Flow cytofluorometric analysis of the uptake of the fluorescent fatty acid pyrene-dodecanoic acid by human peripheral blood cells

The fluorescence activated cell sorter (FACS) was used for measuring the uptake of the fluorescent fatty acid derivative 12-(1-pyrene) dodecanoic acid (P12) by human peripheral blood cells. The results indicate that blood cells differ widely in their ability to take up P12, with polymorphonuclear cells showing the greatest uptake, followed by lymphocytes, platelets, and RBCs. These differences in P12 uptake provide a potential additional parameter for differential cell counting. Using the ability of the FACS to "gate out" nonrelevant cells, it was possible to measure the rate of P12 uptake by each respective cell type even when admixed with other cells. Thus elaborate physical separation procedures could be avoided, and contaminating cells did not influence the results. Differences in P12 uptake were also utilized to separate blood cells into pure subpopulations of specific cell types.     

Continuous spectrofluorometric measurements of uptake by cultured cells of 12-(1-pyrene)-dodecanoic acid from its complex with albumin.

Aqueous dispersions of 12-(1-pyrene)-dodecanoic acid (P12), a medium-chain fatty acid to which the fluorescent probe pyrene has been covalently linked, shows a considerable increase in fluorescence when the probe is introduced into a hydrophobic environment. This enables the uptake of P12 by liposomes and cells to be followed directly in a spectrofluorometer, without separating the cells from the P12-containing medium. In the present study, we show that complexing P12 to albumin produced a very high fluorescence emission intensity. This made direct measurements of the uptake by cells of albumin-bound P12 impossible. Such direct measurements could, however, be made using albumin which had been interacted with trinitrobenzenesulphonic acid (TNBS). The yellow trinitrophenyl (TNP) residues, which were thereby covalently linked to the albumin, quenched the fluorescence of pyrene in the TNP-albumin/P12 complex. Upon release of the P12 molecules from this complex and their subsequent uptake by cells, fluorescence increased. This technique was utilized for the continuous monitoring of the uptake of P12 by different cell types and cells at various stages of maturation.     

Cellular differentiation and I-FABP protein expression modulate fatty acid uptake and diffusion

The effect of cellular differentiation on fatty acid uptake andintracellular diffusion was examined in transfected pluripotent mouseembryonic stem (ES) cells stably expressing intestinal fatty acidbinding protein (I-FABP). Control ES cells, whether differentiated orundifferentiated, did not express I-FABP. The initial rate and maximaluptake of the fluorescent fatty acid,12-(N-methyl)-N-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-octadecanoic acid (NBD-stearic acid), was measured in single cells by kinetic digital fluorescence imaging. I-FABP expression in undifferentiated EScells increased the initial rate and maximal uptake of NBD-stearic acid1.7- and 1.6-fold, respectively, as well as increased its effectiveintracellular diffusion constant(D_eff) 1.8-foldas measured by the fluorescence recovery after photobleachingtechnique. In contrast, ES cell differentiation decreased I-FABPexpression up to 3-fold and decreased the NBD-stearic acid initial rateof uptake, maximal uptake, andD_eff by 10-, 4.7-, and 2-fold, respectively. There were no significant differencesin these parameters between the differentiated control anddifferentiated I-FABP-expressing ES cell lines. In summary,differentiation and expression of I-FABP oppositely modulatedNBD-stearic acid uptake parameters and intracellular diffusion in EScells.

     

Spectrofluorometric measurements of the dispersion state of pyrenedodecanoic acid and its uptake by cultured cells and liposomes

Pyrene dodecanoic acid (P12), a medium-chain fatty acid to which the fluorescent probe pyrene is covalently linked, showed a considerable increase in fluorescence when the probe was introduced into a hydrophobic environment. Also, when closely packed in an aggregate, an energy transfer between two adjacent molecules of pyrene occurred, resulting in a shift of the peak of the emission spectrum from 378 nm ('monomeric') to 475 nm ('excimeric'). These two respective properties were utilized for the following: (a) A spectrofluorometric measurement of the critical micellar concentration (CMC) of the pyrene fatty acid, defined as the concentration at which the 475 nm emission peak appeared as a consequence of the aggregation of P12 molecules in aqueous solution to form micelles; the CMC of P12 was found to be in the range of 1 to 2 microM. (b) The penetration of P12, from an aqueous solution or dispersion, into unilamellar phospholipid vesicles was determined by monitoring the increase of the fluorescence at 378 nm. The fluorescence increase was time-dependent and proportional to the respective concentrations of P12 or phospholipid vesicles. Substituting the neutral phosphatidylcholine with the negatively-charged phosphatidylserine vesicles resulted in a slower rate as well as lesser total uptake of P12. (c) The uptake of P12 by cells was accompanied by an increase in the monomeric fluorescence emission intensity. Using cells in suspension, this could be followed continuously in a spectrofluorometer equipped with a recorder. The uptake was found to be time-dependent and proportional to P12 concentration.     

Influence of chain length of pyrene fatty acids on their uptake and metabolism by Epstein-Barr-virus-transformed lymphoid cell lines from a patient with multisystemic lipid storage myopathy and from control subjects.

The uptake and intracellular metabolism of 4-(1-pyrene)butanoic acid (P4), 10-(1-pyrene)decanoic acid (P10) and 12-(1-pyrene)dodecanoic acid (P12) were investigated in cultured lymphoid cell lines from normal individuals and from a patient with multisystemic lipid storage myopathy (MLSM). The cellular uptake was shown to be dependent on the fatty-acid chain length, but no significant difference in the uptake of pyrene fatty acids was observed between MLSM and control lymphoid cells. After incubation for 1 h the distribution of fluorescent fatty acids taken up by the lymphoid cell lines also differed with the chain length, most of the fluorescence being associated with phospholipid and triacylglycerols. In contrast with P10 and P12, P4 was not incorporated into neutral lipids. When the cells were incubated for 24 h with the pyrene fatty acids, the amount of fluorescent lipids synthesized by the cells was proportional to the fatty acid concentration in the culture medium. After a 24 h incubation in the presence of P10 or P12, at any concentration, the fluorescent triacylglycerol content of MLSM cells was 2-5-fold higher than that of control cells. Concentrations of pyrene fatty acids higher than 40 microM seemed to be more toxic for mutant cells than for control cells. This cytotoxicity was dependent on the fluorescent-fatty-acid chain length (P12 greater than P10 greater than P4). Pulse-chase experiments permitted one to demonstrate the defect in the degradation of endogenously biosynthesized triacylglycerols in MLSM cells (residual activity was around 10-25% of controls on the basis of half-lives and initial rates of P10- or P12-labelled-triacylglycerol catabolism); MLSM lymphoid cells exhibited a mild phenotypic expression of the lipid storage (less severe than that observed in fibroblasts). P4 was not utilized in the synthesis of triacylglycerols, and thus did not accumulate in MLSM cells: this suggests that natural short-chain fatty acids might induce a lesser lipid storage in this disease.     

A live-cell high-throughput screening assay for identification of fatty acid uptake inhibitors

We developed a live-cell high-throughput assay system using the baker's yeast Saccharomyces cerevisiae to screen for chemical compounds that will inhibit fatty acid uptake. The target for the inhibitors is a mammalian fatty acid transport protein (mmFATP2), which is involved in the fatty acid transport and activation pathway. The mmFATP2 was expressed in a S. cerevisiae mutant strain deficient in Fat1p-dependent fatty acid uptake and reduced in long-chain fatty acid activation, fat1Deltafaa1Delta. To detect fatty acid import, a fluorescent fatty acid analog, 4,4-difluoro-5-methyl-4-bora-3a,4a-diaza-s-indacene-3-dodecanoic acid (C1-BODIPY-C12), was incubated with cells expressing FATP2 in a 96-well plate. The mmFATP2-dependent C1-BODIPY-C12 uptake was monitored by measuring intracellular C1-BODIPY-C12 fluorescence on a microtiter plate reader, whereas extracellular fluorescence was quenched by a cell viability dye, trypan blue. Using this high-throughput screening method, we demonstrate that the uptake of the fluorescent fatty acid ligand was effectively competed by the natural fatty acid oleate. Inhibition of uptake was also demonstrated to occur when cells were pretreated with sodium azide or Triacsin C. This yeast live-cell-based assay is rapid to execute, inexpensive to implement, and has adequate sensitivity for high-throughput screening. The assay basis and limitations are discussed.     

Photoaffinity labeling and fatty acid permeation in 3T3-L1 adipocytes

Long chain fatty acid uptake was investigated in 3T3-L1 cells. Differentiation of these cells from fibroblasts to adipocytes was accompanied by an 8.5-fold increase in the rate of oleate uptake. This was saturable in adipocytes with apparent Kt and Vmax values of 78 nM and 16 nmol/min/mg cell protein, respectively. A number of proteins in various subcellular fractions of differentiated cells were labeled with the photoreactive fatty acid 11-m-diazirinophenoxy[11-3H]undecanoate. A 15-kDa cytoplasmic protein was induced upon differentiation to adipocytes. This protein was labeled with the photoreactive fatty acid in cytoplasm isolated from differentiated adipocytes, but not in cytoplasm from undifferentiated, fibroblastic cells. Furthermore, a high affinity fatty acid binding protein of 22 kDa was identified in plasma membranes of undifferentiated cells, and its level of labeling increased 2-fold upon differentiation. These results indicate the usefulness of the photoreactive fatty acid in identifying cellular fatty acid binding proteins, and its potential to elucidate the spatial and temporal distribution of fatty acids in intact cells.     

CMV promotor activity during ES cell differentiation: potential insight into embryonic stem cell differentiation

The activity of the P(CMV IE) promoter was studied during the differentiation of ES cells into neurons. In order to do this, stable embryonic stem (ES) cell lines that express enhanced green fluorescent protein (EGFP) under the control of P(CMV IE) were created and these ES cells were differentiated by aggregation of cells in the presence of retinoic acid (RA). Based on our observations that the activity of P(CMV IE) was highest in undifferentiated cells, and that cell-cell interaction and addition of RA that lead to enhanced cell proliferation also increased expression from P(CMV IE), we hypothesized that the activity of P(CMV IE) was positively regulated in cycling cells. However, when analysis was done at the single cell level it was found that BrdU label and EGFP expression were not correlated. EGFP expression was found to be down-regulated in many cells that were BrdU positive and conversely there were significant numbers of BrdU negative cells that were EGFP positive. Further, P(CMV IE) activity was not observed in cells that were nestin positive or in differentiated neurons, but P(CMV IE) was active in cells with a fibroblast-like morphology. Finally, several proteins present in undifferentiated ES cells were found to bind to regulatory regions of P(CMV IE). These were absent when cells were aggregated in the presence of RA. The above results have implications for expression of transgenes in ES cells as well as providing new insight into the mechanism of lineage restriction.     

Uptake and intracellular degradation of fluorescent sphingomyelin by fibroblasts from normal individuals and a patient with Niemann-Pick disease

Sphingomyelin, labelled with a fluorescent probe, pyrene, in the fatty acyl residue was associated with fetal calf serum; approx. 80% of the sphingomyelin was found in the low- and high-density lipoproteins. This was added to the growth medium of cultured human skin fibroblasts from normal individuals and a patient with Niemann-Pick disease type A, devoid of acid sphingomyelinase activity. The fluorescent sphingomyelin was taken up by both cell types, but only the former degraded it to produce fluorescent ceramide. Differences between normal and Niemann-Pick cells in sphingomyelin content or ceramide production were observed after several hours uptake. A more pronounced difference was noted when cells were incubated for 1 day with fluorescent sphingomyelin and then for two to three days in medium devoid of this compound. Under these conditions, the fluorescence intensity of the Niemann-Pick cells remained practically constant while that of their normal counterparts was almost completely eliminated from the cells. Comparison of fluorescence intensities of these two cell types could be made directly on aqueous suspensions of whole cells or, alternatively, on their lipid extracts. For evaluation of the degradation of fluorescent sphingomyelin to ceramide within the cells, several procedures were developed for the rapid isolation of the latter compound from the total lipid extract. The results suggest that when associated with the constituents of the fetal calf serum, sphingomyelin is taken up by the cells and transported into the lysosomal compartment where it is degraded to ceramide. Use of the fluorescent derivative of sphingomyelin provided a simple and rapid procedure for following the uptake by and degradation within the cultured cells. It also permitted the establishment of differences in the rates of degradation of the fluorescent sphingomyelin by cells with a normal metabolism and others lacking sphingomyelinase (i.e., Niemann-Pick disease type A cells).     

Neuronal differentiation and synapse formation of PC12 and embryonic stem cells on interdigitated microelectrode arrays: contact structures for neuron-to-electrode signal transmission (NEST)

The development of neuron-microelectrode interfaces (neurochips) is highly desirable for the non-invasive recording of the cellular response to neuroactive drugs as well as the electrical stimulation of nervous tissue by implantable electrodes. A prerequisite for neuron-to-electrode signal transmission (NEST) is the formation of synapse-like contacts between the neuronal cell and the conductive surface of a microelectrode array. We attempted synapse formation by neuronal differentiation of rat pheochromocytoma cells (PC12) and blastocyst-derived murine embryonic stem cells (ES-J1) on interdigitated microelectrode arrays that were made of gold (Au), platinum (Pt), or indium tin oxide (ITO). PC12 or ES cells were in vitro differentiated by incubation with nerve growth factor (NGF) and forskolin, or by serum deprivation and treatment with basic fibroblast growth factor (FGF-2), respectively. On top of ITO electrodes, the neuronal cells extended extremely long processes that terminated in pili-like contact structures, which is typical for growth cone formation. ES cells differentiated into neurons as verified by immunofluorescence staining of MAP-2 and developed synapse-like junctions with the ITO electrode surface as indicated by synaptophysin staining. Differentiated PC12 and ES cells showed bona fide morphological characteristics of synaptic growth cones that were unprecedented in tissue culture. Cones formed by PC12 cells could be stimulated with KCI and carbachol as shown by uptake of FM1-43, a fluorescent marker for synaptic vesicle formation. In contrast to Electrical Cell Impedance Spectroscopy (ECIS) recordings, AC impedance spectrometry with differentiated PC12 cells settled on interdigitated microelectrode arrays revealed lower AC impedance than that with undifferentiated cells, indicating that the complex impedance is dependent on ion fluxes at the neuron-to-electrode contact surface.     

High-throughput screening for fatty acid uptake inhibitors in humanized yeast identifies atypical antipsychotic drugs that cause dyslipidemias

Fatty acids are implicated in the development of dyslipidemias, leading to type 2 diabetes and cardiovascular disease. We used a standardized small compound library to screen humanized yeast to identify compounds that inhibit fatty acid transport protein (FATP)-mediated fatty acid uptake into cells. This screening procedure used live yeast cells expressing human FATP2 to identify small compounds that reduced the import of a fluorescent fatty acid analog, 4,4-difluoro-5-methyl-4-bora-3a,4a-diaza-s-indacene-3-dodecanoic acid (C(1)-BODIPY-C(12)). The library used consisted of 2,080 compounds with known biological activities. Of these, approximately 1.8% reduced cell-associated C(1)-BODIPY-C(12) fluorescence and were selected as potential inhibitors of human FATP2-mediated fatty acid uptake. Based on secondary screens, 28 compounds were selected as potential fatty acid uptake inhibitors. Some compounds fell into four groups with similar structural features. The largest group was structurally related to a family of tricyclic, phenothiazine-derived drugs used to treat schizophrenia and related psychiatric disorders, which are also known to cause metabolic side effects, including hypertriglyceridemia. Potential hit compounds were studied for specificity of interaction with human FATP and efficacy in human Caco-2 cells. This study validates this screening system as useful to assess the impact of drugs in preclinical screening for fatty acid uptake.     

Characterization of human monocytic cell line, U937, in taking up acetylated low-density lipoprotein and cholesteryl ester accumulation. A flow cytometric and HPLC study

The uptake of LDL and acetylated LDL and the ability of cholesteryl ester accumulation by cells of a human monocytic cell line, U937, has been characterized by flow cytometric assay using a fluorescent probe, DiI, and by high-performance liquid chromatography (HPLC). The increase of mean fluorescence intensity of U937 incubated with DiI-labeled lipoproteins demonstrates that this cell line could incorporate DiI-AcLDL, as well as DiI-labeled LDL. Competition and saturation studies indicate that the manner of taking up DiI-AcLDL is receptor-mediated. While differentiated U937 incubated with 16 nM phorbol myristate acetate for 24 h took up little DiI-AcLDL, HPLC analysis confirmed that intracellular free and esterified cholesterols significantly increase in the U937 cells incubated with AcLDL or LDL. The ability of mouse peritoneal macrophage to abundantly accumulate at least five kinds of cholesteryl ester were also shown in this analysis. In contrast, in U937 cells, free fatty acids are incorporated into various substances rather than into cholesteryl esters (as revealed by HPLC analysis), so that the cholesterol in AcLDL taken up by U937 cells is not synthesized into cholesteryl esters to any great extent.     

Uptake of a fluorescent-labeled fatty acid by spiroplasma floricola cells

12-(1-pyrene)dodecanoic fatty acid (P12) uptake by Spiroplasma floricola BNR-1 cells was characterized with regard to its kinetics, specificity, metabolism and susceptibility to protein and lipid inhibitors. The uptake process depended on temperature and pH, and exhibited biphasic saturation kinetics with a very low (2.7 M) and a high (37 M) apparent K _m value. Lauric, myristic, palmitic, stearic and oleic fatty acids did not compete with P12 for transport. The fluorescence of P12 was exclusively recovered in the neutral lipid fraction, suggesting that this fatty acid is not further utilized for phospholipid biosynthesis. Valinomycin, carbonylcyanide m-chlorophenyldrazone (CCCP), dicyclohexylcarbodiimide (DCCD), and pronase strongly reduced P12 uptake by cells, but not by membrane vesicles, affecting the high affinity (low K _m) component of the uptake system. Uptake of P12 by cells, as well as by membrane vesicles, was very sensitive to glutaraldehyde, chlorpromazine, phospholipase A₂₁ and ascorbate with FeCl₃, which affected the low affinity (high K _m) component of a transport system. Digitonin stimulated P12 uptake. We suggest that the incorporation of P12 into spiroplasma cell membrane is a two-step process: a high specificity energy-dependent and protease-sensitive binding to the outer surface of membrane, and a low specificity and energy-independent diffusion and partition into the membrane lipid environment.     

Differentiation-dependent sensitivity to apoptogenic factors in PC12 cells

We have investigated the role of the mitochondrial pathway during cell death following serum and nerve growth factor (NGF)/dibutyryl cyclic AMP (Bt(2)cAMP) withdrawal in undifferentiated or NGF/Bt(2)cAMP-differentiated PC12 cells, respectively. Holocytochrome c, Smac/DIABLO, and Omi/HtrA2 are released rapidly following trophic factor deprivation in PC12 cells. Bcl-2 and Akt inhibited this release. The protection, however, persisted longer in differentiated PC12 cells. In differentiated, but not undifferentiated cells, Bcl-2 and Akt also inhibited apoptosis downstream of holocytochrome c release. Thus, undifferentiated PC12 cells showed marked sensitivity to induction of apoptosis by microinjected cytochrome c even in the presence of NGF, Bcl-2, or Akt. In contrast, in differentiated cells these factors suppressed cell death. Consistent with these observations, in vitro processing of procaspase 9 in response to cytochrome c was observed in extracts from undifferentiated but not differentiated cells expressing Akt or Bcl-2. Endogenous caspase 9 was cleaved during cell death, whereas dominant negative caspase 9 inhibited cell death. The results from determining the role of inhibitors of apoptosis (IAPs) suggest that acquisition of inhibition by IAPs is part of the differentiation program. Ubiquitin-DeltaN-AVPI Smac/DIABLO induced cell death in differentiated cells only. c-IAP-2 is unregulated in differentiated cells, whereas X-linked IAP levels decreased in these cells coincident with cell death. Moreover, expressing X-linked IAP rendered undifferentiated cells resistant to microinjected cytochrome c. Overall, the inhibitory regulation, of cell death at the level of release of mitochondrial apoptogenic factors and at post-mitochondrial activation of caspase 9 observed in differentiated PC12 cells, is reduced or absent in the undifferentiated counterparts.     

Effects of fluorophore structure and hydrophobicity on the uptake and metabolism of fluorescent lipid analogs

Cellular transport and metabolism of fatty acids are integral components of lipid metabolism, but the mechanisms and regulation involved are poorly understood. A variety of commercially available fluorescent analogs of fatty acids, are potentially useful probes for the study of lipid metabolism by such techniques as cell sorting and fluorescence microscopy. We have screened a series of fluorescent fatty acids to identify analogs that would reliably simulate the metabolic behavior of natural fatty acids; i.e., similar kinetics of transport, of intracellular movement, and of metabolic fate. The metabolic behavior of these analogs was compared with those of some naturally occurring fatty acids in HepG2 cells, which are a good model of some aspects of hepatic function. Fluorescent analogs containing polar fluorophores yielded the lowest rates of cellular uptake and conversion to acylated lipid products. Similarly, fluorescent analogs with the fluorophore located near the carboxylic acid group were poorly metabolized. Fatty acid analogs containing anthracene or pyrene at the n-terminus of the acyl chain were the most extensively incorporated into cellular lipids. The types and amounts of labeled lipid products formed from these analogs and from natural fatty acids were similar. Pyrene-labeled analogs have spectral properties that can be measured fluorometrically at very low concentrations. Therefore, we compared the cellular metabolism of 12-(1-pyrenyl)dodecanoic acid with those of palmitic and oleic acids.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential effect of dexamethasone on the regulation of phospholipase A2 and prostanoid synthesis in undifferentiated and phorbolester-differentiated U937 cells

The human undifferentiated histiocytic cell-line U937 can be induced to differentiate by incubation with 12-0-tetradecanoylphorbol-13-acetate (TPA) into macrophage-like cells. Dexamethasone reduced the prostaglandin production in TPA-differentiated U937 cells dose dependently, whereas undifferentiated U937 cells were dexamethasone insensitive. Concomitantly phospholipase A2, the enzyme liberating the prostaglandin precursor arachidonic acid, was inhibited by dexamethasone in TPA-differentiated but not in undifferentiated U937 cells. The activity of lysophosphatide acyltransferase, the key enzyme of fatty acid reacylation into phospholipids, remained unchanged both in undifferentiated and TPA-differentiated U937 cells. The data suggest that responsiveness to glucocorticoid-dependent regulation of prostanoid synthesis is acquired by cells of the monocyte-macrophage lineage late in differentiation.     

Real-Time Tracking of BODIPY-C12 Long-Chain Fatty Acid in Human Term Placenta Reveals Unique Lipid Dynamics in Cytotrophoblast Cells

While the human placenta must provide selected long-chain fatty acids to support the developing fetal brain, little is known about the mechanisms underlying the transport process. We tracked the movement of the fluorescently labeled long-chain fatty acid analogue, BODIPY-C₁₂, across the cell layers of living explants of human term placenta. Although all layers took up the fatty acid, rapid esterification of long-chain fatty acids and incorporation into lipid droplets was exclusive to the inner layer cytotrophoblast cells rather than the expected outer syncytiotrophoblast layer. Cytotrophoblast is a progenitor cell layer previously relegated to a repair role. As isolated cytotrophoblasts differentiated into syncytialized cells in culture, they weakened their lipid processing capacity. Syncytializing cells suppress previously active genes that regulate fatty-acid uptake (SLC27A2/FATP2, FABP4, ACSL5) and lipid metabolism (GPAT3, LPCAT3). We speculate that cytotrophoblast performs a previously unrecognized role in regulating placental fatty acid uptake and metabolism.     

Uptake of long-chain fatty acids in HepG2 cells involves caveolae: analysis of a novel pathway

We investigated the role of caveolae in uptake and intracellular trafficking of long chain fatty acids (LCFA) in HepG2 human hepatoma cells. The uptake of [(3)H]oleic acid and [(3)H]stearic acid into HepG2 cells was measured by radioactive assays and internalization of the non-metabolizable fluorescent fatty acid 12-(N-methyl)-N-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino] (12-NBD) stearate into single HepG2 cells was semi-quantitatively assessed by laser scanning microscopy. The initial rate of [(3)H]oleic acid uptake (V(0)) in HepG2 cells exhibited saturable transport kinetics with increasing concentrations of free oleic acid (V(max) 854 +/- 46 pmol mg protein(-1) min(-1), K(m) 100 +/- 14 nmol/l). While inhibition of clathrin coated pits did not influence LCFA uptake in HepG2, inhibition of caveolae formation by filipin III, cyclodextrin, and caveolin-1 antisense oligonucleotides resulted in reduction of [(3)H]oleic acid uptake by 54%, 45%, and 23%, respectively. Furthermore, filipin III inhibited the uptake of [(3)H]stearic acid and its fluorescent derivative 12-NBD stearate by 44% and 50%, respectively. Transfection studies with alpha-caveolin-1/cyanofluorescent protein chimeras showed significant colocalization of caveolae and internalized 12-NBD stearate. In conclusion, these data suggest a significant role for caveolae mediated uptake and intracellular trafficking of LCFA in HepG2 cells.     

A facile method for direct determination of phospholipase A2 activity in intact cells

The fluorescent phospholipid analogue 1-acyl-2-(N-4-nitrobenzo-2-oxa-1,3- diazole)aminocaproylphosphatidylcholine (C6-NBD-PC), which incorporates into cell membranes, is employed as a substrate for phospholipase A2 (PLA2) in intact cells. The interaction of this substrate with the cells produces only one fluorescent product; the fatty acid C6-NBD-FA, which does not incorporate into other lipids, and is not further metabolized. The product, a hydrophilic fatty acid, is separated from the substrate by aqueous: organic solvent phase separation. Using this method, the fatty acid produced is fully recovered and its amount, as measured by its fluorescence intensity, is a direct measure of the cell membrane PLA2 activity.     

Casein phosphopeptides modulate proliferation and apoptosis in HT-29 cell line through their interaction with voltage-operated L-type calcium channels

At the intestinal level, proliferation and apoptosis are modulated by the extracellular calcium concentration; thus, dietary calcium may exert a chemoprotective role on normal differentiated intestinal cells, while it may behave as a carcinogenesis promoter in transformed cells. Calcium in milk is associated with casein and casein phosphopeptides (CPPs), hence is preserved from precipitation. CPPs were demonstrated to induce uptake of extracellular calcium ions by in vitro intestinal tumor HT-29 cells but only upon differentiation. Here, the hypothesis that CPPs could differently affect proliferation and apoptosis in undifferentiated and differentiated HT-29 cells through their binding with calcium ions was investigated. Results showed that CPPs protect differentiated intestinal cells from calcium overload toxicity and prevent their apoptosis favoring proliferation while inducing apoptosis in undifferentiated tumor cells. The CPP effect on undifferentiated HT-29 cells, similar to that exerted by ethyleneglycol-O, O'-bis(2-aminoethyl)-N, N, N', N'-tetraacetic acid (EGTA), is presumably due to the ability in binding the extracellular calcium. The effect on differentiated HT-29 cells is coupled to the interaction of CPPs with the voltage-operated L-type calcium channels, known to activate calcium entry into the cells under depolarization and to exert a mitogenic effect: the use of an agonist potentiates the cell response to CPPs, while the antagonists abolish the response to CPPs (36% of examined cells) or reduce both the percentage of responsive cells and the increase of intracellular calcium concentration. Taken together, these results confirm the potentialities of CPPs as nutraceuticals/functional food and also as modulators of cellular processes connected to the expression of a cancer phenotype.