting their origin from whole chromosomes. Almost 50% of all CS-induced MN showed one centromeric signal and were assumed to contain one single chromosome. Only 4.5% of all MN did not show any signal and 23-28% showed telomeric signals only, thus containing acentric fragments. Based on the experimental data from FISH the distribution of the DNA content of CS-induced MN was calculated assuming random breakage of chromosomes, and random combination of chromosomes and chromosome fragments. Good agreement between calculated MN distributions and distributions measured by flow cytometry was obtained. By sorting MN with distinct DNA content and hybridization of the sorted MN with the centromeric probe, regions in the MN distribution containing mainly MN with single whole chromosomes could be demonstrated

The calcium signal in human neutrophils and its relation to exocytosis investigated by patch-clamp capacitance and Fura-2 measurements.

Intracellular calcium ([Ca2+]i) and exocytosis of human neutrophils were investigated with patch-clamp capacitance and Fura-2 fluorescence measurements. Intracellular application of GTP gamma S induces a calcium transient and exocytosis. The onset of degranulation occurs at the time where the maximal [Ca2+]i is reached. Despite the close correlation in time, buffering [Ca2+]i at the resting level or at approximately 2 microM leaves the extent and the time course of degranulation unchanged. The decay of the calcium transient is due to diffusional equilibration between the cytosol and the pipette volume. GTP gamma S activates no cellular mechanisms for Ca2+ reuptake or extrusion. The endogenous calcium buffer capacity can be estimated to be as low as that of approximately 90 microM Fura-2. Stimulation with fMLP also induces degranulation and a calcium transient. The decay of fMLP-induced calcium transients is much faster than that of GTP gamma S-induced transients and is independent of diffusion indicating that fMLP also induces rapid reuptake or extrusion of Ca2+. Degranulation but not the calcium transient requires the presence of intracellular GTP. Different signalling pathways appear to be involved in GTP gamma S- and fMLP-stimulated calcium signals. The intracellular calcium release is not an essential signal to initiate exocytosis in neutrophils

Evidence that repair and expression of potentially lethal damage cause the variations in cell survival after X irratiation observed through the cell cycle in Ehrlich ascites tumor cells.

The survival of synchronously growing Ehrlich ascites tumor cells (EAT cells) was measured after X irradiation in various stages of the cell cycle. Cells at the beginning of S or in G2 + M phase showed a high level of killing, whereas cells irradiated in G1 or in the middle of S phase were more resistant. These changes resulted fom a change in the survival curve shoulder width (D(q)) as cells passed through the cell cycle, and the mean lethal dose (D(0)) remained practically unchanged (0.8 ± 0.05 Gy). When synchronization of the cell population was further sharpened using nocodazole, exponential survival curves were obtained at the beginning of S phase and at mitosis with a D(0) = 0.8 Gy. When cells (in all stages) were incubated in balanced salt solution for 6 hr after irradiation, repair of potentially lethal damage (PLD) was observed, resulting in an increase in D(q), while D(0) remained constant. Treatment of the cells after irradiation with either caffeine (2-6 mM) or β-arabinofuranosyladenine (β-araA) (60-100 μM) or hypertonic medium resulted in an expression of PLD and reduced the D(q) of the survival curve, which approached or reached an exponential line with D(0) = 0.8 ± 0.1 Gy. We measured the rate of the loss of sensitivity of these treatments that we assume reflects the rate of repair of PLD. For caffeine (6 mM) treatment (S cells, 5 hr) we found a repair time constant (t50) or about 1 hr, similar to that observed for repair of PLD in growth medium containing 0.5 μg/ml aphidicolin. With hypertonic treatment we detected two repair components, a fast one that restored the slope of the survival curve, and a slow one with a t50 of about 1 hr that restored the shoulder of the survival curve. PLD induced by irradiating in G1 phase was repaired when cells were arrested for some hours either in G1 phase or in the subsequent mitosis but was not repaired if the cells were arrested in S phase. PLD induced in S or G2 + M phase was repaired only when the cells were arrested in the cell cycle before division. Results indicate that the shoulder width D(q) of the survival curve in cells irradiated at various stages of the cell cycle results from repair of PLD. This repair of PLD probably takes place in the interval between irradiation and the next S phase or mitosis and is therefore minimal for cells irradiated at the G1/S border or in mitosis (D(q) = 0). PLD still unrepaired when the cells reach these phases is assumed to be expressed, as was found for PLD repaired in cells incubated in balanced salt solution, for some hours after irradiation. We therefore suggest that the variations observed in cell survival through the cell cycle might reflect variations in the final amount of PLD either repaired or expressed as the cells progress through the various stages of the cell cycle

GTPγS-induced calcium transients and exocytosis in human neutrophils.

Exocytosis and intracellular free calcium ([Ca2+]in) were simultaneously recorded in single human neutrophils using patch-clamp capacitance measurements and the fura-2 fluorescence ratio method. Intracellular application of guanosine-5′-O(3-thiotriphosphate) (GTPγS) stimulates both exocytosis and a calcium transient. The calcium transient starts to develop after a lag phase of ∼40s and normally appears to trigger the onset of exocytosis indicated by the beginning of the capacitance increase. After this delay [Ca2+]in increases from ∼150 nM to ∼600 nM with a sigmoidal time course. The peak concentration is reached within ∼30 s but the main increase occurs during ∼ 3s. [Ca2+]in subsequently decays within 1–2 min to a level which is close to the resting value. This calcium transient is due to calcium release from inositoltrisphosphate-sensitive intracellular stores. Exocytosis also occurs if the calcium transient is abolished by intracellular EGTA but the lag phase is markedly prolonged. The GTPγS-induced calcium transient is very similar to that observed after stimulation with N-formyl-methionyl-leucyl-phenylalanine. The interplay between guanine nucleotides, [Ca2+]in and exocytosis in neutrophils closely resembles previous results obtained in mast cells suggesting a similar regulation of exocytosis in both cell types