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J Storch

Publications and source records attributed to J Storch.

At least 55 records · Page 3Linked to original sources
PubMed1992

Mechanism of the spontaneous transfer of unconjugated bilirubin between small unilamellar phosphatidylcholine vesicles.

Unconjugated bilirubin (bilirubin-IX alpha), the hydrophobic end product of heme degradation, is esterified in the hepatocyte endoplasmic reticulum to water-soluble conjugates prior to excretion in bile. To characterize the process of intracellular bilirubin transport, the kinetic and thermodynamic activation parameters for the spontaneous transfer of bilirubin between small unilamellar egg lecithin vesicles were determined. Bilirubin-IX alpha was added to donor vesicles labeled with the fluorescent phospholipid probe, (5-(dimethylamino)naphthalene-1-sulfonyl) dipalmitoyl-L-alpha-phosphatidylethanolamine (dansyl-PE). When bound to the donor vesicles, bilirubin quenches the dansyl probe fluorescence through resonance energy transfer. The movement of bilirubin from dansyl-labeled donor vesicles to unlabeled acceptor vesicles was monitored directly by the reemergence of dansyl fluorescence over time. Vesicle fusion and intervesicle transfer of the dansyl-PE probe were excluded by quasielastic light scattering and fluorescence resonance energy transfer studies. Stopped-flow analysis demonstrated that the transfer of bilirubin was described by a single-exponential function with a mean half-time of 2.0 +/- 0.1 ms (+/- SD) at 37 degrees C. The rate of bilirubin transfer was independent of acceptor vesicle concentration and decreased with increasing buffer ionic strength, indicating that intermembrane transfer occurred via aqueous diffusion, rather than vesicle collisions. The free energy of activation (delta G++) for the dissociation of bilirubin from donor vesicles was 14.2 kcal.mol-1. These studies suggest that bilirubin is associated with phospholipid bilayers at the membrane-water interface. We postulate that the movement of unconjugated bilirubin between intracellular membranes occurs via spontaneous transfer through the aqueous phase.

Bilirubin↗
PubMed1992

Free fatty acid transfer from rat liver fatty acid-binding protein to phospholipid vesicles. Effect of ligand and solution properties.

Fatty acid binding proteins (FABP) are a family of 14-15-kDa proteins found in many mammalian cell types in high abundance. Although their precise physiological role remains hypothetical, the transfer of free fatty acids (ffa) to intracellular membrane sites is believed to be an important function of FABP. To better understand the role of FABP in this process, we have examined how the rate of ffa transfer from liver FABP (L-FABP) to model membranes is influenced by variations in ffa structure and properties of the aqueous phase. The rate of transfer of fluorescent anthroyloxy ffa to model acceptor membranes was monitored using a resonance energy transfer assay. The results show that a monounsaturated ffa transfers 2-fold more rapidly than a saturated ffa of equivalent chain length, and a two-carbon increase in acyl chain length results in a 3-fold decrease in transfer rate. The transfer rate decreases logarithmically with increasing ionic strength, suggesting that the aqueous solubility of the ffa is an important determinant of its dissociation rate from L-FABP. Fatty acid binding and the relative partition of n-(9-anthroloxy) ffa to L-FABP as compared with phospholipid membranes both decrease as pH decreases, indicating that ionized but not protonated ffa bind to L-FABP. The rate of ffa transfer from L-FABP to membranes increases approximately 4-fold with increasing pH, suggesting that ionization of the ffa carboxyl group is also an important determinant of the transfer process. Analysis of the dependence of the transfer rate on temperature demonstrates that the delta G++ of the activated state for ffa transfer arises from both enthalpic and entropic processes. These studies demonstrate that the rate of transfer of long chain ffa from L-FABP to membranes is substantially affected by aqueous phase variables as well as properties of the ffa ligand itself.

Animals↗
PubMed1991

Direct determination of free fatty acid transport across the adipocyte plasma membrane using quantitative fluorescence microscopy.

Movement of free fatty acids (FFA) across the plasma membrane has been directly measured for the first time, using fluorescent FFA analogs and quantitative fluorescence microscopy. The rate of short chain FFA (less than or equal to 12 carbons) transport from the extracellular medium into intracellular lipid droplets of 3T3F442A adipocytes was more than 40-fold faster than long chain FFA (16 and 18 carbons). The membrane-impermeable amino reagent 4,4'-diisothiocyanostilbene-2,2'-disulfonate, inhibited greater than or equal to 50% of the long chain FFA transport but had no effect on short chain FFA transport. Oleic acid (2 microM) inhibited 90% of the fluorescent oleate transport but had no effect on the 11-carbon analog. These results indicate that a large fraction of long chain FFA uptake is mediated by a plasma membrane protein (s).

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1991

IgE and IgG are synergistic in antigen-mediated release of thromboxane from human lung macrophages.

The mechanism of IgE-mediated release of thromboxane A2 from human lung macrophages has been studied using a monoclonal chimeric human/mouse IgE antibody and its specific antigen. The cells could be sensitized at 37 degrees C but not at 4 degrees C by incubation with IgE, and released a significant amount of thromboxane A2 (TXA2), measured as the stable hydrolysis product TXB2, in response to an anti-chimeric IgE antibody. In contrast, stimulation of IgE-sensitized macrophages with the specific antigen produced less than 10% of this response. A similar time course for the release of TXB2 and the formation of inositol monophosphate in the presence of LiCl was observed. Cleavage of the Fc domain of the anti-chimeric IgE antibody substantially eliminated its capacity to stimulate IgE-sensitized cells. However, the weak or undetectable response to chimeric IgE plus specific antigen was substantially potentiated by an antigen-specific chimeric IgG antibody. IgG-sensitized macrophages did not respond to antigen challenge by the release of TXB2. Preincubation of the cells with a monoclonal antibody against the low affinity receptor for IgE (Fc epsilon RII/CD23) did not prevent IgE sensitization. We conclude that cell-bound IgE antibody cannot induce the release of TXB2 but has fixed antigen which then must interact with specific IgG antibody and IgG receptors to induce mediator release.

Antigen-Antibody Complex↗
PubMed1991

Fatty acid uptake and metabolism in a human intestinal cell line (Caco-2): comparison of apical and basolateral incubation.

Free fatty acids can enter the enterocyte via the apical or basolateral plasma membrane. We have used the Caco-2 intestinal cell line to examine the polarity of free fatty acid uptake and metabolism in the enterocyte. Differentiated Caco-2 cells form polarized monolayers with tight junctions, and express the small intestine-specific enzymes sucrase and alkaline phosphatase. Cells were grown on permeable polycarbonate Transwell filters, thus allowing separate access to the apical and basolateral compartments. Total uptake of [3H]palmitate bound to bovine serum albumin (palmitate-BSA 4:1) was twofold higher (P less than 0.05 or less) at the apical surface than at the basolateral surface. The relative apical and basolateral membrane surface areas of the Caco-2 cells, as measured by partition of the fluorophore trimethylammonium-diphenylhexatriene TMA-DPH), was found to be 1:3. Thus, apical fatty acid uptake was sixfold higher than basolateral uptake per unit surface area. Analysis of metabolites after incubation with submicellar concentrations of [3H]palmitate showed that the triacylglycerol to phospholipid (TG:PL) ratio was higher for fatty acid added to the apical as compared to the basolateral compartment (20% at 60 min, P less than 0.025). Little fatty acid oxidation was observed. Preincubation with albumin-bound palmitate, alone or with monoolein, increased the incorporation of both apical and basolateral free fatty acids into TG. The results suggest that the net uptake of long-chain free fatty acids across the apical plasma membrane is greater than uptake across the basolateral membrane. In addition, a small increase in the TG:PL ratio for apically, compared to basolaterally, added free fatty acids suggests that polarity of metabolism occurs to a limited extent in Caco-2 enterocytes.

Blotting, Western↗
PubMed1990

Fatty acid binding sites of rodent adipocyte and heart fatty acid binding proteins: characterization using fluorescent fatty acids.

Murine adipocyte and rat heart fatty acid binding proteins (FABP) are closely related members of a family of cytosolic proteins which bind long-chain free fatty acids (ffa). The physical and chemical characteristics of the fatty acid binding sites of these proteins were studied using a series of fluorescent analogues of stearic acid (18:0) with an anthracene moiety covalently attached at seven different positions along the length of the hydrocarbon chain (AOffa). Previously, we used these probes to investigate the binding site of rat liver FABP (L-FABP) [Storch et al. (1989) J. Biol. Chem. 264, 8708-8713]. Here we extend those studies to adipocyte and heart FABP, two members of the FABP family which share a high degree of sequence homology with each other (62% identity) but which are less homologous with L-FABP (approximately 30%). The results show that the fluorescence emission spectra of AOffa bound to adipocyte FABP (A-FABP) are blue-shifted relative to heart FABP (H-FABP), indicating that AOffa bound to A-FABP are held in a more constrained configuration. For both proteins, constraint on the bound ffa probe is highest at the midportion of the acyl chain. Ffa are bound in a hydrophobic environment in both proteins. Excited-state lifetimes and fluorescence quantum yields suggest that the binding site of H-FABP is more hydrophobic than that of A-FABP. Nevertheless, acrylamide quenching experiments indicate that ffa bound to H-FABP are more accessible to the aqueous environment than are A-FABP-bound ffa.(ABSTRACT TRUNCATED AT 250 WORDS)

Adipose Tissue↗
PubMed1990

Transfer of fluorescent fatty acids from liver and heart fatty acid-binding proteins to model membranes.

Fatty acid binding proteins (FABP) are a family of 14-15 kDa proteins found in high abundance in many mammalian cell types. The physiological functions of the FABP remain unknown. It is also not known whether each FABP has a unique function, or whether all FABP function in a similar manner in their respective tissues. In this report the rate of transfer of anthroyloxy-labeled free fatty acid (ffa) from FABP to phospholipid bilayers is monitored using a fluorescence resonance energy transfer assay. A comparison is made between heart muscle FABP and liver FABP, and the results show that the rate of ffa transfer from the heart protein is an order of magnitude greater than the rate of transfer from the liver protein. Ffa transfer rates from both liver and heart FABP are independent of acceptor concentration and composition, suggesting that, at least in the case of model membrane acceptor vesicles, the mechanism of transfer is via aqueous diffusion rather than via collision of FABP with membranes. Since the rate of ffa transfer is likely to be important to cellular ffa traffic, these studies suggest that heart FABP may function differently within the myocyte than does liver FABP within the hepatocyte.

Animals↗
PubMed1990

The purification and characterization of a fatty acid binding protein specific to pig (Sus domesticus) adipose tissue.

Western-blot analysis using antiserum to 3T3-L1-cell fatty acid binding protein (FABP) revealed that pig adipose tissue contains a 15 kDa protein immunologically similar to the murine protein. This 15 kDa protein was purified from pig adipose tissue by sequential application of Sephadex G-50 gel filtration, cation exchange and covalent chromatography on Thiol-Sepharose-4B. The purity of the pig protein was established by two-dimensional polyacrylamide-gel electrophoresis. Isoelectric focusing indicated that the pig adipose FABP (a-FABP) exists with two charge isoforms (pI 5.1 and 5.2), both of which persist after delipidation. The N-terminus of the purified pig a-FABP was blocked; however, cleavage with CNBr allowed recovery of a 12-amino-acid peptide which was identical with the murine a-FABP sequence (residues 36-48) at 10 of 12 positions. The pig a-FABP bound 12-(9-anthroyloxy)oleic acid saturably and stoichiometrically, with an apparent dissociation constant of 1.0 microM. Northern-blot analysis using the cDNA for the murine 3T3-L1 FABP revealed that the pig a-FABP was expressed exclusively in adipose tissue.

Adipose Tissue↗
PubMed1990

Thromboxane release by lymphokine-differentiated U937 human monocytic cells: response to platelet-activating factor (PAF) and chemotactic peptide (FMLP) but not to low affinity IGE-receptor (Fc epsilon RII/CD23) occupation.

The primary objective of this study was to explore if the CD23 antigen is a functional low affinity IgE receptor on macrophages for the release of thromboxane B2 (TXB2). The responsiveness of U937 monocytic cells and their macrophage-like inducible forms to platelet-activating factor (Paf), the chemotactic peptide fMLP, and low affinity IgE-receptor occupation was examined. Differentiation of U937 cells by phorbol myristate acetate (PMA) and a cancer cell line (HBT 5637) conditioned medium (5637-CM), but not INFg or IL4, resulted in a macrophage-like cell line which released TXB2. A high basal release of TXB2 with no significant response to Paf or fMLP challenge was seen following culture of cells with PMA. In 5637-CM-differentiated cells, Paf and fMLP induced a rapid release of TXB2, about 10 fold above basal activity. There was a slow Ca-independent response to short-term treatment with PMA and a rapid Ca-dependent response to the ionophore A23187. Both stimulants acted synergistically on TXB2 synthesis in 5637-CM differentiated cells. Although low affinity receptors for IgE (Fc epsilon RII/CD23) were induced by 5637-CM, no TXB2 was released in response to soluble or latex-bound IgE-antigen complexes or to anti-Fc epsilon RII/CD23-antibodies. IL4 and to a lesser extent INFg both induced Fc epsilon RII/CD23 receptor expression, but inhibited release of TXB2 in response to Paf, fMLP, or PMA. We conclude that the functional receptors for IgE on mature macrophages are most probably not Fc epsilon RII/CD23.

Antibodies, Anti-Idiotypic↗
PubMed1990

Resistance to the pore-forming protein of cytotoxic T cells: comparison of target cell membrane rigidity.

Cytotoxic T lymphocytes (CTL) release from their granules a 70 kDa protein, called PFP, perforin or cytolysin, which inserts into the target cell plasma membrane in its monomeric form. Here it polymerizes into a macromolecular complex forming pores as large as 20 nm. Although purified PFP/perforin can effectively lyze all target cells tested. CTL are refractory to lysis. The mechanism underlying the resistance of CTL is currently unknown. This study represents a search for membrane structural properties that could confer resistance to CTL against PFP/perforin-mediated lysis. The fluorescent dye merocyanine 540 was used to measure the lipid head group packing of CTL and several target cells, and 1-[4-(trimethylamine)phenyl]-6-phenylhexa-1,3,5-triene was used to estimate the fluidity of the membrane hydrocarbon region. The resistance against PFP/perforin-mediated lysis was determined by the 51Cr release assay. A comparison of the membrane rigidity with cell resistance led to the conclusion that the membrane lipid structure cannot account for the unusually high resistance of CTL. In particular, the resistant CTL line CTLL-2 has a lipid head group packing that is looser than that of Yac-1, and the sensitive target cells Jy-25 and EL-4 have membrane acyl chains that are less fluid than those of the effector CTLL-R8.

Animals↗
PubMed1989

3-[p-(6-phenyl)-1,3,5-hexatrienyl]phenylpropionic acid (PA-DPH): characterization as a fluorescent membrane probe and binding to fatty acid binding proteins.

The negatively charged fluorophore 3-[p-(6-phenyl)-1,3,5-hexatrienyl]phenylpropionic acid (PA-DPH) was characterized by comparison with its parent compound DPH, and with cationic trimethylammonium-DPH (TMA-DPH). The molar absorption coefficient of PA-DPH (60,000 cm-1.mol-1) as well as its quantum yield (0.7) and fluorescence lifetime (5 ns) in fluid phase membranes are intermediate between DPH and TMA-DPH. Steady-state fluorescence polarization studies show that PA-DPH detects the phase transition of both neutral and anionic bilayers. In fluid phase membranes the absolute values of PA-DPH polarization are considerably higher than DPH and somewhat lower than TMA-DPH. The results suggest that like TMA-DPH, PA-DPH is anchored to the surface of the membrane by its charge, but that it is probing a region somewhat deeper along the bilayer normal. PA-DPH binds to rat hepatic fatty acid binding protein (hFABP) and bovine serum albumin at PA-DPH/protein molar ratios of 1.5:1 and at least 6:1, respectively. Native oleic acid competes with PA-DPH for binding to both proteins, suggesting that the two ligands compete for similar binding sites. The affinity of PA-DPH for hFABP is similar to that of oleic acid. Thus, PA-DPH should be useful both as an anionic fluorescent membrane probe and a long-chain free fatty acid analogue.

Carrier Proteins↗
PubMed1989

Plasma membrane lipid order and composition during adipocyte differentiation of 3T3F442A cells. Studies in intact cells with 1-[4-(trimethylamino)phenyl]-6-phenylhexatriene.

The plasma membrane lipid order of 3T3F442A cells was examined during the course of adipocyte differentiation by measuring the fluorescence polarization of 1-[4-(trimethylamino)phenyl]-6-phenylhexatriene. This cationic fluorophore labels the plasma membrane but does not rapidly redistribute to intracellular organellar membranes and can, therefore, be used to specifically probe the plasma membrane of intact cells. Studies with whole cells demonstrated that the plasma membrane of 3T3F442A cells becomes less ordered during the course of adipocyte conversion and that this alteration begins relatively early during the differentiation process. In addition, the lipid order of plasma membranes isolated from adipocyte-stage cells was found to be lower than the lipid order of the early, fibroblast-stage cells. Analysis of membrane lipid composition suggests that the molecular bases for the decrease in adipocyte plasma membrane lipid order are a large increase in the level of monounsaturated phospholipid acyl chains and a decrease in the molar ratio of cholesterol to phospholipid. The alteration in plasma membrane lipid composition may be specifically required for integral membrane protein function, since the differentiation-dependent fatty acid desaturase activity is known to be maintained even in the absence of triacylglycerol accumulation.

Adipose Tissue↗
PubMed1989

Studies of the fatty acid-binding site of rat liver fatty acid-binding protein using fluorescent fatty acids.

Rat liver fatty acid-binding protein (FABP) is a 14.3-kDa cytosolic protein which binds long chain free fatty acids (ffa) and is believed to participate in intracellular movement and/or distribution of ffa. In the studies described here fluorescently labeled ffa were used to examine the physical nature of the ffa-binding site on FABP. The fluorescent analogues were 16- and 18-carbon ffa with an anthracene moiety covalently attached at eight different points along the length of the hydrocarbon chain (AOffa). Emission maxima of all FABP-bound AOffa were found to be considerably blue-shifted with respect to emission of phospholipid membrane-bound AOffa, suggesting a high degree of motional constraint for protein-bound ffa. Large fluorescence quantum yields and long excited state life-times indicate that the FABP-binding site for ffa is highly hydrophobic. Analysis of rotational correlation times for the FABP-bound AOffa suggest that the ffa are tightly bound to the protein. Variation of the quantum yield with attachment site suggests that the carboxylic acid group of the fatty acyl chain is located near the aqueous surface of the FABP. The rest of the ffa hydrocarbon chain is buried within the protein in a hydrophobic pocket and is particularly constrained at the midportion of the acyl chain.

Animals↗
PubMed1989

Primary treatment of regional and disseminated pancreatic cancer with hexamethylmelamine, mitomycin C and 5-fluorouracil infusion.

Hexamethylmelamine, mitomycin C and 5-fluorouracil infusion (HexMF) achieved a median survival of 9 months for the 45 patients with either metastatic stage III or unresectable stage II carcinoma of the pancreas. Fifteen percent survived 2 years. Of 32 patients with measurable tumors, 7 had partial and 3 had minor responses (31%); an additional 44% has stable disease for 3 months or more. Response was associated with a 17-month median survival. These findings are indications for further evaluation of both dosage-intensive 5-fluorouracil infusions alone and HexMF as an alternative to streptozotocin- or adriamycin-containing regimens. Patients with nonmeasurable disease are candidates for survival-oriented phase III studies.

Altretamine↗
PubMed1988

Structurally distinct plasma membrane regions give rise to extracellular membrane vesicles in normal and transformed lymphocytes.

Shedding of extracellular membranes from the cell surface may be one of the means through which cells communicate with one another. In an attempt to elucidate whether cell surface exfoliation is a directed or random process, we investigated the membrane lipid and protein composition and membrane lipid order of shed extracellular membranes and of plasma membranes from which they arose in normal circulating lymphocytes and in the B-lymphoblastoid cell lines Raji, WI HF2 729 and the T-lymphoblastoid cell line Jurkat. Extracellular membranes derived from transformed cell lines were more rigid as assessed by steady state polarization of 1,6-diphenylhexatriene (DPH) and were highly enriched in cholesterol when compared with the corresponding plasma membrane. The extracellular membranes from normal lymphocytes, on the other hand, were more fluid and contained more polyunsaturated acyl chains than did the plasma membranes from these cells. Our results suggest that extracellular membranes are shed from specialized regions of the lymphocyte plasma membrane and that membrane exfoliation is likely to be a directed event.

Animals↗
PubMed1988

Influence of platelet activating factor and a nonmetabolizable analogue on superoxide production by bone marrow derived macrophages.

Serum-free cultured macrophages could be stimulated for lucigenin-dependent chemiluminescence by platelet activating factor (PAF) and phorbol myristate acetate (PMA). Stimulation with PMA resulted in a desensitization against PAF, whereas prestimulation with PAF had no influence on a following response caused by PMA. The PAF analogue, 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (Et-18-OCH3), did not induce chemiluminescence by itself and desensitized the cells against PAF, like substimulating concentrations of PAF. PAF and PMA responsiveness was rapidly modulated in a similar manner during adherence of the cells to polystyrene tubes. At higher concentrations, Et-18-OCH3 as well as lysophosphatidylcholines potentiated PMA-induced chemiluminescence. The PAF analogue was most effective. Although PMA-induced chemiluminescence was stimulated at least 5-fold by Et-18-OCH3, this compound increased the PMA-induced activation of protein kinase C only 1.39-fold. The priming effect of Et-18-OCH3 was not reduced in the absence of extracellular Ca2+ and after cell membrane depolarisation.

Bone Marrow Cells↗