Nuts and volts: searching for ways to zap EMI effects.
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Data Collection↗
Biomedical subjects
J Storch
Publications and source records attributed to J Storch.
EMC(electromagnetic compatibility): how to manage the challenge.
The significant changes in health care and telecommunications has produced an emergence of new and exciting technologies. Clinical engineers must continue to safeguard patient safety by adequately refurbishing their arsenal of technical knowledge of electromagnetic compatibility (EMC). This document examines the methodology and the tools for managing the technical changes in the telecommunications and biomedical equipment areas.
Data Collection↗
Nuts and volts. Searching for ways to zap EMI (electromagnetic interference) effects.
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Attitude of Health Personnel↗
Fatty acid transfer in taurodeoxycholate mixed micelles.
Dietary triacylglycerol is acted upon by lipolytic enzymes in the stomach and the proximal small intestine, releasing fatty acids and monoacylglycerol as the ultimate products. These digestive products are solubilized by bile released from the gall bladder, resulting in the formation of two product phases-vesicles and micelles-depending upon the concentration of bile in the small intestine. Absorption of lipid is thought to occur from these two phases. We have previously examined the rate and mechanism of long-chain fatty acid transfer between unilamellar vesicles [Kleinfeld, A. M., & Storch, J. (1993) Biochemistry 32, 2053-2061]. In order to begin to assess the relative contributions of micellar vs vesicular phases in the absorption of dietary lipid, a simple model system was designed to investigate the transfer of fatty acid and monoacylglycerol between micelles. A fluorescence self-quenching assay was used to monitor the transfer of fluorescent anthroyloxy-labeled lipids from donor micelles to acceptor micelles. The mechanism of fatty acid transfer was found to be a combination of diffusional and collisional processes, with the latter dominating at high micelle concentrations. The rate of diffusional transfer of fatty acid and monoacylglycerol analogues was approximately 30-fold greater from micelles than vesicles. Intermicellar and intervesicular rates of transfer were 3-fold greater for fatty acids as compared with monoacylglycerol. The results suggest that uptake of the products of intestinal lipase hydrolysis is more efficient from micellar than vesicular phases. Nevertheless, fatty acid and monoacylglycerol transfer from unilamellar vesicles could account, in part, for the relatively efficient uptake of dietary lipid observed in conditions of intestinal bile salt insufficiency.
Fatty Acids, Nonesterified↗
Binding site polarity and ligand affinity of homologous fatty acid-binding proteins from animals with different body temperatures.
Binding affinity and binding-pocket polarity is determined for intracellular fatty acid-binding protein (FABP) from aerobic muscle of Chaenocephalus aceratus, the Antarctic icefish, and from rat heart. FABPs bind fatty acids via weak-bond forces (both ionic and hydrophobic), and these bond forces are temperature sensitive, yet FABPs are present in animals whose body temperatures range over nearly 40 degrees C. To investigate FABP's sensitivity to body temperature, fatty acid binding affinity (Kd) was determined for both rat heart-FABP and icefish heart-FABP at two physiological temperatures (0 degrees C or 37 degrees C). Saturated and unsaturated fatty acids (16:0 and 16:1), delivered in model membranes (liposomes) whose composition is typical of either Antarctic fish (16:0/22:6 phosphatidylcholine) or mammals (bovine-heart phosphatidylcholine) were examined. Incubation at 0 degree C or 37 degrees C dose not significantly affect Kd for rat heart FABP, regardless of liposome composition or fatty acid ligand (Kd = 0.686 +/- 0.127 - 1.129 +/- 0.356 microM at 0 degree C, 0.775 +/- 0.307 - 1.605 +/- 0.427 microM at 37 degrees C). Incubation temperature significantly affects icefish FABP's affinity for 16:1 (0.626 +/- 0.093 microM at 37 degrees C vs. 1.896 +/- 0.343 microM at 0 degree C for fatty acid presented in Antarctic fish liposomes; 0.331 +/- 0.101 microM at 37 degrees C vs. 0.949 +/- 0.121 microM at 0 degree C for bovine heart liposomes) but not 16:0. Kd is not significantly different between FABPs under any set of conditions (with one exception: Kd is significantly lower in rat FABP vs. icefish FABP for 16:0 at 0 degree C for fatty acids delivered in bovine heart liposomes). Although Kd values are largely equivalent between the two FABPs, relative contributions from ionic vs. hydrophobic weak-bond forces are different between the two animals. Rat heart FABP has a binding pocket that is significantly more nonpolar than that of icefish FABP (as measured by quantum yield of the bound fluorescent fatty-acid analogue (PA-DPH); Q = 0.067 +/- 0.008 vs. 0.034 +/- 0.005 at 0 degree C, 0.030 +/- 0.003 vs. 0.019 +/- 0.002 at 37 degrees C). This suggests that rat-heart FABP realizes a micromolar Kd with a greater reliance upon hydrophobic interactions than does icefish FABP.
Animals↗
Fatty acid transfer from liver and intestinal fatty acid-binding proteins to membranes occurs by different mechanisms.
Intestinal absorptive cells contain high levels of expression of two homologous fatty acid-binding proteins (FABP), liver FABP (L-FABP), and intestinal FABP (I-FABP). Both bind long chain fatty acids with relatively high affinity. The functional distinction, if any, between these two proteins remains unknown. It is often hypothesized that FABP are important in intracellular transport of fatty acids. To assess whether fatty acid transport properties might differ between the two enterocyte FABPs, we examined the rate and mechanism of transfer of fluorescent anthroyloxy fatty acids (AOFA) from these proteins to model membranes using a resonance energy transfer assay. The results show that the absolute rate of AOFA transfer from I-FABP is faster than from L-FABP. Moreover, the apparent mechanism of fatty acid transfer is different between the two proteins. The rate of AOFA transfer from I-FABP is independent of ionic strength, directly dependent on the concentration of acceptor membrane vesicles, and dramatically regulated by the lipid composition of the membranes. These data strongly suggest that fatty acid transfer from I-FABP to membranes occurs by direct collisional interaction of the protein with the phospholipid bilayer. In contrast, the characteristics of fatty acid transfer from L-FABP are consistent with an aqueous diffusion-mediated process. Thus the two enterocyte FABPs may perform different functions within the intestinal absorptive cell in the regulation of fatty acid transport and utilization. It is hypothesized that L-FABP may act as a cytosolic buffer for fatty acids, maintaining the unbound fatty acid concentration, whereas I-FABP may be involved in the uptake and/or specific targeting of fatty acid to subcellular membrane sites.
Animals↗
Role of portal region lysine residues in electrostatic interactions between heart fatty acid binding protein and phospholipid membranes.
The structure of heart fatty acid binding protein (HFABP) is a flattened beta-barrel comprising 10 antiparallel beta-sheets capped by two alpha-helical segments. The helical cap region is hypothesized to behave as a portal "lid" for the entry and release of ligand from the binding pocket. The transfer of fatty acid from HFABP is thought to occur via effective collisional interactions with membranes, and these interactions are enhanced when transfer is to membranes of net negative charge, thus implying that specific basic residues on the surface of HFABP may govern the transfer process [Wootan, M. G., & Storch, J. (1994) J. Biol. Chem. 269, 10517-10523]. To directly examine the role of charged lysine residues on the HFABP surface in specific interactions with membranes, chemical modification and selective mutagenesis of HFABP were used. All surface lysine residues were neutralized by acetylation of recombinant HFABP with acetic anhydride. In addition, seven mutant HFABPs were generated that resulted in charge alterations in five distinct sites of HFABP. Modification of the protein did not significantly alter the structural or ligand binding properties of HFABP, as assessed by circular dichroism, fluorescence quantum yield, and ligand binding analyses. By using a resonance energy transfer assay, transfer of 2-(9-anthroyloxy)palmitate (2AP) from acetylated HFABP to membranes was significantly slower than transfer from native HFABP. In addition, in distinct contrast to transfer from native protein, the 2AP transfer rate from acetylated HFABP was not increased to acceptor membranes of increased negative charge. Transfer of 2AP from HFABP mutants involving K22, located on alpha-helix I (alpha-I) of the helical cap region, was 3-fold slower than transfer from wild-type protein, whereas rates from a mutant involving the K59 residue, located on the beta 2-turn of the barrel near the helical cap, were 2-fold faster than those of wild type. A double mutant involving K22 and K59 resulted in transfer rates identical to those of wild type, indicating that at least two domains are involved in determining the overall rate of ligand transfer. In addition, 2AP transfer rates from HFABP mutated at position 22 were totally unaffected by the charge characteristics of acceptor membranes, in marked contrast to wild type and other members of the mutant series. Further, by introducing a positive charge to alpha-helix II (alpha-II) of the helical cap region, 2AP transfer rates increased by 4-fold and properties of HFABP transfer began to approach those seen for AFABP, another member of the FABP family thought to transfer ligand via collisional interactions with membranes, which has a lysine residue in the alpha-II helix. These studies demonstrate that the helical cap region of HFABP may play an important role in governing ionic interactions between binding protein and membranes.
Acetylation↗
Fatty acid uptake by Caco-2 human intestinal cells.
The Caco-2 human enterocytic cell line was used to study the kinetics and mechanism of intestinal long chain fatty acid uptake. Initial rates of palmitate (16:0), oleate (18:1), and octanoate (8:0) uptake were determined for adherent cells at greater than 7 days confluence. Uptake of long chain 18:1 and 16:0 by cells grown on coverslips was saturable with an apparent Km of 0.3 microM, but also included a notable diffusive component. Uptake of short chain 8:0, on the other hand, was linear up to 10 microM. Cells grown on permeable Transwell filters were used to study uptake at the apical versus the basolateral membrane. Uptake of long chain (18:1 and 16:0), but not short chain (8:0), fatty acid was saturable at both surfaces with a similar Km of 0.3 microM. In addition, long chain but not short chain fatty acid uptake was competitively inhibitable. Western blot analysis demonstrated that Caco-2 cells express a protein immunoreactive with antibodies to the rat liver plasma membrane fatty acid binding protein (FABPpm), which is thought to be involved in long chain fatty acid transport. Nevertheless, long chain fatty acid uptake was not inhibited by pretreatment of the cells with an FABPpm antibody, nor by pretreatment with two proteases. These data support a saturable component in the transport of long chain but not short chain fatty acids by human intestinal epithelial cells, which may involve an as yet unknown plasma membrane protein.
Animals↗
Surface lysine residues modulate the collisional transfer of fatty acid from adipocyte fatty acid binding protein to membranes.
The transfer of unesterified fatty acids (FA) from adipocyte fatty acid binding protein (A-FABP) to phospholipid membranes is proposed to occur via a collisional mechanism involving transient ionic and hydrophobic interactions [Wootan & Storch (1994) J. Biol. Chem. 269, 10517-10523]. In particular, it was suggested that membrane acidic phospholipids might specifically interact with basic residues on the surface of A-FABP. Here we addressed whether lysine residues on the surface of the protein are involved in this collisional transfer mechanism. Recombinant A-FABP was acetylated to neutralize all positively charged surface lysine residues. Protein fluorescence, CD spectra, and chemical denaturant data indicate that acetylation did not substantially alter the conformational integrity of the protein, and nearly identical affinities were obtained for binding of the fluorescently labeled FA [12-(9-anthroyloxy)oleate] to native and acetylated protein. Transfer of 2-(9-anthroyloxy)palmitate (2AP) from acetylated A-FABP to small unilamellar vesicles (SUV) was 35-fold slower than from native protein. In addition, whereas the 2AP transfer rate from native A-FABP was directly dependent on SUV concentration, 2AP transfer from acetylated protein was independent on the concentration of acceptor membranes. Factors which alter aqueous-phase solubility of FA, such as ionic strength and acyl chain length and saturation, affected the AOFA transfer rate from acetylated but not native A-FABP. Finally, an increase in the negative charge density of the acceptor SUV resulted in a marked increase in the rate of transfer from native A-FABP but did not increase the rate from acetylated A-FABP.(ABSTRACT TRUNCATED AT 250 WORDS)
Acetylation↗
Regulation of fluorescent fatty acid transfer from adipocyte and heart fatty acid binding proteins by acceptor membrane lipid composition and structure.
Adipocyte and heart fatty acid binding proteins (A-FABP and H-FABP) are closely related members of the FABP family. Unlike the more distantly related liver FABP, these FABP have been proposed to transfer free fatty acids to model membranes by a collisional mechanism (Wootan, M. G., Bernlohr, D. A., and Storch, J. (1993) Biochemistry 32, 8622-8627; Kim, H. K., and Storch, J. (1992) J. Biol. Chem. 267, 20051-20056). Collisional transfer requires that the acceptor membranes interact with FABP during the transfer process. We, therefore, examined whether the acceptor membrane structure and lipid composition regulate the rate of anthroyloxy-labeled palmitate (2AP) transfer from A- and H-FABP, using a fluorescence resonance energy transfer assay. The results showed that 2AP transfer from A- and H-FABP was more rapid to acceptor vesicles containing acidic phospholipids and was slower to positively charged membranes. In addition, the rate of 2AP transfer from A- and H-FABP was enhanced by unsaturation of the phosphatidylcholine acyl chains and was slowed by the presence of cholesterol or sphingomyelin in the acceptor membranes. These latter changes were small but of a similar magnitude and together suggest that fatty acid transfer from A- and H-FABP was slower to membranes of greater lipid order. Since transfer by an aqueous diffusion mechanism would be unaffected by acceptor membrane properties, these studies strengthen the hypothesis that free fatty acid transfer from A- and H-FABP to membranes occurs via a collisional mechanism.
Adipocytes↗
Activation of human blood lymphocytes by house dust mite protein and Mycobacterium tuberculosis purified protein derivative: effects of interferon-gamma, interleukin-4, dexamethasone and cyclosporin A.
Peripheral blood mononuclear cells (PBMC) from nonatopic donors sensitive to Mycobacterium tuberculosis purified protein derivative (PPD) and from atopic donors sensitive to PPD and house dust mite antigen (HDM), were stimulated in vitro to proliferate in response to exogenous IL-2. In the presence of exogenous IFN-gamma, the response of cells from atopic donors to PPD was either unaffected or slightly enhanced, whereas the response to HDM was inhibited in a dose-dependent manner. The response of cells from nonatopic donors to PPD remained unchanged or was only slightly inhibited and this was not dose-dependent. Exogenous IL-4 did not reverse the inhibitory effects of IFN-gamma and a neutralizing antibody to IL-4 did not inhibit the proliferative response of cells to HDM and IL-2. Dexamethasone inhibited the IL-2-mediated response of cells from atopic donors stimulated with HDM, whereas the IL-2-mediated response of cells induced by PPD was either unchanged or stimulated by dexamethasone. Cyclosporin A inhibited the response of cells to both HDM and PPD. These results suggest that IFN-gamma can exert a selective effect on the response of T lymphocytes to different antigens and that it is possible to identify compounds able to regulate this activity.
Animals↗
Mechanism of fluorescent fatty acid transfer from adipocyte fatty acid binding protein to membranes.
Adipocyte fatty acid binding protein (A-FABP) is a 15-kDa protein found in high abundance in the cytosol of adipose cells. To better understand the role of this protein in intracellular free fatty acid (ffa) transport, the mechanism of ffa transfer from A-FABP to model membranes was examined by monitoring the transfer of fluorescent anthroyloxy ffa (AOffa) to small unilamellar phospholipid vesicles, using a resonance energy transfer assay. Structural features of ffa that increase aqueous solubility, such as shorter chain length and unsaturation, did not increase the AOffa transfer rate. In addition, solution conditions that increase the aqueous solubility of ffa, such as decreasing ionic strength and increasing pH, had little effect on AOffa transfer from A-FABP to membranes. These results suggest that AOffa do not transfer through the aqueous phase. The small entropic contribution to the free energy of the transfer process provides further evidence that AOffa may not travel through the surrounding aqueous environment when transferred from A-FABP to phospholipid membranes. Finally, the rate of AOffa transfer from A-FABP was directly dependent on the concentration of the acceptor membranes. These studies suggest that AOffa transfer from A-FABP to phospholipid vesicles may occur via transient collisional interactions between the protein and membranes.(ABSTRACT TRUNCATED AT 250 WORDS)
Adipose Tissue↗
Effect of phospholipid headgroup composition on the transfer of fluorescent long-chain free fatty acids between membranes.
The transfer of long-chain anthroyloxy-labeled-free fatty acids (AOffa) between small unilamellar vesicles (SUV) was studied using a fluorescence energy transfer assay. Donor SUV were labeled with AOffa, and acceptor SUV contained the nonexchangeable quencher NBD-phosphatidylethanolamine. Donor and acceptor membranes were mixed using a stopped-flow apparatus, and intermembrane transfer was monitored by the decrease in AO fluorescence with time. The effect of donor membrane phospholipid headgroup composition on AOffa transfer was examined by incorporating phosphatidylethanolamine (PE), phosphatidic acid (PA), or phosphatidylglycerol (PG) into donor SUV otherwise composed of phosphatidylcholine (PC). Addition of 25 mol% of either of the negatively charged phospholipids (PA or PG) resulted in an increase in the rate of AOffa transfer, whereas addition of zwitterionic PE had no effect on transfer rate. The transfer kinetics were in all cases best described by a biexponential process, and it was found that the addition of PA caused an increase in the fraction of AOffa which transfer at the fast rate. This was likely due in large part to the asymmetric distribution of AOffa in these vesicles, with more fatty acid in the outer hemileaflet. This in turn may be due to the asymmetric distribution of PA between the inner and outer hemileaflets. Thus the increased AOffa transfer rate from negatively charged vesicles may be caused by charge repulsion between ffa and negatively charged headgroups. This increase in transfer rate was maximized at pH 9 as compared to pH 7, further suggesting that the increased rate of intermembrane transfer may arise because of charge repulsion. Finally, it was shown that decreasing the membrane surface potential by increasing the ionic strength caused the rate of AOffa transfer from PA-containing vesicles and PC vesicles to become identical. The results demonstrate that the ionic character of the donor membrane bilayer is an important determinant of the transfer rate of long-chain fatty acids between membranes.
Fatty Acids, Nonesterified↗
Fatty acid esterification during differentiation of the human intestinal cell line Caco-2.
The Caco-2 human intestinal cell line was used to examine fatty acid esterification during development of the enterocytic phenotype. Acyl-CoA synthetase activity increased approximately 40%, and the incorporation of palmitic acid into triacylglycerol relative to phosphatidylcholine increased nearly 2-fold during Caco-2 differentiation. A rate-limiting enzyme activity in the glycerol 3-phosphate pathway of triacylglycerol synthesis, glycerol-3-phosphate acyltransferase, was at levels comparable with rat jejunum and remained unchanged during differentiation. In contrast, the activity of monoacylglycerol acyltransferase, which is unique to the monoacylglycerol pathway of triacylglycerol synthesis, was present at < 7% of the levels in rat jejunum. Further analysis of the glycerol 3-phosphate pathway showed that the rate-limiting enzyme activities for diacylglycerol conversion to triacylglycerol, diacylglycerol acyltransferase, and phosphatidylcholine, CTP:phosphocholine cytidylyltransferase, increased 2-3-fold and decreased approximately 40%, respectively, during Caco-2 differentiation. In addition, a 2-fold increase in cellular diacylglycerol mass was observed during enterocytic conversion. These data indicate that fatty acid esterification to triacylglycerol in Caco-2 cells occurs primarily via the glycerol 3-phosphate pathway. Furthermore, the differentiation-dependent increase in fatty acid esterification to triacylglycerol relative to phosphatidylcholine appears to result from increased utilization of diacylglycerol to synthesize triacylglycerol and a concomitant decrease in diacylglycerol utilization for phosphatidylcholine synthesis.
Acyltransferases↗
Transfer of long-chain fluorescent fatty acids between small and large unilamellar vesicles.
Transfer of 12-(9-anthroyloxy)stearic acid (12AS) was measured between small unilamellar vesicles (SUV) and between large unilamellar vesicles (LUV), over a temperature range of 5-50 degrees C. The results of this study clearly establish the biexponential nature of the time dependence of the transfer in a variety of vesicle types and confirm our previous results using egg phosphatidylcholine (EPC) SUV at 25 degrees C (Storch & Kleinfeld, 1986). In our previous study we developed a kinetic model of the transfer process and concluded that the observed time dependence of the transfer of long-chain 12-(9-anthroyloxy) fatty acids (AOFA) was due to transbilayer flip-flop that was much slower than the rate at which the fatty acids (FA) move from the vesicle and into the surrounding aqueous phase (the off step). In the present study, experimental and theoretical advances have allowed us to examine, in detail, predictions of the kinetic model that critically depend upon the slow rate of flip-flop. The current results verify these predictions and demonstrate that slow AOFA flip-flop is rate limiting in at least three different vesicle systems and at all temperatures studied. Moreover, both flip-flop and the off rate constants were almost an order of magnitude smaller in EPC-LUV than in EPC-SUV. Flip-flop was found to be asymmetric (the rate constant for transfer from the inner to outer hemileaflet of the bilayer is approximately twice that from the outer to inner hemileaflet) in SUV but virtually symmetric in LUV. The temperature dependence of transfer was used to determine the thermodynamic activation potentials for the flip-flop and off rate constants.(ABSTRACT TRUNCATED AT 250 WORDS)
Dimyristoylphosphatidylcholine↗
Stimulation of phospholipase-D in Epstein Barr virus-transformed human B-cells.
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B-Lymphocytes↗
Nutritional control of fatty acid esterification in differentiating Caco-2 intestinal cells is mediated by cellular diacylglycerol concentrations.
The Caco-2 human intestinal cell line was used to investigate the effects of exogenous lipid on fatty acid esterification in differentiating intestinal absorptive cells. Preincubation of Caco-2 cells with either palmitate or palmitate plus 2-monoolein resulted in greater utilization of subsequently added fatty acid for triacylglycerol relative to phosphatidylcholine synthesis. Despite this lipid-induced alteration in metabolism, the activities of acyl-CoA synthetase, glycerol-3-phosphate acyltransferase and diacylglycerol acyltransferase were unchanged. In addition, monoacylglycerol acyltransferase activity was nearly undetectable, even after preincubation with 2-monoolein. The intracellular diacylglycerol concentrations were, however, increased with greater lipid substrate availability. These studies indicate that, under conditions of increased dietary lipid, intestinal fatty acid esterification via the glycerol-3-phosphate pathway is modulated by cellular diacylglycerol concentrations.
Acyltransferases↗
Mechanism of free fatty acid transfer from rat heart fatty acid-binding protein to phospholipid membranes. Evidence for a collisional process.
Fatty acid binding proteins (FABP) are a family of low molecular weight proteins found in many tissues that actively utilize free fatty acids (ffa). FABP would be expected to have a particularly important role in the heart, where over 80% of energy requirements are derived from oxidation of long chain fatty acids. The precise physiological function of heart FABP (H-FABP) has not been definitively identified, although it is thought to play a role in intracellular ffa transport. To examine the possible role of H-FABP in cardiac myocyte transfer of ffa, we examined the transfer of fluorescent anthroyloxy ffa (AOffa) from H-FABP to model phospholipid membranes, using a resonance energy transfer assay. In contrast to previous observations of ffa transfer from liver FABP and from membranes, transfer from H-FABP to membranes appears to occur by a different mechanism. AO-palmitate (16:0) transfer was 1.5-fold slower than AO-stearate (18:0) transfer, and mono-unsaturation did not affect the transfer rate. The AOffa transfer rate from H-FABP increased with increasing ionic strength and decreased slightly between pH 7 and 9. These results suggest that the rate of ffa transfer from H-FABP to membranes is independent of the ffa aqueous solubility. Thermodynamic analysis showed that the free energy of activation for the ffa transfer process arises primarily from an enthalpic component, with only a small entropic contribution, again suggesting the lack of an aqueous phase route of ffa delivery. Finally, the ffa transfer rate was found to be directly dependent on the concentration of acceptor membranes. These data therefore suggest that transfer of AOffa from H-FABP to membranes may occur via collisional interactions between the protein and membranes.
Animals↗