Neodiprion sertifer defoliation causes long-term systemic changes of oxidative enzyme activities in Scots pine needles

The aim of the experiments reported here was to study possible long-term effects of Neodiprion sertifer Geoff. (Hymenoptera: Diprionidae) herbivory, or artificial defoliation, on oxidative enzyme activities in Scots pine (Pinus sylvestris L.) needles as a consequence of induced defense responses. During year 1 (the first season), defoliation by N. sertifer, which feeds on previous season’s needles, did not result in statistically significant changes in polyphenol oxidase activity in the current year’s needles. In contrast, defoliation did lead to increased peroxidase activity in those needles. In the second season (year 2) N. sertifer defoliation of pine seedlings, also defoliated in the previous season either by larvae or artificially, resulted in a decrease of peroxidase activity in the current year’s needles. No significant differences between treatments carried out in year 1 were found in year 2 for peroxidase activity in the previous year’s needles. However, defoliation in year 1 by N. sertifer resulted in decreased needle consumption, and higher mortality of larvae, in year two. These results indicate the existence of long-term changes in needle oxidative enzyme activities as a consequence of N. sertifer feeding.     

Calcium entry through receptor-operated channels in bovine pulmonary artery endothelial cells

The activation of endothelial cells by endothelium-dependent vasodilators has been investigated using bioassay, patch clamp and 45Ca flux methods. Cultured pulmonary artery endothelial cells have been demonstrated to release EDRF in response to thrombin, bradykinin, ATP and the calcium ionophore A23187. The resting membrane potential of the endothelial cells was -56 mV and the cells were depolarized by increasing extracellular K+ or by the addition of (0.1-1.0 mM)Ba2+ to the bathing solution. The electrophysiological properties of the cultured endothelial cells suggest that the membrane potential is maintained by an inward rectifying K+ channel with a mean single channel conductance of 35.6 pS. The absence of a depolarization-activated inward current and the reduction of 45Ca influx with high K+ solution suggests that there are no functional voltage-dependent calcium or sodium channels. Thrombin and bradykinin were shown to evoke not only an inward current (carried by Na+ and Ca2+) but also an increase in 45Ca influx suggesting that the increase in intracellular calcium necessary for EDRF release is mediated by an opening of a receptor operated channel. High doses of thrombin and bradykinin induced intracellular calcium release, however, at low doses of thrombin no intracellular calcium release was observed. We propose that the increased cytosolic calcium concentration in endothelial cells induced by endothelium dependent vasodilators is due to the influx of Ca2+ through a receptor operated ion channel and to a lesser degree to intracellular release of calcium from a yet undefined intracellular store.     

DNA damage and repair in the differentiation of stem cells and cells of connective cell lineages: A trigger or a complication?

The review summarizes literature data on the role of DNA breaks and DNA repair in the differentiation of pluripotent stem cells (PSC) and connective cell lineages. PSC, including embryonic stem cells (ESC) and induced pluripotent stem cells (iPSC), are rapidly dividing cells with highly active DNA damage response (DDR) mechanisms to ensure the stability and integrity of the DNA. In PSCs, the most common DDR mechanism is error-free homologous recombination (HR) that is primarily active during the S phase of the cell cycle, whereas in quiescent, slow-dividing or non-dividing tissue progenitors and terminally differentiated cells, errorprone non-homologous end joining (NHEJ) mechanism of the double-strand break (DSB) repair is dominating. Thus, it seems that reprogramming and differentiation induce DNA strand breaks in stem cells which itself may trigger the differentiation process. Somatic cell reprogramming to iPSCs is preceded by a transient increase of the DSBs induced presumably by the caspase-dependent DNase or reactive oxygen species. In general, pluripotent stem cells possess stronger DNA repair systems compared to differentiated cells. Nonetheless, during a prolonged cell culture propagation, DNA breaks can accumulate due to the DNA polymerase stalling. Consequently, the DNA damage might trigger the differentiation of stem cells or replicative senescence of somatic cells. The differentiation process per se is often accompanied by a decrease in the DNA repair capacity. Thus, the differentiation might be triggered by DNA breaks, alternatively, the breaks can be a consequence of the decay in the DNA repair capacity of differentiated cells.Key words: DNA breaks, DNA repair, differentiation, stem cells, connective tissue     

Establishment of two new multi-drug resistant variants of the human tumor line Hep-2

Two multi-drug resistant variants of the human carcinoma line Hep-2 have been selected by adaptation to progressively increasing concentrations of adriamycin. In comparison to the wild-type Hep-2 cells, the variant lines both showed approximately 100-fold resistance to adriamycin, 10 to 20-fold resistance to the vinca alkaloids but only 2–3 fold resistance to VP-16 and VM-26. There was essentially no difference between wild-type and variant cells in regard to sensitivity to threosulfan and 5-fluorouracil. The drug-resistant phenotype is stable for at least 3 months in the absence of drug, and is partially reversible by concomitant treatment with Verapamil. Chromosomal abnormalities consistent with gene amplification were observed in one of the variant lines. Sensitivity of variant cells to adriamycin was enhanced following trypsin-EDTA treatment.     

Use of microcarriers to isolate and culture pulmonary microvascular endothelium

Microcarriers of known diameter can be used to collect endothelial cells from microvessels of the same or slightly smaller internal diameter. The procedure is illustrated by collection of endothelial cells from rabbit pulmonary pre-capillary vessels. The lungs are perfused free of blood with physiological saline and then cold (4 °C) saline (containing EDTA, 0.02 %, and microcarriers 600/ml; 40–60 μm diameter) is perfused via the pulmonary artery. The direction of flow is reversed periodically to collect the bead-cell harvest from the arterial side. Cold shock and EDTA cause the endothelial cells to detach from the vessels under conditions such that the cells remain attached to the microcarriers. The selective attachment to microcarriers is apparently aided by the tight fit of the beads within vessels of the same diameter. Beads do not emerge on the venous side, all being trapped at the pre-capillary level. Electron microscopic examination of lungs fixed during the perfusion shows that the beads lodge in terminal arterioles and pre-capillary vessels (approximately 40–60 μm in diameter, with one, sometimes incomplete, muscle layer). Endothelial cells recovered on microcarriers can be allowed to migrate on to flasks and back on to beads. The resultant cultures have an endothelial morphology and possess high levels of angiotensin coverting enzyme and carboxypeptidase N activity.