The anti-tumor alkylphospholipid perifosine is internalized by an ATP-dependent translocase activity across the plasma membrane of human KB carcinoma cells

Perifosine is a promising anticancer alkylphospholipid (ALP) that induces apoptosis in tumor cells. Here we report evidences against a role of endocytosis in perifosine uptake by human KB carcinoma cells. We have generated a KB cell line resistant to perifosine (KB PER(R) clone10), which shows cross-resistance to the ALPs miltefosine and edelfosine, a marked impairment in the uptake of (14)C-perifosine at both 37 degrees C and 4 degrees C, and no signs for active efflux of the drug. KB PER(R) clone10 cells show a similar rate of raft-dependent endocytosis with respect to the parental cells, and silencing of both clathrin and dynamin in the latter causes only minor changes in the rate of perifosine uptake. Perifosine uptake is a temperature- and ATP-dependent, N-ethylmaleimide- and orthovanadate-sensitive process in parental cells. Accumulation of (14)C-perifosine and the fluorescent phospholipid analogue 6-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)aminocaproyl]-phosphatidylethanolamine (NBD-PE) is inhibited by perifosine in a concentration-dependent manner in parental cells. Moreover, NBD-PE accumulation is slower in PER(R) clone10 cells and correlated with phosphatidylserine exposure in their plasma membrane surface. Together, all these data suggest a role of plasma membrane translocation by a putative phospholipid translocase, rather than endocytosis, as the true mechanism for ALPs uptake in KB carcinoma cells.     

Anticancer mechanisms and clinical application of alkylphospholipids

Synthetic alkylphospholipids (ALPs), such as edelfosine, miltefosine, perifosine, erucylphosphocholine and erufosine, represent a relatively new class of structurally related antitumor agents that act on cell membranes rather than on DNA. They selectively target proliferating (tumor) cells, inducing growth arrest and apoptosis, and are potent sensitizers of conventional chemo- and radiotherapy. ALPs easily insert in the outer leaflet of the plasma membrane and cross the membrane via an ATP-dependent CDC50a-containing ‘flippase’ complex (in carcinoma cells), or are internalized by lipid raft-dependent endocytosis (in lymphoma/leukemic cells). ALPs resist catabolic degradation, therefore accumulate in the cell and interfere with lipid-dependent survival signaling pathways, notably PI3K-Akt and Raf-Erk1/2, and de novo phospholipid biosynthesis. At the same time, stress pathways (e.g. stress-activated protein kinase/JNK) are activated to promote apoptosis. In many preclinical and clinical studies, perifosine was the most effective ALP, mainly because it inhibits Akt activity potently and consistently, also in vivo. This property is successfully exploited clinically in highly malignant tumors, such as multiple myeloma and neuroblastoma, in which a tyrosine kinase receptor/Akt pathway is amplified. In such cases, perifosine therapy is most effective in combination with conventional anticancer regimens or with rapamycin-type mTOR inhibitors, and may overcome resistance to these agents. This article is part of a Special Issue entitled Phospholipids and Phospholipid Metabolism.     

Perifosine induces cell cycle arrest and apoptosis in human hepatocellular carcinoma cell lines by blockade of Akt phosphorylation

Hepatocellular carcinoma (HCC) is one of the most common solid cancers, representing the third cause of cancer-related death among cirrhotic patients. Treatment of advanced HCC has become a very active area of research. Perifosine, a new synthetic alkylphospholipid Akt inhibitor, has shown anti-tumor activity by inhibition of Akt phosphorylation. In this study, the effect of perifosine on the cell proliferation and apoptosis in hepatoma cells has been investigated. Cell growth inhibition was detected by MTT assay, cell cycle was analyzed by flow cytometry, AnnexinV-FITC apoptosis detection kit was used to detect cell apoptosis, and protein expression was examined by Western blotting analysis. Our present studies showed that Akt phosphorylation was inhibited by perifosine in HepG2 and Bel-7402 human hepatocellular carcinoma cells. Perifosine inhibited the growth of HepG2 cells and Bel-7402 cells in a dose-dependent manner, and arrested cell cycle progression at the G₂ phase. Apoptosis induction became more effective with increasing perifosine concentration. The caspase cascade and its downstream effectors, Poly (ADP-ribose) polymerase (PARP), were also activated simultaneously upon perifosine treatment. The proapoptotic effect of perifosine was in part depending on regulation of the phosphorylation level of ERK and JNK. Perifosine cotreatment substantially increased cytotoxic effects of cisplatin in HepG2 cells. Down-regulating the expression of Bcl-2 and up-regulating the level of Bax may be the potential mechanism for this synergistic effect. Our findings suggest that the small molecule Akt inhibitor perifosine shows substantial anti-tumor activity in human hepatoma cancer cell lines, and is a good candidate for treatment combinations with classical cytostatic compounds in hepatocellular carcinoma.     

Validation of an in vitro screen for phospholipidosis using a high-content biology platform

Several cationic amphiphilic drugs cause local or systemic phospholipidosis (PLD) after chronic exposure in preclinical species. PLD is characterized by the accumulation of drug, phospholipid, and concentric lamellar bodies in cellular lysosomes. We have developed a fluorescence-based in vitro screen that is predictive of PLD using the Cellomics ArrayScan high-content screening platform, which captures and analyzes images from 96-well cell culture microtiter plates using multichannel fluorescence microscopy. I-13.35 adherent mouse spleen macrophage cells were cultured with drug and a fluorescently tagged phospholipid, N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (NBD-PE). Drug concentrations were used in a range from 1 to $100 μmol/L. After 24 h incubations, the cells were fixed with formalin. NBD-PE uptake was quantified in controls and treated cells. Nuclei were identified by Hoechst 33258 staining and dead cells were identified using ethidium homodimer-2 incorporation. Thus, confounding accumulation of NBD-PE due to cytotoxicity that produces false-positive results at high concentrations was eliminated from quantitation by ethidium staining and employing cell gating (dead cell rejection). The assay was found to be both sensitive and selective in that 26 of 28 positive, phospholipidogenic controls and 8 of 8 negative, nonphospholipidogenic controls were correctly called.     

Perifosine Induces Cell Apoptosis in Human Osteosarcoma Cells: New Implication for Osteosarcoma Therapy?

Despite the advances of adjuvant chemotherapy and significant improvement of survival, the prognosis for patients with osteosarcoma is generally poor. The search for more effective anti-osteosarcoma agents is necessary and urgent. Here we report that perifosine induces cell apoptosis and growth inhibition in cultured human osteosarcoma cells. Perifosine blocks Akt/mTOR complex 1 (mTORC1) signaling, while promoting caspase-3, c-Jun N-terminal kinases (JNK), and p53 activation. Further, perifosine inhibits survivin expression probably by disrupting its association with heat shock protein-90 (HSP-90). These signaling changes together were responsible for a marked increase of osteosarcoma cell apoptosis and growth inhibition. Finally, we found that a low dose of perifosine enhanced etoposide- or doxorubicin-induced anti-OS cells activity. The results together suggest that perifosine might be used as a novel and effective anti-osteosarcoma agent.     

Dissolved Organic Nitrogen Release and Bacterial Activity in the Upper Layers of the Atlantic Ocean

The variability of the percentage of extracellular dissolved organic nitrogen (DON) release (PER), along with the relationship between DON release and bacterioplankton activity, was examined during five oceanographic cruises, carried out in the upwelling region of the NW Iberian Peninsula, the SW Bay of Biscay, and a latitudinal transect in the Atlantic Ocean (50°N–35°S). Rates of nitrogen uptake, DON release, and bacterial production were measured at 66 stations and sampled between August 1998 and October 2000. The percentage of DON release relative to the gross uptake of ammonium (PER^NH4+) ranged from 3 to 46%, whereas that relative to total nitrogen (NH₄ ⁺ + NO₃ ⁻ + urea) gross uptake (PER^total) varied between 21 and 82%. The highest values for both PER^NH4+ and PER^total were found in oligotrophic oceanic waters (<0.25 mg chlorophyll a m⁻³). In oceanic environments, a positive relationship was found between nitrogen uptake and DON release rates, with a log–log linear regression slope significantly lower than 1, suggesting an inverse relationship between PER and gross nitrogen uptake rates. In contrast, in areas with higher productivity levels (>6 mg chlorophyll a m⁻³), such as the continental shelf off the NW Iberian Peninsula, PER held constant as nitrogen uptake increased. These results suggest the dominance of different processes controlling DON release in oceanic and neritic zones. DON release rates accounted for less than 15% of the variability observed in bacterial production rates, suggesting a weak response of bacterioplankton to phytoplankton on short time scales (hours). Furthermore, nitrogen budgets showed an excess of DON release in relation to bacterial requirements.     

Lipopolysaccharide effects on sensitive and resistant variant chinese hamster ovary cell lines

Chinese hamster ovary (CHO · K1 · PRO) cell growth was inhibited by addition of a gram-negative bacterial lipopolysaccharide (LPS) to the cell culture medium. Growth inhibition began after three or four days of incubation, was dose-dependent up to a maximum at an LPS concentration of 500 μg/ml and was accompanied by cell shape changes and enhanced cytoplasmic vacuolization. Formation of bizarre CHO · K1 · PRO cell shapes and vacuole formation were most pronounced after seven days of incubation with LPS and could be observed by light and electron microscopy. An LPS-resistant cell population was obtained by intermittent in vitro exposure to high levels of LPS; these variant cells or clones derived from them failed to display growth inhibition in the presence of LPS. A clone from the LPS-resistant variant population showed altered cell properties compared to the parental cell line which included changes in cell morphology, adhesion, and endocytosis. Parental cells were markedly density-inhibited, whereas the variant clone exhibited considerable growth after confluency. The LPS-resistant variant cells showed a more elongated morphology than the parental line. No significant differences were observed between rates of detachment of parental and variant cells when sparse cultures of either line were removed from tissue culture dishes by ethylenediaminetetracetate (EDTA). However, at confluency approximately 100% of the variant cells versus 35% of the parental cells were removed by EDTA in one hour. Measurements of ¹²⁵I-ferritin uptake by parental and variant cells showed approximately twenty-fold and twofold increases, respectively, in uptake induced by LPS when compared to untreated control cultures.     

Mechanism of resistance to ricin toxin in selected mouse lymphoma cell lines

The role of impaired toxin uptake in conferring cellular resistance to the plant toxin RCA_II (ricin) has been examined using a murine BW5 147 lymphoma line and a toxin-resistant variant (BW5 147 Ric^R.3) selected by repeated exposure to RCA_II. The toxin-resistant variant is 250 times more resistant to RCA_II in long-term growth experiments and 1,000 times more resistant in short-term protein synthesis assays. Experiments with ferritin-conjugated ¹²⁵I-labeled RCA_II (ferritin-¹²⁵I-RCA_II) indicated that toxin binding to sensitive and resistant cells is similar at low toxin concentrations where maximum differential cytotoxicity occurs but that major difference exist with respect to toxin uptake. In sensitive cells toxin is internalized via endocytosis, and as seen previously in other systems subsequent rupture of some of the toxin-containing endocytotic vesicles releases toxin into the cytoplasm, where it inhibits protein synthesis. The process of toxin transfer to the cytoplasm is presumed to account for the one-hour lag before toxin-induced inhibition of protein synthesis can be detected. Endocytotic uptake of toxin is impaired in resistant BW5147Ric^R.3 cells, and they are unaffected by toxin concentrations that inhibit protein synthesis and kill sensitive parental cells. Killing of resistant cells at low toxin concentrations was accomplished by encapsulating RCA_II into lipid vesicles capable of fusing with the plasma membrane. Direct introduction of toxin into resistant cells using lipid vesicles as carriers produced rapid inhibition (< 15 min) of protein synthesis and eliminated the lag in toxin action seen in sensitive cells exposed to free toxin. These findings are discussed in relation to the mechanism of toxin action and proposals that toxin activity requires structural modification of the toxin molecule at the cell surface before transport into the cell.     

Targeting the PI3K/Akt Cell Survival Pathway to Induce Cell Death of HIV-1 Infected Macrophages with Alkylphospholipid Compounds

Background

HIV-1 infected macrophages and microglia are long-lived viral reservoirs persistently producing viral progenies. HIV-1 infection extends the life span of macrophages by promoting the stress-induced activation of the PI3K/Akt cell survival pathway. Importantly, various cancers also display the PI3K/Akt activation for long-term cell survival and outgrowth, and Akt inhibitors have been extensively searched as anti-cancer agents. This led us to investigate whether Akt inhibitors could antagonize long-term survival and cytoprotective phenotype of HIV-1 infected macrophages.

Principal Findings

Here, we examined the effect of one such class of drugs, alkylphospholipids (ALPs), on cell death and Akt pathway signals in human macrophages and a human microglial cell line, CHME5, infected with HIV-1 BaL or transduced with HIV-1 vector, respectively. Our findings revealed that the ALPs, perifosine and edelfosine, specifically induced the death of HIV-1 infected primary human macrophages and CHME5 cells. Furthermore, these two compounds reduced phosphorylation of both Akt and GSK3β, a downstream substrate of Akt, in the transduced CHME5 cells. Additionally, we observed that perifosine effectively reduced viral production in HIV-1 infected primary human macrophages. These observations demonstrate that the ALP compounds tested are able to promote cell death in both HIV-1 infected macrophages and HIV-1 expressing CHME5 cells by inhibiting the action of the PI3K/Akt pathway, ultimately restricting viral production from the infected cells.

Significance

This study suggests that Akt inhibitors, such as ALP compounds, may serve as potential anti-HIV-1 agents specifically targeting long-living HIV-1 macrophages and microglia reservoirs.     

Modification of phospholipid metabolism in dibutyryl-cAMP-mediated morphological conversion of CHO cells.

The cellular uptake and incorporation of [1-¹⁴C]palmitic acid and ³²P into lipids of Chinese hamster ovary cells, clone K₁ (CHO-K₁) have been investigated under conditions where the cells are converted from the compact, epithelial-like shape to the elongated fibroblast-like morphology by N⁶,O^2′-dibutyryl adenosine 3′:5′-phosphate. The primary alteration in lipid metabolism accompanying the morphological conversion to the fibroblast form was an increased incorporation of lipid precursors into all phospholipid classes and a decreased incorporation into the “neutral” lipid fraction. These results reflect the cells' need for phospholipid precursors when the membrane expands to form the fibroblast shape. When the fibroblast-shaped cells were allowed to revert to the epithelial shape, lipid metabolism was similar to that found in untreated cells.     

Enhancing perifosine's anticancer efficacy by preventing autophagy

Our long-term research goal is to develop efficacious regimens for cancer therapy through our understanding of cancer biology and drug mechanisms. Perifosine is an alkylphospholipid exhibiting antitumor activity and is currently being tested in clinical trials. Its activity is partly associated with its ability to inhibit Akt activity. In an effort to understand the mechanism by which perifosine exerts its anticancer activity, our recent work shows that perifosine, in addition to inhibition of Akt, inhibits mTOR signaling through a different mechanism than classical mTOR inhibitors such as rapamycin via facilitating the degradation of major components in the mTOR axis including mTOR, raptor and rictor. Accordingly, perifosine substantially induces autophagy in addition to apoptosis. The combination of perifosine with a lysosomal inhibitor enhances apoptosis and inhibition of the growth of xenografts in nude mice, suggesting that perifosine-induced autophagy protects cells from undergoing apoptosis. Thus, our findings highlight a novel mechanism accounting for perifosine's anticancer activity involving degradation-mediated mTOR inhibition and also suggest a potential strategy to enhance perifosine's anticancer efficacy by preventing autophagy.     

Fluid endocytosis in isolated rat parenchymal and non-parenchymal liver cells

Fluid endocytosis in rat liver parenchymal (hepatocytes) and non-parenchymal cells was studied by measuring uptake of [¹²⁵I]polyvinylpyrrolidone (PVP). Radioactive sucrose preparations were also tested but turned out to be unsuitable because of impurities of radioactive glucose and fructose. Fluid endocytosis was temperature dependent without any transition temperature. The rate of endocytosis was inhibited by inhibitors of the glycolytic and the respiratory pathway. Colchicine, but not cytochalasin B, inhibited the uptake of [¹²⁵I]PVP in hepatocytes. Therefore, intact microtubuli, but not microfilaments may be required for normal rate of fluid endocytosis in hepatocytes. Colchicine reduced the rate of fluid endocytosis in the non-parenchymal liver cells. Subcellular fractionation by isopycnic centrifugation in sucrose gradients indicated that [¹²⁵I]PVP taken up by the hepatocytes accumulated in the lysosomes. The rate of uptake expressed as volume of fluid internalized per unit time (endocytic index) was calculated to 0.08 μl/h/10⁶ cells for hepatocytes and 0.07 μl/h/10⁶ cells for non-parenchymal liver cells.     

Perifosine sensitizes UVB-induced apoptosis in skin cells: New implication of skin cancer prevention?

We demonstrate here that a relative low dose of perifosine significantly enhanced UVB-induced apoptosis in skin cells (keratinocytes and fibroblasts), associated with a significant increase of reactive oxygen species (ROS) and ceramide production as well as multiple perturbations of diverse cell signaling pathways, shifting to a significant pro-apoptosis outcomes. Perifosine inhibited UVB-induced pro-survival Akt/mammalian target of rapamycin (mTOR) and ERK activation, while facilitating pro-apoptotic AMP-activated protein kinas (AMPK), c-Jun-NH(2)-kinase (JNK), and p53 activation; these signaling changes together promoted a striking increase in skin cell apoptosis and a significantly reduced amount of DNA damages. Our results suggest that perifosine may represent a novel skin cancer prevention strategy.     

Targeted delivery of paclitaxel using folate-conjugated heparin-poly(β-benzyl-l-aspartate) self-assembled nanoparticles

A self-assembled nanoparticulate system composed of a folate-conjugated heparin-poly(β-benzyl-l-aspartate) (HP) amphiphilic copolymer was proposed for targeted delivery of the antineoplastic drug paclitaxel (PTX). PTX was incorporated into three types of heparin-based nanoparticles, including HP, folate-conjugated HP (FHP), and folate-polyethylene glycol (PEG)-conjugated HP (FPHP), using a simple dialysis method. The PTX-loaded nanoparticles were then characterized according to particle size (140-190 nm) and size distribution, drug-loading content and efficiency, and in vitro release behavior. In the cellular uptake study using KB cells positive for the folate-receptor (FR), FHP and FPHP nanoparticles showed a much higher cellular uptake than did unconjugated HP nanoparticles. Specifically, when the PEG spacer was inserted between the folate ligand and heparin backbone, FPHP nanoparticles had a greater cellular uptake than did FHP nanoparticles. The in vitro cytotoxicity of PTX-loaded HP, FHP, and FPHP nanoparticles was studied in KB cells and FR-negative A549 cells. Compared with the cytotoxicity in A549 cells, PTX-loaded FHP and FPHP nanoparticles exhibited more potent cytotoxicity in KB cells than did PTX-loaded HP nanoparticles and free-PTX, suggesting that the presence of folate enhanced intracellular uptake via FR-mediated endocytosis. In addition, FPHP nanoparticles exhibited much greater cytotoxicity in KB cells than did FHP nanoparticles. These results suggest that PTX-loaded folate-conjugated HP nanoparticles are a potentially useful delivery system for cancer cells positive for the folate-receptor.     

Testosterone induces a rapid stimulation of endocytosis,amino acid and hexose transport in mouse kidney cortex

Testosterone induced a rapid (<1 min) stimulation of endocytosis, amino acid and hexose transport, measured by the temperature-sensitive uptake of HRP, ¹⁴C-AIB and ³H-DG, in mouse kidney cortex slices. The hormonal increment in uptake persisted for at least 60–120 min, showed time-, energy-, and Na⁺-dependence, and varied with substrate and testosterone concentration. Testosterone was maximally effective at 10^?8 to 10^?7 M. Peroxidase histochemistry indicated that the hormonal increase in HRP uptake is restricted to proximal tubules. Testosterone was more effective than DHT, whereas cyproterone acetate, androsterone and dexamethasone had little or no stimulating effect on this uptake. Kidney slices from androgen-insensitive $\text{tfm}\text{Y}$ mice did not respond to testosterone. The rapid increase in endocytosis, amino acid and hexose transport may represent a direct, receptor-mediated response of the surface membrane of target cells to testosterone.     

Quality Control of Plasma Membrane Proteins by Saccharomyces cerevisiae Nedd4-Like Ubiquitin Ligase Rsp5p under Environmental Stress Conditions

In Saccharomyces cerevisiae, when a rich nitrogen source such as ammonium is added to the culture medium, the general amino acid permease Gap1p is ubiquitinated by the yeast Nedd4-like ubiquitin ligase Rsp5p, followed by its endocytosis to the vacuole. The arrestin-like Bul1/2p adaptors for Rsp5p specifically mediate this process. In this study, to investigate the downregulation of Gap1p in response to environmental stresses, we determined the intracellular trafficking of Gap1p under various stress conditions. An increase in the extracellular ethanol concentration induced ubiquitination and trafficking of Gap1p from the plasma membrane to the vacuole in wild-type cells, whereas Gap1p remained stable on the plasma membrane under the same conditions in rsp5^A401E and Δend3 cells. A ¹⁴C-labeled citrulline uptake assay using a nonubiquitinated form of Gap1p (Gap1p^K9R/K16R) revealed that ethanol stress caused a dramatic decrease of Gap1p activity. These results suggest that Gap1p is inactivated and ubiquitinated by Rsp5p for endocytosis when S. cerevisiae cells are exposed to a high concentration of ethanol. It is noteworthy that this endocytosis occurs in a Bul1/2p-independent manner, whereas ammonium-triggered downregulation of Gap1p was almost completely inhibited in Δbul1/2 cells. We also found that other environmental stresses, such as high temperature, H₂O₂, and LiCl, also promoted endocytosis of Gap1p. Similar intracellular trafficking caused by ethanol occurred in other plasma membrane proteins (Agp1p, Tat2p, and Gnp1p). Our findings suggest that stress-induced quality control is a common process requiring Rsp5p for plasma membrane proteins in yeast.     

Epidermal growth factor stimulates fluid phase endocytosis in human fibroblasts through a signal generated at the cell surface

We have investigated the stimulation of fluid phase endocytosis by epidermal growth factor (EGF) in normal human fibroblasts using ¹²⁵I-labeled polyvinylpyrrolidone (¹²⁵I-PVP) as a fluid phase marker. We found that EGF initially induced a thereefold increase in the rate of ¹²⁵I-PVP uptake. This initial burst of fluid uptake terminated within 10 min. Thereafter, the rate of fluie uptake in EGF-treated cells was approximately 40% higher than in control cells. To identify the cellular site of EGF action in stimulating fluid phase endocytosis, we examined the kinetics of the induction of this response as well as the kinetics of cell surface binding and internalization of ¹²⁵I-EGF. Although there was no detectable lag between binding of EGF to the cell surface and its internalization, the kinetics of the two processes were quite different. Significantly, the kinetics of induction of ¹²⁵I-PVP uptake matched the kinetics of binding of ¹²⁵I-EGF to its cell surface receptors, indicating that the signal for the increase in fluid phase endocytosis is generated at the cell surface. To determine if EGF-stimulated fluid phase endocytosis was related to EGF-stimulated endocytosis of its own receptor, we compared the EGF dose dependency and time course of the two processes. Although the stimulated endocytosis of the EGF receptor was not saturable with respect to the concentration of EGF used, the stimulation of fluid phase endocytosis was half maximal at an EGF concentration of 1 ng/ml and saturated at a concentration of 5 ng/ml. Also, the stimulation of fluid phase endocytosis was sevenfold greater initially after adding EGF than after a 30-min continuous incubation with the hormone, whereas the enhanced clearance of the EGF receptor did not change during this time period. We conclude that the EGF-stimulated increase in fluid phase endocytosis is not directly coupled to EGF-stimulated endocytosis of its own receptor but instead to a separate signal generated at the cell surface.     

Clonal selection by fluorescence redistribution after photobleaching (FRAP)—A “fast” lateral mobility fibroblast mutant (E7G1)

Fluorescence redistribution after photobleaching (FRAP) was utilized to select a “fast” lateral mobility clone from Kirsten murine sarcoma virus-transformed 3T3 (KMSV-3T3) fibroblasts. The clone, E7G1, demonstrated a lateral mobility for membrane wheat germ agglutinin (WGA) and succinylated concanavalin A (sCon A) receptors of (2.1 ± 1.6) × 10⁻⁹ cm²/s and (2.7 ± 2.3) × 10⁻⁹ cm²/s, respectively. These mobilities were approximately equivalent to phospholipid mobility (2.8 ± 1.9 × 10⁻⁹ cm²/s). The fast mobility phenotype is observed when the cells are unattached and spherical. Upon attachment, the mobility decreases to (0.19 ± 0.19) × 10⁻¹⁰ cm²/s. In addition, the ability of Con A to initiate global modulation was completely lost in spread as well as spherical cells in the E7G1 fast mobility clone. A comparison of F-actin patterns between untransformed Balb/c fibroblasts and the E7G1-transformed line suggests a correlation between well-developed stress fiber assemblies and the ability to induce global modulation. The fast mobility clone was stable for at least 23 passages.     

The Significance of Zooplankton in the Cycling of Phosphorus in Lakes of Different Trophic Categories

The species composition, number, biomass and phosphorus excretion rate (PER) of zooplankton (Crustacea and Rotatoria) in 8 mesotrophic and eutrophic lakes were studied. PER were calculated using specific expenditures on metabolism estimated from the oxygen uptake, phosphorus content in zooplankton and field observations of number and biomass of planktonic Rotatoria and Crustacea. The zooplankton biomass and PER increased from mesotrophic to eutrophic lakes. The values of PER were compared with primary production and spring phosphorus concentration. The relative significance of zooplankton in phosphorus cycling declined with increasing trophic category of lakes.