The effects of acclimation and rearing conditions on the response of tropical and temperate populations ofDrosophila melanogaster andD. simulans to a temperature gradient (Diptera: Drosophilidae)

The effects of rearing and acclimation on the response of adultDrosophila to temperature were investigated in a gradient.D. melanogaster flies preferred a higher mean temperature and were distributed over a wider range of temperatures thanD. simulans flies. Acclimating adults at different temperatures for a week did not influence the response of either species. Adults reared at 28°C as immatures had a lower mean preference than those reared at cooler temperatures, suggesting that flies compensated for the effects of rearing conditions. Adults from tropical and temperate populations ofD. melanogaster andD. simulans did not differ in the mean temperature they preferred in a gradient, suggesting little genetic divergence for this trait within species. The species differences and environmental responses may be related to changes in optimal physiological conditions for the flies.     

Membrane function and vascular reactivity

This communication examines the possibility that nitric oxide (NO) production by endothelial cells results from changes in cell membrane fluidity. Lysophosphatidylcholine (LPC) alters fluidity of the endothelial cell membranes causing vascular relaxation. Through membrane alterations LPC influences function of a number of membrane receptors and modulates enzyme activity. As a result of detergent action, lysophosphatidylcholine (LPC) causes activation of guanylate cyclase, stimulates syalytransferase and regulates protein kinase C activity. It has already been demonstrated that ionic detergents, such as Triton X-100 also cause vascular relaxation, possibly induced by NO production from endothelial cells. It is postulated that production of nitric oxide results from changes in membrane viscosity; this may represent a mechanism for its regulation in biological systems.     

Fusion protein mediated prodrug activation (FMPA) in vivo

A two component system, consisting of a fusion protein and an appropriate prodrug, suited to perform selective tumor therapy in vivo, is presented. The fusion protein, owing to its humanized carcinoembryonic antigen (CEA)-specific variable region, specifically binds to CEA-expressing tumors and has an enzymatic activity comparable to human β-glucuronidase. The prodrug is a nontoxic glucuronide-spacer-derivative of doxorubicin decomposing to doxorubicin by enzymatic deglucuronidation. In vivo studies in nude mice bearing human CEA-expressing tumor xenografts revealed that 7 d after injection of 20 mg/kg fusion protein, a high specificity ratio (>100:1) was obtained between tumor and plasma. Injection of 250 mg/kg of prodrug at d 7 resulted in tumor therapeutic effects superior to conventional chemotherapy without any detectable toxicity. These superior therapeutic effects that were observed using established human tumor xenografts can be explained by the approx 10-fold higher drug concentrations found in tumors of mice treated with fusion protein and prodrug than in those treated with the maximal tolerable dose of drug alone.     

Na+, K+, Cl− cotransport and its regulation in Ehrlich ascites Tumor cells. Ca2+/Calmodulin and protein kinase C dependent pathways

Summary Net Cl^– uptake as well as unidirectional³⁶Cl influx during regulatory volume increase (RVI) require external K⁺. Half-maximal rate of bumetanide-sensitive³⁶Cl uptake is attained at about 3.3mm external K⁺. The bumetanide-sensitive K⁺ influx found during RVI is strongly dependent on both Na⁺ and Cl^–. The bumetanide-sensitive unidirectional Na⁺ influx during RVI is dependent on K⁺ as well as on Cl^–. The cotransporter activated during RVI in Ehrlich cells, therefore, seems to transport Na⁺, K⁺ and Cl^–. In the presence of ouabain and Ba⁺ the stoichiometry of the bumetanide-sensitive net fluxes can be measured at 1.0 Na⁺, 0.8 K⁺, 2.0 Cl^– or approximately 1 : Na, 1 : K, 2 : Cl. Under these circumstances the K⁺ and Cl^– flux ratios (influx/efflux) for the bumetanide-sensitive component were estimated at 1.34 ±0.08 and 1.82 ± 0.15 which should be compared to the gradient for the Na⁺, K⁺, 2Cl^– cotransport system at 1.75 ± 0.24.Addition of sucrose to hypertonicity causes the Ehrlich cells to shrink with no signs of RVI, whereas shrinkage with hypertonic standard medium (all extracellular ion concentrations increased) results in a RVI response towards the original cell volume. Under both conditions a bumetanide-sensitive unidirectional K⁺ influx is activated. During hypotonic conditions a small bumetanide-sensitive K⁺ influx is observed, indicating that the cotransport system is already activated.The cotransport is activated 10–15 fold by bradykinin, an agonist which stimulates phospholipase C resulting in release of internal Ca²⁺ and activation of protein kinase C.The anti-calmodulin drug pimozide inhibits most of the bumetanide-sensitive K⁺ influx during RVI. The cotransporter can be activated by the phorbol ester TPA. These results indicate that the stimulation of the Na⁺, K⁺, Cl^– cotransport involves both Ca²⁺/calmodulin and protein kinase C.     

Volume regulation in human fibroblasts: role of Ca2+ and 5-lipoxygenase products in the activation of the Cl− efflux

Trypsinized human skin fibroblasts in suspension perform regulatory volume decrease (RVD) after cell swelling in hypotonic medium. During RVD, ³⁶Cl^– efflux is dramatically increased and the cell membrane is depolarized, indicating the activation of Cl^– channels. This activation of Cl^– channels depends on extracellular as well as on intracellular Ca²⁺. The swelling-induced Cl^– efflux and the RVD response are inhibited by the 5-lipoxygenase inhibitor ETH 615-139. Finally, following hypotonic treatment, cellular pH decreases. The pH decrease does not involve the Cl^–/HCO ₃ ^– exchange because it is independent of the external Cl^– concentration.T. Mastrocola was recipient of a scientific fellowship from the Italian Consiglio Nazionale delle Ricerche (C.N.R.). This work was supported by Progetto Finalizzato Ingegneria Genetica, C.N.R., Roma, and by the Danish Natural Research Council.     

Regulation of taurine transport in Ehrlich ascites tumor cells

Summary Taurine influx is inhibited and taurine efflux accelerated when the cell membrane of Ehrlich ascites tumor cells is depolarized. Taurine influx is inhibited at acid pH partly due to the concomitant depolarization of the cell membrane partly due to a reduced availability of negatively charged free carrier. These results are in agreement with a 2Na, 1Cl, 1taurine cotransport system which is sensitive to the membrane potential due to a negatively charged empty carrier. Taurine efflux from Ehrlich cells is stimulated by addition of LTD4 and by swelling in hypotonic medium. Cell swelling in hypotonic medium is known to result in stimulation of the leukotriene synthesis and depolarization of the cell membrane. The taurine efflux, activated by cell swelling, is dramatically reduced when the phospholipase A₂ is inhibited indirectly by addition of the anti-calmodulin drug pimozide, or directly by addition of RO 31-4639. The inhibition is in both cases lifted by addition of LTD₄. The swelling-induced taurine efflux is also inhibited by addition of the 5-lipoxygenase inhibitors ETH 615-139 and NDGA. It is concluded that the swelling-induced activation of the taurine leak pathway involves a release of arachidonic acid from the membrane phospholipids and an increased oxidation of arachidonic acid into leukotrienes via the 5-lipoxygenase pathway. LTD₄ seems to act as a second messenger for the swelling induced activation of the taurine leak pathway either directly or indirectly via its activation of the Cl^– channels, i.e., via a depolarization of the cell membrane.     

The influence of lecithin on plasma choline concentrations in triathletes and adolescent runners during exercise

An investigation was carried out on the effect of lecithin (phosphatidylcholine, 90%) on the plasma choline concentrations during continuous strain in 10 top level triathletes (4 women and 6 men), trial I, and 13 excellent adolescent runners (3 girls and 10 boys), trial II. Venous blood, collected before and immediately after the race, was separated and plasma was assayed by an improved high performance liquid chromatography method for choline. Each study comprised three experiments. In trial I the triathletes performed two periods of bicycle exercise each lasting 2 h at an average speed of 35 km · h^–1, and in the second study (trial II) the subjects were subjected to severe physical stress on two occasions during cross-country races of durations between 30–60 min according to their ages. The participants received either a placebo or 0.2 g lecithin · kg body mass^–1, 1 h before each exercise. As a control the same dose of lecithin was administered without any exercise (both trials I and II). Bicycle exercise without lecithin supply decreased plasma choline concentrations in all the triathletes, on average by 16.9% (P0.01). When lecithin was given before exercise, average plasma choline concentrations remained at the same level as the initial values. The supply of lecithin without exercise led to a significant increase of the plasma choline concentrations, on average by 26.9% (P0.01). In trial II, when running without a supply of lecithin, the mean plasma choline concentrations in the adolescent runners remained stable which may have been due to the duration of the physical stress. When lecithin was given before exercise, plasma choline concentrations increased, on average by 18.9% (P0.01). The administration of lecithin without exercise led in these participants to an increase in plasma choline concentrations, on average by 54% (P0.001).It was found from the present study that a combination of both lecithin intake and hard physical stress prevented in most subjects a decrease in plasma choline and this could affect performance.     

Phosphorylation and activation of human cdc25-C by cdc2--cyclin B and its involvement in the self-amplification of MPF at mitosis.

We have investigated the mechanisms responsible for the sudden activation of the cdc2-cyclin B protein kinase before mitosis. It has been found previously that cdc25 is the tyrosine phosphatase responsible for dephosphorylating and activating cdc2-cyclin B. In Xenopus eggs and early embryos a cdc25 homologue undergoes periodic phosphorylation and activation. Here we show that the catalytic activity of human cdc25-C phosphatase is also activated directly by phosphorylation in mitotic cells. Phosphorylation of cdc25-C in mitotic HeLa extracts or by cdc2-cyclin B increases its catalytic activity. cdc25-C is not a substrate of the cyclin A-associated kinases. cdc25-C is able to activate cdc2-cyclin B1 in Xenopus egg extracts and to induce Xenopus oocyte maturation, but only after stable thiophosphorylation. This demonstrates that phosphorylation of cdc25-C is required for the activation of cdc2-cyclin B and entry into M-phase. Together, these studies offer a plausible explanation for the rapid activation of cdc2-cyclin B at the onset of mitosis and the self-amplification of MPF observed in vivo.     

The unexpected long period of elevated CH4 emissions from an inundated fen meadow ended only with the occurrence of cattail (Typha latifolia)

Drainage and agricultural use transform natural peatlands from a net carbon (C) sink to a net C source. Rewetting of peatlands, despite of high methane (CH₄) emissions, holds the potential to mitigate climate change by greatly reducing CO₂ emissions. However, the time span for this transition is unknown because most studies are limited to a few years. Especially, nonpermanent open water areas often created after rewetting, are highly productive. Here, we present 14 consecutive years of CH₄ flux measurements following rewetting of a formerly long-term drained peatland in the Peene valley. Measurements were made at two rewetted sites (non-inundated vs. inundated) using manual chambers. During the study period, significant differences in measured CH₄ emissions occurred. In general, these differences overlapped with stages of ecosystem transition from a cultivated grassland to a polytrophic lake dominated by emergent helophytes, but could also be additionally explained by other variables. This transition started with a rapid vegetation shift from dying cultivated grasses to open water floating and submerged hydrophytes and significantly increased CH₄ emissions. Since 2008, helophytes have gradually spread from the shoreline into the open water area, especially in drier years. This process was periodically delayed by exceptional inundation and eventually resulted in the inundated site being covered by emergent helophytes. While the period between 2009 and 2015 showed exceptionally high CH₄ emissions, these decreased significantly after cattail and other emergent helophytes became dominant at the inundated site. Therefore, CH₄ emissions declined only after 10 years of transition following rewetting, potentially reaching a new steady state. Overall, this study highlights the importance of an integrative approach to understand the shallow lakes CH₄ biogeochemistry, encompassing the entire area with its mosaic of different vegetation forms. This should be ideally done through a study design including proper measurement site allocation as well as long-term measurements.