Impact of UV radiation on DOM transformation on molecular level using FT-ICR-MS and PARAFAC.

Dissolved organic matter (DOM) is an omnipresent constituent of natural water bodies. Reuse and transformation of DOM compounds in the water column is driven by physicochemical and biological processes leading to the production of refractory DOM. Typically, breakdown of DOM chemical compounds into smaller or more condensed fragments is triggered by ultraviolet (UV) radiation. Here, we present a study on the photodegradation of DOM produced during an incubation experiment with a natural microbial community. At the end of the first incubation without UV irradiation, the samples from 3 mesocosms were filtered to remove microbes and particles and continuously exposed to UV radiation (280-365 nm). We investigated DOM in depth via monitoring of dissolved organic carbon (DOC) concentrations, DOM molecular characterization by Fourier-Transform Ion Cyclotron Resonance Mass Spectrometry (FT-ICR-MS) and excitation emission matrix spectroscopy (EEMS). Analysis of variance indicated no significant differences in the DOC concentration between treatments. Main peaks in the fluorescent DOM (FDOM) were photo-bleached by UV radiation, and an increase in the fluorescent intensity of selected peaks was observed on irradiated samples toward the end of the experiment. Parallel factor analysis (PARAFAC) indicated the presence of three main components in all treatments: C1 (Marine humic M), C2 (Bacterial produced humic C), C3 (Tyrosine), and an additional component in the dark incubation of mesocosm 3, C4 (Tryptophan). Despite an intensive filtration protocol through 0.7, 0.2 and 0.1 µm filters, low bacterial abundances were determined (<2.5 × 10-3 cells mL-1). We observed a direct correlation between structural indices and the intensity of PARAFAC components. Average double bond equivalent and aromaticity were strongly positively correlated with PARAFAC components C1 and C2 for one or more mesocosm. Moreover, FT-ICR-MS showed that under the tested conditions, the refractory character of the DOM assessed as the similarity to a deep ocean DOM reference did not increase on molecular level. Thus, mechanisms other than photochemical transformations of relatively recent DOM are likely necessary to facilitate long-term stability of DOM in the oceans.

Long-term stability of marine dissolved organic carbon emerges from a neutral network of compounds and microbes.

Dissolved organic carbon (DOC) is the main energy source for marine heterotrophic microorganisms, but a small fraction of DOC resists microbial degradation and accumulates in the ocean. The reason behind this recalcitrance is unknown. We test whether the long-term stability of DOC requires the existence of structurally refractory molecules, using a mechanistic model comprising a diverse network of microbe-substrate interactions. Model experiments reproduce three salient observations, even when all DOC compounds are equally degradable: (i) >15% of an initial DOC pulse resists degradation, but is consumed by microbes if concentrated, (ii) the modelled deep-sea DOC reaches stable concentrations of 30-40 mmolC/m3, and (iii) the mean age of deep-sea DOC is several times the age of deep water with a wide range from <100 to >10,000 years. We conclude that while structurally-recalcitrant molecules exist, they are not required in the model to explain either the amount or longevity of DOC.

Climate-driven shifts in sediment chemistry enhance methane production in northern lakes.

Freshwater ecosystems are a major source of methane (CH4), contributing 0.65 Pg (in CO2 equivalents) yr-1 towards global carbon emissions and offsetting ~25% of the terrestrial carbon sink. Most freshwater CH4 emissions come from littoral sediments, where large quantities of plant material are decomposed. Climate change is predicted to shift plant community composition, and thus change the quality of inputs into detrital food webs, with the potential to affect CH4 production. Here we find that variation in phenol availability from decomposing organic matter underlies large differences in CH4 production in lake sediments. Production is at least 400-times higher from sediments composed of macrophyte litter compared to terrestrial sources because of inhibition of methanogenesis by phenol leachates. Our results now suggest that earth system models and carbon budgets should consider the effects of plant communities on sediment chemistry and ultimately CH4 emissions at a global scale.

Differential responses of marine, mesohaline and oligohaline bacterial communities to the addition of terrigenous carbon.

In response to global warming, increasing quantities of tDOM are transported through estuaries from land to the sea. In this study, we investigated microbial responses to increased tDOM concentrations in three salinity regimes (salinity: 32, 7 and 3) characteristic of the Baltic Sea. Mesocosm experiments performed in May and November revealed low (0-6%) dissolved organic carbon (DOC) utilisation. Molecular DOM analyses using ultrahigh-resolution mass spectrometry identified the terrigenous signal in the tDOM manipulation, but the molecular changes in DOM levels over the course of the experiment were subtle. However, tDOM had significant stimulatory effects on bacterial production in the oligohaline mesocosms. The shift in the bacterial community composition was especially prominent in the tDOM-amended marine and mesohaline mesocosms, but not in the oligohaline mesocosms after 7 and 11 days of incubation. These results suggested the inherent ability of oligohaline bacterial communities to adapt to high tDOM concentrations and therefore to use tDOM. The higher rates of bacterial activity and DOC removal in mesocosms containing UV-pretreated tDOM supported the increased bioavailability of photoinduced, modified tDOM. The overall low rates of microbial tDOM utilisation highlights the importance of abiotic factors in determining the distribution and dynamics of tDOM in estuaries.

Hydrogen retention in beryllium: concentration effect and nanocrystalline growth.

We herein report on the formation of BeD2 nanocrystalline domes on the surface of a beryllium sample exposed to energetic deuterium ions. A polycrystalline beryllium sample was exposed to D ions at 2 keV/atom leading to laterally averaged deuterium areal densities up to 3.5 10(17) D cm(-2), and studied using nuclear reaction analysis, Raman microscopy, atomic force microscopy, optical microscopy and quantum calculations. Incorporating D in beryllium generates a tensile stress that reaches a plateau at ≈1.5 10(17) D cm(-2). For values higher than 2.0 10(17) cm(-2), we observed the growth of ≈90 nm high dendrites, covering up to 10% of the surface in some zones of the sample when the deuterium concentration was 3 × 10(17) D cm(-2). These dendrites are composed of crystalline BeD2, as evidenced by Raman microscopy and quantum calculations. They are candidates to explain low temperature thermal desorption spectroscopy peaks observed when bombarding Be samples with D ions with fluencies higher than 1.2 10(17) D cm(-2).

What's in an EEM? Molecular signatures associated with dissolved organic fluorescence in boreal Canada.

Dissolved organic matter (DOM) is a master variable in aquatic systems. Modern fluorescence techniques couple measurements of excitation emission matrix (EEM) spectra and parallel factor analysis (PARAFAC) to determine fluorescent DOM (FDOM) components and DOM quality. However, the molecular signatures associated with PARAFAC components are poorly defined. In the current study we characterized river water samples from boreal Québec, Canada, using EEM/PARAFAC analysis and ultrahigh resolution mass spectrometry (FTICR-MS). Spearman's correlation of FTICR-MS peak and PARAFAC component relative intensities determined the molecular families associated with 6 PARAFAC components. Molecular families associated with PARAFAC components numbered from 39 to 572 FTICR-MS derived elemental formulas. Detailed molecular properties for each of the classical humic- and protein-like FDOM components are presented. FTICR-MS formulas assigned to PARAFAC components represented 39% of the total number of formulas identified and 59% of total FTICR-MS peak intensities, and included significant numbers compounds that are highly unlikely to fluoresce. Thus, fluorescence measurements offer insight into the biogeochemical cycling of a large proportion of the DOM pool, including a broad suite of unseen molecules that apparently follow the same gradients as FDOM in the environment.

The dark side of stem cells: triggering cancer progression by cell fusion.

The phenomenon of cell fusion plays a crucial role in a plethora of physiological processes, including fertilization, wound healing, and tissue regeneration. In addition to this, cell fusion also takes place during pathophysiological processes such as virus entry into host cells and cancer. Particularly in cancer, cell fusion has been linked to a number of properties being associated with the progression of the disease including an increased proliferation rate, an enhanced metastatogenic behavior, an increased drug resistance and an increased resistance towards apoptosis. Although the process of cell fusion including the molecules to be involved-in is not completely understood in higher organisms, recent data revealed that chronic inflammation seems to be strong mediator. Since tumor tissue resembles chronically inflamed tissue, it can be concluded that cell fusion between recruited macrophages, bone marrow-derived cells (BMDCs), and tumor (stem) cells should be a common phenomenon in cancer. In the present review, we will summarize how a chronic inflamed microenvironment could originate in cancerous tissues, the role of M2-polarized tumor associated macrophages (M2-TAMs) within this process and how fusion between macrophages and BMDCs will trigger cancer progression. A particular emphasis will be drawn on recurrence cancer stem cells (rCSCs), which will play a pivotal role in "oncogenic resistance" and which might originate from fusion events between tumor (stem) cells and BMDCs.

Nonlinear evolution of surface morphology under shadowing.

Fluorocarbon thin-film deposition is studied, which shows an anomalous high dynamic growth exponent and therefore does not fit in any universal class of fractal surface growth models. A detailed analysis of the nonlinear behavior of the surface morphology evolution is carried out, quantifying several features of the shadowing instability. A synergy effect with the Kardar-Parisi-Zhang nonlinearity, which couple the large scales induced by shadowing with intermediate scales, may explain the anomalous high growth exponent.

Characterization and analysis of migration patterns of dentospheres derived from periodontal tissue and the palate.

BACKGROUND AND OBJECTIVE: Stem cells derived from periodontal and palatal tissues may be useful for regenerative therapies of periodontal tissues. In addition to the use of single periodontium-derived stem cells (pdSCs) and palatal-derived stem cells (paldSCs), the application of pdSC and paldSC dentospheres, providing a pool of vital stem cells, may be a useful approach. As cell migration is a prerequisite for stem cells to regenerate a three-dimensional tissue environment, we characterized pdSCs and paldSCs and investigated the migratory activity of dentospheres within a three-dimensional environment. We also investigated the capacity of the dentospheres to grow on zirconium dioxide surfaces. MATERIAL AND METHODS: The capacity of pdSCs and paldSCs to differentiate into the neuronal and osteogenic lineages was proved by RT-PCR and immunohistochemistry through the detection of specific lineage markers, such as alkaline phosphatase, glutamate decarboxylase 1 (also known as GAD67, the 67-kDa isoform of glutamate decarboxylase), neurofilament-M and ß-III-tubulin. The expression profile of surface molecules on pdSCs and paldSCs was analyzed by flow cytometry. Adhesion and growth of pdSC/paldSC dentospheres on zirconium dioxide surfaces were determined using confocal laser-scanning microscopy. The migratory behavior of the cells was analyzed using a three-dimensional collagen matrix migration assay. RESULTS: Both pdSCs and paldSCs were positive for epidermal growth factor receptor, CC chemokine receptor 2 and CXC chemokine receptor 4 expression and were able to grow on zirconium dioxide surfaces. Cell-migration experiments revealed that both stem-cell populations responded similarly to epidermal growth factor (EGF), monocyte chemotactic protein 1 (MCP-1) and stromal cell-derived factor 1alpha (SDF-1α). Stimulation with EGF resulted in an increased migratory activity of both stem-cell types, whereas the locomotory behavior of the cells was impaired by both MCP-1 and SDF-1α. CONCLUSION: Dentospheres represent a pool of vital pdSCs/paldSCs. As a result of the migratory activity demonstrated, along with the capacity to grow on zirconium dioxide surfaces, dentospheres may be useful for regenerative purposes in periodontal tissues.

Morphological characterization of periodontium-derived human stem cells.

The aim of this study has been to characterize adult human somatic periodontium-derived stem cells (PDSCS) isolated from human periodontium and to follow their differentiation after cell culture. PDSCS were isolated from human periodontal tissue and cultured as spheres in serum-free medium. After 10 days the primary spheres were dissociated and the secondary spheres sub-cultured for another 1-2 weeks. Cells from different time points were analyzed, and immunohistochemical and electron microscopic investigations carried out. Histological analysis showed differentiation of spheres deriving from the PDSCS with central production of extracellular matrix beginning 3 days after sub-culturing. Isolated PDSCS developed pseudopodia which contained actin. Tubulin was found in the central portion of the cells. Pseudopodia between different cells anastomosed, indicating intercellular transport. Immunostaining for osteopontin demonstrated a positive reaction in primary spheres and within extracellular matrix vesicles after sub-culturing. In cell culture under serum-free conditions human PDSCS form spheres which are capable of producing extracellular matrix. Further investigations have do be carried out to investigate the capability of these cells to differentiate into osteogenic progenitor cells.

Expression of early placenta insulin-like growth factor in breast cancer cells provides an autocrine loop that predominantly enhances invasiveness and motility.

Early placenta insulin-like growth factor (EPIL) is expressed by a subpopulation of the Her2-positive SKBR3 breast cancer cell line displaying high motility and transendothelial invasiveness in vitro, as recently shown by our group. As a consequence of this, we established cellular models by generating an EPIL-overexpressing SKBR3 cell line, knocked down EPIL by adding specific small interfering RNA (siRNA) to those cells and produced EPIL-enriched and depleted serum-free culture media. EPIL-expressing cells as well as EPIL-induced SKBR3 cells acquired a high capacity for transendothelial invasiveness. We observed a thin and outspread morphology caused by enhanced formation of lamellipodia, i.e. protrusions in the initial phase of motility. In parallel, Her2-positive MDAHer2 breast cancer cells also showed increased invasiveness when induced by EPIL-conditioned medium. A downstream signaling impact of EPIL could be observed in the form of reduced phosphorylation of Her2, erk1/2 and akt, while phospholipase Cgamma1 phophorylation remained unaffected. As an in vivo model for highly motile tumor cells, Paget's disease of the nipple showed simultaneous EPIL and Her2 expression upon immunohistochemical examination using specific antibodies. Such experimental data have been translated to a clinical setting by using a prognostic tissue microarray established from 603 breast cancer cases. Survival data analysis found a significant association between expression levels of EPIL and 5-year overall survival that was dose dependent: EPIL (negative) 84%, EPIL (moderately positive) 77%, EPIL (strongly positive) 48% (P < 0.005). One particular subgroup (7.6% of the cases with full clinical records) that comprised tumors simultaneously expressing EPIL and Her2 represented patients with the poorest 5-year overall survival. The results suggested that EPIL might be a cancer cell-produced growth factor that influences lateral Her2 signaling. Moreover, EPIL may be induced by factors apart from Her2 and may independently provide signaling for cancer invasion and motility.

Propofol-induced calcium signalling and actin reorganization within breast carcinoma cells.

BACKGROUND AND OBJECTIVE: MDA-MB-468 breast carcinoma cells respond to non-volatile anaesthetics such as propofol with an increased migration. Here we investigated the relationship between GABA-A receptor modulators, the mode of calcium oscillation and actin reorganization with regard to breast carcinoma cell migration. METHODS: Expression of the GABA-A receptor was determined by Western blot analysis. Calcium-imaging experiments of individual MDA-MB-468 cells as well as visualization of the F-actin distribution were performed by confocal laser scanning microscopy. Cell migration was investigated in a three-dimensional collagen matrix by time-lapse video microscopy. The GABA agonist propofol was used in a final concentration of 6 microg mL(-1). GABA-A receptor antagonist bicuculline (50 micromol) and selective L-type calcium channel blocker verapamil (5 micromol) were used to modulate the propofol effects. RESULTS: A functional GABA-A receptor is expressed by MDA-MB-468 cells. Activation with propofol resulted in sustained increased intracellular calcium concentrations concomitant with actin reorganization and induction of migration in MDA-MB-468 cells. These propofol effects were completely blocked by verapamil. Spontaneous migration of MDA-MB-468 cells (64.4 +/- 7.0%) was significantly increased by propofol to 85.0 +/- 5.0%. MDA-MB-468 cells co-treated with propofol and verapamil showed a migratory activity of 63.0 +/- 2.0% indicating that verapamil blocked the propofol effect. Similar results were achieved with the GABA-A receptor inhibitor bicuculline (control: 56.3 +/- 8.5%; propofol: 80.5 +/- 7.1%; propofol + bicuculline: 52.5 +/- 8.6%). CONCLUSION: Activation of GABA-A receptor by propofol correlated with an increased migration of MDA-MB-468 breast carcinoma cells, mediated by calcium influx via L-type calcium channels and reorganization of the actin cytoskeleton.

Antitumour effects of PLC-gamma1-(SH2)2-TAT fusion proteins on EGFR/c-erbB-2-positive breast cancer cells.

Due to its pivotal role in the growth factor-mediated tumour cell migration, the adaptor protein phospholipase C-gamma1 (PLC-gamma1) is an appropriate target to block ultimately the spreading of EGFR/c-erbB-2-positive tumour cells, thereby minimising metastasis formation. Here, we present an approach to block PLC-gamma1 activity by using protein-based PLC-gamma1 inhibitors consisting of PLC-gamma1 SH2 domains, which were fused to the TAT-transduction domain to ensure a high protein transduction efficiency. Two proteins were generated containing one PLC-gamma1-SH2-domain (PS1-TAT) or two PLC-gamma1-SH2 domains (PS2-TAT). PS2-TAT treatment of the EGFR/c-erbB-2-positive cell line MDA-HER2 resulted in a reduction of the EGF-mediated PLC-gamma1 tyrosine phosphorylation of about 30%, concomitant with a complete abrogation of the EGF-driven calcium influx. In addition to this, long-term PS2-TAT treatment both reduces the EGF-mediated migration of about 75% combined with a markedly decreased time locomotion of single MDA-HER2 cells as well as decreases the proliferation of MDA-HER2 cells by about 50%. Due to its antitumoral capacity on EGFR/c-erbB-2-positive breast cancer cells, we conclude from our results that the protein-based PLC-gamma1 inhibitor PS2-TAT may be a means for novel adjuvant antitumour strategies to minimise metastasis formation because of the blockade of cell migration and proliferation.

Realtime visualization of tumor cell/endothelial cell interactions during transmigration across the endothelial barrier.

PURPOSE: In cancer the blood-borne spread of tumor cells leads to the formation of secondary tumors at distant loci whereby the extravasation of tumor cells is a prerequisite step during hematogenous metastasis. Here, we describe a novel in vitro realtime model which shows the complete sequence of the extravasation process. METHODS: We developed an in vitro system allowing us to monitor the sequence of extravasation events of tumor cell clusters across a monolayer of human umbilical cord endothelial cells (HUVEC). Fluorescence markers and laser scanning confocal microscopy were used to visualize the interactions between tumor cells and endothelium. RESULTS: Our model indicates that the extravasation of tumor cell clusters derived from the invasive human bladder carcinoma cell line T24 occurs in a relatively short time-frame up to 4 h after adhesion to the endothelium. We demonstrate that the vascular endothelium is irreversibly damaged at the site of tumor cell extravasation. CONCLUSION: Realtime laser scanning confocal microscopy leads to a better understanding of the complex and dynamic cell-to-cell and cell-to-matrix interactions during the extravasation process.

Semaphorin4F interacts with the synapse-associated protein SAP90/PSD-95.

Semaphorins are a family of secreted and membrane-associated proteins involved in growth cone guidance during development. Here, we describe the interaction of Semaphorin4F (Sema4F) with the post-synaptic density protein SAP90/PSD-95. Using the yeast two-hybrid system and coprecipitation assays we were able to show an interaction between the extreme C-terminus of Sema4F and the PDZ domains of SAP90/PSD-95. Heterologous coexpression of a chimeric EphrinB1/Semaphorin4F protein with SAP90/PSD-95 in COS cells leads to translocation of SAP90/PSD-95 from the cytosol to the membrane. Deletion analysis shows that this translocation activity of Sema4F is completely dependent on the presence of the last three C-terminal amino acids. In addition, Sema4F immunoreactivity is present in synaptosome fractions and enriched in post-synaptic density fractions. Consistently, in cultured hippocampal neurons, we demonstrate punctate colocalization of Sema4F and SAP90/PSD-95 in dendrites, furthermore we found colocalization of Sema4F with synapsin1 suggesting a synaptic localization. Our data implicate a new functional context for semaphorins at glutamatergic synapses.

Estrogen receptor (ER)-alpha, but not ER-beta, mediates regulation of the insulin-like growth factor I gene by antiestrogens.

The importance of insulin-like growth factor I (IGF-I) on maintenance of skeletal integrity has been widely recognized. Although osteoblasts secrete some IGF-I, the liver is the primary endocrine source for IGF-I. We have studied the regulation of the human IGF-I promoter in the hepatocyte cell line Hep3B, and we have shown that the IGF-I promoter, when co-transfected in Hep3B cells together with an estrogen receptor (ER)-alpha expression vector, was transcriptionally regulated by raloxifene or raloxifene-like molecules but not by 17beta-estradiol and 4(OH)-tamoxifen. The induction mediated by raloxifene is antagonized by 17beta-estradiol and mediated selectively by ER-alpha, but not by ER-beta. Transfer of IGF-I promoter sequences from -733 to -65 or from -375 to -65 to a minimal Fos promoter resulted in a comparable responsiveness to raloxifene. This region contains two CAAT/enhancer-binding protein sites and an activator protein 1 site, both of which have been shown to be involved in estrogen receptor-mediated transactivation. When the CAAT/enhancer-binding protein sites were mutated in a construct bearing the sequence from -375 to -65 in front of the minimal Fos promoter, raloxifene induction was reduced, whereas mutation of the other elements did not affect induction. In addition, using chimeric proteins, we delineated the domains of ER-alpha that confer to ER-alpha transactivation abilities on the IGF-I promoter that are not exhibited by ER-beta. These data shed new light on the mechanism of action of antiestrogens and might help explain, at least in part, the bone-protective effects observed for some antiestrogens in ovariectomized animals.

Accelerated assessing of antisense RNA efficacy using a chimeric enhanced green fluorescent protein-antisense RNA-producing vector.

The selection of suitable parts of a gene as antisense RNA sequences is largely a matter of trial and error and, as a consequence, a rather time-consuming process. In this study, we present a rapid and reproducible method to bypass this protracted procedure by using a chimeric enhanced green fluorescent protein (EGFP)-antisense RNA-producing vector. The combination of a reporter gene and antisense RNA allows easy measurement by flow cytometry of antisense RNA efficacy in successfully transfected cells shortly after transfection. Four chimeric EGFP-p185c-erbB-2-antisense RNA vectors were constructed and transfected into the p185-c-erbB-2-overexpressing cell line SKBR3. Within 1 week, we were able to estimate the inhibitory capacities of the different antisense RNA sequences used in this study. Our results strongly suggest that a chimeric EGFP-antisense RNA vector is an appropriate tool to expedite the laboratory work and time in screening the efficacy of antisense RNA strategies.

Lessons from tumor and immunocompetent cells. The quantitative engagement of ligand-receptor interactions modulates stop-and-go behavior as well as proliferation.

The four main cell functions, proliferation, apoptosis, differentiation and migration, are tightly regulated by external signals that initiate intracellular signal transduction pathways and determine the cellular behaviour. The concentration and composition of such external signals are at least important for the decision of cells as to which function has to be executed. Interleukin-8 is a well known inducing signal for neutrophil granulocyte migration, while the epidermal growth factor is an inducing signal for breast carcinoma cell migration. Depending on the concentrations of interleukin-8, the neutrophil granulocytes are capable of migration. However, at high concentration of interleukin-8 the migratory activity of each single cell is reduced, indicating that high concentrations of the chemokine inhibit migration and promote the performance of other cell functions. Concerning breast carcinoma cells, the epidermal growth factor is not only an inducer of migration but also an inhibitor of proliferation. These two examples provide evidence for a dose dependent action of external signals for several cell functions in parallel. This versatility of the effects of one ligand might be based on several intracellular signal transduction pathways that are turned on. For the dose-dependent differences of the effect of interleukin-8 we propose a two wheel model of an inositolphosphate-mediated, ATP-independent release of calcium from intracellular stores and a cyclic AMP-mediated, ATP-dependent uptake of calcium into the endoplasmatic reticulum.

Lessons from tumor and immunocompetent cells. The quantitative engagement of ligand-receptor interactions modulates stop-and-go behavior as well as proliferation

The four main cell functions, proliferation, apoptosis, differentiation and migration, are tightly regulated by external signals that initiate intracellular signal transduction pathways and determine the cellular behaviour. The concentration and composition of such external signals are at least important for the decision of cells as to which function has to be executed. Interleukin-8 is a well known inducing signal for neutrophil granulocyte migration, while the epidermal growth factor is an inducing signal for breast carcinoma cell migration. Depending on the concentrations of interleukin-8, the neutrophil granulocytes are capable of migration. However, at high concentration of interleukin-8 the migratory activity of each single cell is reduced, indicating that high concentrations of the chemokine inhibit migration and promote the performance of other cell functions. Concerning breast carcinoma cells, the epidermal growth factor is not only an inducer of migration but also an inhibitor of proliferation. These two examples provide evidence for a dose dependent action of external signals for several cell functions in parallel. This versatility of the effects of one ligand might be based on several intracellular signal transduction pathways that are turned on. For the dose-dependent differences of the effect of interleukin-8 we propose a two wheel model of an inositolphosphate-mediated, ATP-independent release of calcium from intracellular stores and a cyclic AMP-mediated, ATP-dependent uptake of calcium into the endoplasmatic reticulum.