Effect of phenylalanine- or tryptophan-loaded liposomes on the rheological properties of AA and SS erythrocytes

Phenylalanine or tryptophan was incorporated into AA and SS red blood cells by a liposomal transport system which was previously shown by Kumpati to inhibit and reverse sickling of intact SS red blood cells in vitro. In the present study, the effect of phenylalanine or tryptophan incorporation on the rheological properties was evaluated. The incorporation of phenylalanine or tryptophan into red blood cells decreased the viscosity of deoxy SS red blood cells which reached a level close to that for normal red blood cells due to the antisickling effect. These results demonstrate that this liposomal transport system which transferred phenylalanine or tryptophan into intact red cells and did not have any adverse effect on red cell metabolism or function did correct the viscosity of deoxy SS red cells by its antisickling effect. This method may have significant therapeutic implications in the treatment of sickle cell disease.     

Differential destabilization of membranes by tryptophan and phenylalanine during freezing: the roles of lipid composition and membrane fusion

The stability of cellular membranes during dehydration can be strongly influenced by the partitioning of amphiphilic solutes from the aqueous phase into the membranes. The effects of partitioning on membrane stability depend in a complex manner on the structural properties of the amphiphiles and on membrane lipid composition. Here, we have investigated the effects of the amphiphilic aromatic amino acids Trp and Phe on membrane stability during freezing. Both amino acids were cryotoxic to isolated chloroplast thylakoid membranes and to large unilamellar liposomes, but Trp had a much stronger effect than Phe. In liposomes, both amino acids induced solute leakage and membrane fusion during freezing. The presence of the chloroplast galactolipids monogalactosyldiacylglycerol or digalactosyldiacylglycerol in egg phosphatidylcholine (EPC) membranes reduced leakage from liposomes during freezing in the presence of up to 5 mM Trp, as compared to membranes composed of pure EPC. The presence of the nonbilayer-forming lipid phosphatidylethanolamine increased leakage. Membrane fusion followed a similar trend, but was dramatically reduced when the anthracycline antibiotic daunomycin was incorporated into the membranes. Daunomycin has been shown to stabilize the bilayer phase of membranes in the presence of nonbilayer lipids and was therefore expected to reduce fusion. Surprisingly, this had only a small influence on leakage. Collectively, these data indicate that Trp and Phe induce solute leakage from liposomes during freezing by a mechanism that is largely independent of fusion events.     

Differential destabilization of membranes by tryptophan and phenylalanine during freezing: the roles of lipid composition and membrane fusion

The stability of cellular membranes during dehydration can be strongly influenced by the partitioning of amphiphilic solutes from the aqueous phase into the membranes. The effects of partitioning on membrane stability depend in a complex manner on the structural properties of the amphiphiles and on membrane lipid composition. Here, we have investigated the effects of the amphiphilic aromatic amino acids Trp and Phe on membrane stability during freezing. Both amino acids were cryotoxic to isolated chloroplast thylakoid membranes and to large unilamellar liposomes, but Trp had a much stronger effect than Phe. In liposomes, both amino acids induced solute leakage and membrane fusion during freezing. The presence of the chloroplast galactolipids monogalactosyldiacylglycerol or digalactosyldiacylglycerol in egg phosphatidylcholine (EPC) membranes reduced leakage from liposomes during freezing in the presence of up to 5 mM Trp, as compared to membranes composed of pure EPC. The presence of the nonbilayer-forming lipid phosphatidylethanolamine increased leakage. Membrane fusion followed a similar trend, but was dramatically reduced when the anthracycline antibiotic daunomycin was incorporated into the membranes. Daunomycin has been shown to stabilize the bilayer phase of membranes in the presence of nonbilayer lipids and was therefore expected to reduce fusion. Surprisingly, this had only a small influence on leakage. Collectively, these data indicate that Trp and Phe induce solute leakage from liposomes during freezing by a mechanism that is largely independent of fusion events.     

Centrifugal and chromatographic analyses of tryptophan and tyrosine uptake by red blood cells and GLUT1 proteoliposomes with permeability estimates and observations on dihydrocytochalasin B

We analyzed transport into liposomes and proteoliposomes, separated the free and internalized radioactively labeled substrates by size-exclusion chromatography (SEC) and observed a net influx owing to nonfacilitated diffusion across the lipid bilayers during the separation. The permeabilities (10(-9) cm/s) of glucose transporter (GLUT1) proteoliposomes were estimated to be 4.6, 1.0, 1.4 and 2.1 for D-glucose, L-glucose, L-Tyr and L-Trp, respectively; 15, 3.3, 5.1 and 2.1 times higher than the corresponding permeabilities of liposomes. These values indicated that GLUT1 did not transport Tyr or Trp, or transported Tyr, and only Tyr, slowly. This interpretation was supported by further analyses. Dihydrocytochalasin B inhibited the transport of Tyr and, partially, Trp into human red blood cells (centrifugal analyses). It did not inhibit Tyr and Trp influx into GLUT1 proteoliposomes, but partitioned strongly into the bilayers and seemed to make them fragile. The GLUT1 inhibitor cytochalasin B and the GLUT1 substrate 2-deoxy-D-glucose did not inhibit Tyr transport into the cells. Upon immobilized biomembrane affinity chromatography, Trp decreased the cytochalasin B retardation by GLUT1 only at levels far above the physiological Trp concentration. Ethanol (commonly added to aqueous solutions for enhancing a compound's solubility) halved the retardation at 4% (v/v) concentration. Drastic modification of the SEC method is required to allow permeability measurements with nonlabeled and highly permeable substrates.     

Mutational analysis and membrane-interactions of the beta-sheet-like N-terminal domain of the pediocin-like antimicrobial peptide sakacin P

To gain insight into how the N-terminal three-stranded beta-sheet-like domain in pediocin-like antimicrobial peptides positions itself on membranes, residues in the well-conserved (Y)YGNGV-motif in the domain were substituted and the effect of the substitutions on antimicrobial activity and binding of peptides to liposomes was determined. Peptide-liposome interactions were detected by measuring tryptophan-fluorescence upon exposing liposomes to peptides in which a tryptophan residue had been introduced in the N-terminal domain. The results revealed that the N-terminal domain associates readily with anionic liposomes, but not with neutral liposomes. The electrostatic interactions between peptides and liposomes facilitated the penetration of some of the peptide residues into the liposomes. Measuring the antimicrobial activity of the mutated peptides revealed that the Tyr2Leu and Tyr3Leu mutations resulted in about a 10-fold reduction in activity, whereas the Tyr2Trp, Tyr2Phe, Tyr3Trp and Tyr3Phe mutations were tolerated fairly well, especially the mutations in position 3. The Val7Ile mutation did not have a marked detrimental effect on the activity. The Gly6Ala mutation was highly detrimental, consistent with Gly6 being in one of the turns in the beta-sheet-like N-terminal domain, whereas the Gly4Ala mutation was tolerated fairly well. All mutations involving Asn5, including the conservative mutations Asn5Gln and Asn5Asp, were very deleterious. Thus, both the polar amide group on the side chain of Asn5 and its exact position in space were crucial for the peptides to be fully active. Taken together, the results are consistent with Val7 positioning itself in the hydrophobic core of target membranes, thus forcing most of the other residues in the N-terminal domain into the membrane interface region: Tyr3 and Asn5 in the lower half with their side chains pointing downward and approaching the hydrophobic core, Tyr2, Gly4 and His8 and 12 in the upper half, Lys1 near the middle of the interface region, and the side chain of Lys11 pointing out toward the membrane surface.     

D-Glucose-recognition and phlorizin-binding sites in human sodium/D-glucose cotransporter 1 (hSGLT1): a tryptophan scanning study

In order to gain a better understanding of the structure-function relation in hSGLT1, single Trp residues were introduced into a functional hSGLT1 mutant devoid of Trps at positions that previously had been postulated to be involved in sugar recognition/translocation and/or phlorizin binding. The mutant proteins were expressed in Pichia pastoris, purified, and reconstituted into liposomes. In transport experiments the putative sugar binding site mutants W457hSGLT1 and W460hSGLT1 showed a drastic decrease in affinity toward alpha-methyl-d-glucopyranoside with Km values of 13.3 and 5.26 mM compared to 0.4 mM of the Trp-less hSGLT1. In addition, a strong decrease in the inhibitory effect of phlorizin was observed. In Trp fluorescence studies the position of the emission maxima of the mutants, their sensitivity to N-bromosuccinimide oxidation, and their interaction with water soluble quenchers demonstrate that Trp457 and Trp460 are in contact with the hydrophilic extravesicular environment. In both mutants Trp fluorescence was quenched significantly, but differently, by various glucose analogues. They also show significant protection by d-glucose and phlorizin against acrylamide, KI, or TCE quenching. W602hSGLT1 and W609hSGLT1, the putative aglucone binding site mutants, exhibit normal sugar and phlorizin affinity, and show fluorescence properties which indicate that these residues are located in a very hydrophilic environment. Phlorizin and phloretin, but not d-glucose, protect both mutants against collisional quenchers. Depth-calculations using the parallax method suggest a location of Trp457 and Trp460 at an average distance of 10.8 A and 7.4 A from the center of the bilayer, while Trp602 and Trp609 are located outside the membrane. These results suggest that in the native carrier residues Gln at position 457 and Thr at position 460 reside in a hydrophilic access pathway extending 5-7 A into the membrane to which sugars as well as the sugar moiety of inhibitory glucosides bind. Residues Phe602 and Phe609 contribute by their hydrophobic aromatic residues toward binding of the aglucone part of phlorizin. Thereby in the phlorizin-carrier complex a close vicinity between these two subdomains of the transporter is established creating a phlorizin binding pocket with the previously estimated dimensions of 10 x 17 x 7 A.     

Amino acid analogues: uptake, pool formation and incorporation of phenylalanine and two halogenated derivatives in cultured mammalian cells.

HeLa cells take up Phe and two of its ring halogenated derivatives (pFPhe and pClPhe) with rpaidity, concentrating them against the external medium both at 4 and 37 degrees C. The majority of amino acid (greater than 90%) is accumulated without energy expenditures at 4 degrees C, and can be quickly discharged by normal cell washing procedures in saline. At 37 degrees C the freely-diffusible (FDP) pool is accompanied by another which develops more slowly and cannot diffuse out freely during washings with saline but is extractable with trichloracetic acid (the slowly-diffusible pool, SDP, or more conventionally, the acid-soluble pool). Both of the analogues produced larger pools of the latter type than Phe itself from external concentrations ranging from 10(-5) to 10(-3) M. The incorporation of pFPhe into proteins over these same concentrations ranged from 30 to 90--95% of Phe incorporation, whereas pClPhe showed negligible incorporation. From these and similar analyses it can be concluded that amino acid pools form largely independently of protein synthesis, but bear a close relationship with the external amino acid concentration. The fraction of total uptake into cellular pools entering the SDP was relatively constant over a wide range of external concentrations. pFPhe incorporation into cellular proteins produced the same labelling distribution of Phe. It appears to ener all proteins, the vast majority of which have similar half-lives and turnover rates to Phe proteins. In competition, little or no interference was experienced between the analogue and Phe in uptake and pool formation until excessive amounts of one or the other were present (50--100x). By contrast, incorporation of pFPhe into protein was markedly reduced by the presence of Phe. However, the development of normal or large pools of pFPhe or Phe in cells prior to 3H-Phe incorporation did not affect the linear incorporation pattern of the radioisotope into protein. The relationship of pools to protein synthesis is discussed, and it is concluded that, although the SDP could contain potential precursor molecules for protein synthesis, it does not usually act as the direct supplier of amino acid for protein synthesis. Alternative explanations for precursor supply are discussed.     

Substitution of Trp1242 of TM17 alters substrate specificity of human multidrug resistance protein 3

Multidrug resistance protein 3 (MRP3) is an ATP-dependent transporter of 17beta-estradiol 17beta(d-glucuronide) (E(2)17betaG), leukotriene C(4) (LTC(4)), methotrexate, and the bile salts taurocholate and glycocholate. In the present study, the role of a highly conserved Trp residue at position 1242 on MRP3 transport function was examined by expressing wild-type MRP3 and Ala-, Cys-, Phe-, Tyr-, and Pro-substituted mutants in human embryonic kidney 293T cells. Four MRP3-Trp(1242) mutants showed significantly increased E(2)17betaG uptake, whereas transport by the Pro mutant was undetectable. Similarly, the Pro mutant did not transport LTC(4). By comparison, LTC(4) transport by the Ala, Cys, Phe, and Tyr mutants was reduced by approximately 35%. The Ala, Cys, Phe, and Tyr mutants all showed greatly reduced methotrexate and leucovorin transport, except the Tyr mutant, which transported leucovorin at levels comparable with wild-type MRP3. In contrast, the MRP3-Trp(1242) substitutions did not significantly affect taurocholate transport or taurocholate and glycocholate inhibition of E(2)17betaG uptake. Thus Trp(1242) substitutions markedly alter the substrate specificity of MRP3 but leave bile salt binding and transport intact.     

Liposomes as a means to introduce fragment A of diphtheria toxin into cells

The incorporation of fragment A of diphtheria toxin into liposomes is described. The intracellular delivery of the entrapped toxin, as evidenced by the inhibition of protein synthesis by a human lymphoblastoid cell line could be demonstrated with liposomes that contained phosphatidylethanolamine or phosphatidylserine in addition to phosphatidylcholine and cholesterol. Free fragment A, either alone or added to empty liposomes of any composition, did not affect protein synthesis, even when present in considerably higher concentrations than the liposome-entrapped form.     

Interaction between the critical aromatic amino acid residues Tyr(352) and Phe(504) in the yeast Gal2 transporter

Three critical aromatic sites have been identified in the yeast galactose transporter Gal2: Tyr(352) at the extracellular boundary of putative transmembrane segment (TM) 7, Tyr(446) in the middle of TM10 and Phe(504) in the middle of TM12. The relationship between these sites was investigated by random mutagenesis of each combination of two of the three residues. Galactose transport-positive clones selected by plate assays encoded Tyr(446) and specific combinations of aromatic residues at sites 352 and 504. Double-site mutants containing aromatic residues at these latter two positions showed either essentially full galactose transport activity (Phe(352)Trp(504) and Trp(352)Trp(504)) or no significant activity (Phe(352)Tyr(504) and Trp(352)Tyr(504)), whereas single-site mutants showed markedly reduced activity. These results are indicative of a specific interaction between sites 352 and 504 of Gal2.     

Residues Leu261, Trp264, and Phe265 account for apolipoprotein E-induced dyslipidemia and affect the formation of apolipoprotein E-containing high-density lipoprotein

Overexpression of apolipoprotein E (apoE) induces hypertriglyceridemia in apoE-deficient mice, which is abrogated by deletion of the carboxy-terminal segment of residues 260-299. We have used adenovirus-mediated gene transfer in apoE-/- and apoA-I-/- mice to test the effect of three sets of apoE mutations within the region of residues 261-265 on the induction of hypertriglyceridemia, the esterification of cholesterol of very low-density lipoprotein (VLDL) and high-density lipoprotein (HDL), and the formation of spherical or discoidal apoE-containing HDL. A single-amino acid substitution (apoE4[Phe265Ala]) induced hypertriglyceridemia in apoE-/- or apoA-I-/- mice, promoted the accumulation of free cholesterol in the very low-density lipoprotein (VLDL) and HDL region, and decreased HDL cholesterol levels. A double substitution (apoE4[Leu261Ala/Trp264Ala]) induced milder hypertriglyceridemia and increased HDL cholesterol levels. A triple substitution (apoE4[Leu261Ala/Trp264Ala/Phe265Ala] or apoE2[Leu261Ala/Trp264Ala/Phe265Ala]) did not induce hypertriglyceridemia and increased greatly the HDL cholesterol levels. Electron microscopy (EM) analysis of the HDL fractions showed that apoE4[Leu261Ala/Trp264Ala/Phe265Ala] and apoE2[Leu261Ala/Trp264Ala/Phe265Ala] contained spherical HDL, apoE4[Leu261Ala/Trp264Ala] contained mostly spherical and few discoidal HDL particles, and apoE4[Phe265Ala] contained discoidal HDL. We conclude that residues Leu261, Trp264, and Phe265 play an important role in apoE-induced hypertriglyceridemia, the accumulation of free cholesterol in VLDL and HDL, and the formation of discoidal HDL. Substitution of these residues with Ala improves the apoE functions by preventing hypertriglyceridemia and promoting formation of spherical apoE-containing HDL.     

Redesign of cytochrome c peroxidase into a manganese peroxidase: role of tryptophans in peroxidase activity.

Trp191Phe and Trp51Phe mutations have been introduced into an engineered cytochrome c peroxidase (CcP) containing a Mn(II)-binding site reported previously (MnCcP; see Yeung, B. K.-S., et al. (1997) Chem. Biol. 5, 215-221). The goal of the present study is to elucidate the role of tryptophans in peroxidase activity since CcP contains both Trp51 and Trp191 while manganese peroxidase (MnP) contains phenylalanine residues at the corresponding positions. The presence of Trp191 in CcP allows formation of a unique high-valent intermediate containing a ferryl oxo and tryptophan radical called compound I'. The absence of a tryptophan residue at this position in MnP is the main reason for the formation of an intermediate called compound I which contains a ferryl oxo and porphyrin pi-cation radical. In this study, we showed that introduction of the Trp191Phe mutation to MnCcP did not improve MnP activity (specific activity: MnCcP, 0.750 micromol min-1 mg-1; MnCcP(W191F), 0.560 micromol min-1 mg-1. k(cat)/K(m): MnCcP, 0.0517 s-1 mM-1; MnCcP(W191F), 0.0568 s-1 mM-1) despite the fact that introduction of the same mutation to WTCcP caused the formation of a transient compound I (decay rate, 60 s-1). However, introducing both the Trp191Phe and Trp51Phe mutations not only resulted in a longer lived compound I in WTCcP (decay rate, 18 s-1), but also significantly improved MnP activity in MnCcP (MnCcP(W51F, W191F): specific activity, 8.0 micromol min-1 mg-1; k(cat)/K(m), 0. 599 s-1 mM-1). The increase in activity can be attributed to the Trp51Phe mutation since MnCcP(W51F) showed significantly increased MnP activity relative to MnCcP (specific activity, 3.2 micromol min-1 mg-1; k(cat)/K(m), 0.325 s-1 mM-1). As with MnP, the activity of MnCcP(W51F, W191F) was found to increase with decreasing pH. Our results demonstrate that, while the Trp191Phe and Trp51Phe mutations both play important roles in stabilizing compound I, only the Trp51Phe mutation contributes significantly to increasing the MnP activity because this mutation increases the reactivity of compound II, whose oxidation of Mn(II) is the rate-determining step in the reaction mechanism.     

Sodium-independent low-affinity D-glucose transport by human sodium/D-glucose cotransporter 1: critical role of tryptophan 561

Although there is no evidence of significant Na-independent glucose flux in tissues naturally expressing SGLT1, previous kinetic and biophysical studies suggest that sodium/d-glucose cotransporter 1 (hSGLT1) can facilitate sodium-independent d-glucose transport and may contain more than one sugar binding site. In this work, we analyze the kinetic properties and conformational states of isolated hSGLT1 reconstituted in liposomes by transport and fluorescence studies in the absence of sodium. In the transport studies with hSGLT1, significant sodium-independent phlorizin inhibitable alpha-methyl d-glucopyranoside (alpha-MDG) uptake was observed which amounted to approximately 20% of the uptake observed in the presence of a sodium gradient. The apparent affinity constant for alpha-MDG was thereby 3.4 +/- 0.5 mM, a value approximately 10-fold higher than that in the presence of sodium. In the absence of sodium, various sugars significantly decreased the intrinsic Trp fluorescence of hSGLT1 in proteoliposomes exhibiting the following sequence of affinities: alpha-MDG > d-glucose approximately d-galactose > 6-deoxy-d-glucose > 2-deoxy-d-glucose > d-allose. Furthermore, significant protection effects of d-glucose or phlorizin against potassium iodide, acrylamide, or trichloroethanol quenching were observed. To locate the Trps involved in this reaction, we generated mutants in which all Trps were sequentially substituted with Phe. None of the replacements significantly affected sodium-dependent uptake. Uptake in the absence of sodium and typical fluorescence changes depended, however, on the presence of Trp at position 561. This Trp residue is conserved in all known SGLT1 forms (except Vibrio parahaemolyticus SGLT) and all SGLT isoforms in humans (except hSGLT3). If all these data are taken into consideration, it seems that Trp-561 in hSGLT1 forms part of a low-affinity sodium-independent binding and/or translocation site for d-glucose. The rate of sodium-independent translocation via hSGLT1 seems, however, to be tightly regulated in the intact cell by yet unknown factors.     

Transport of exogenous fluorescent phosphatidylserine analogue to the Golgi apparatus in cultured fibroblasts

We have examined intracellular transport and metabolism of the fluorescent analogue of phosphatidylserine, 1-palmitoyl-2-(N-[12[(7-nitrobenz-2-oxa-1,3-diazole-4-yl)amino] dodecanoyl])-phosphatidylserine ([palmitoyl-C12-NBD]-PS) in cultured fibroblasts. When monolayer cultures were incubated with liposomes containing (palmitoyl-C12-NBD)-PS at 37 degrees C, fluorescent PS was transported to the Golgi apparatus. NBD-containing analogues of phosphatidylcholine, phosphatidylethanolamine (PE), or phosphatidic acid did not accumulate in the Golgi apparatus under the same experimental conditions. We suggest that the transport is not due to endocytosis, but is the result of incorporation and trans-bilayer movement of the (palmitoyl-C12-NBD)-PS at the plasma membrane followed by translocation of the lipid from plasma membrane to the Golgi apparatus via nonvesicular mechanisms. Uptake of fluorescent PS was inhibited by depletion of cellular ATP and was blocked by structural analogues of the lipid or by pretreatment of cells with glutaraldehyde or N-ethylmaleimide. After incorporation into the cell, fluorescent PS was metabolized to fluorescent PE. The intracellular distribution of fluorescence changed during the conversion. In addition to the Golgi apparatus, mitochondria also became labeled.     

Incorporation of human liver and placental alkaline phosphatases into liposomes and membranes is via phosphatidylinositol

As assessed by incorporation into liposomes and by adsorption to octyl-Sepharose, the integrity of the membrane anchor for the purified tetrameric forms of alkaline phosphatase from human liver and placenta was intact. Any treatment that resulted in a dimeric enzyme precluded incorporation and adsorption. An intact anchor also allowed incorporation into red cell ghosts. The addition of hydrophobic proteins inhibited incorporation into liposomes to varying degrees. Alkaline phosphatase was 100% releasable from liposomes and red cell ghosts by a phospholipase C specific for phosphatidylinositol. There was no appreciable difference in the rates of release of placental and liver alkaline phosphatases, although both were approximately 250 x slower in liposomes and 100 x slower in red cell ghosts than the enzyme's release from a suspension of cultured osteosarcoma cells. Both enzymes were released by phosphatidylinositol phospholipase C as dimers and would not reincorporate or adsorb to octyl-Sepharose. However, the enzyme incorporated, resolubilized by Triton X-100, and cleansed of the detergent by butanol treatment was tetrameric by gradient gel electrophoresis, was hydrophobic, and could reincorporate into fresh liposomes. A monoclonal antibody to liver alkaline phosphatase inhibited the enzyme's incorporation into liposomes, and abolished its release from liposomes and its conversion to dimers by phosphatidylinositol phospholipase C.     

Red blood cells under flow show maximal ATP release for specific hematocrit

ATP release by red blood cells (RBCs) under shear stress (SS) plays a pivotal role in endothelial biochemical signaling cascades. The aim of this study is to investigate through numerical simulation how RBC spatiotemporal organization depends on flow and geometrical conditions to generate ATP patterns. Numerical simulations were conducted in a straight channel by considering both plasma and explicit presence of RBCs, their shape deformation and cell-cell interaction, and ATP release by RBCs. Two ATP release pathways through cell membrane are taken into account: pannexin 1 channel, sensitive to SS, and cystic fibrosis transmembrane conductance regulator, which responds to cell deformation. Several flow and hematocrit conditions are explored. The problem is solved by the lattice Boltzmann method. Application of SS to the RBC suspension triggers a nontrivial spatial RBC organization and ATP patterns. ATP localizes preferentially in the vicinity of the cell-free layer close to channel wall. Conditions for maximal ATP release per cell are identified, which depend on vessel size and hematocrit Ht. Increasing further Ht beyond optimum enhances the total ATP release but should degrade oxygen transport capacity, a compromise between an efficient ATP release and minimal blood dissipation. Moreover, ATP is boosted in capillaries, suggesting a vasomotor activity coordination throughout the resistance network.     

Inhibition of cell proliferation by putative metabolites and non-degradable analogs of methotrexate-gamma-dimyristoylphosphatidylethanolamine

Previous investigations have shown that untargeted liposomes, in which methotrexate is anchored to the lipid bilayers as methotrexate-gamma-dimyristoylphosphatidylethanolamine (methotrexate-gamma-DMPE), can inhibit in vitro cell proliferation. To test the possibility that this inhibition may involve extracellular metabolism of methotrexate-gamma-DMPE, we have degraded it chemically (dilute alkali) or enzymatically (phospholipase A2, phospholipase C, phospholipase C plus phosphatase), and assayed the products using human lymphoblastoid T cells or a subline that has a defective methotrexate transport system. Neither methotrexate-gamma-(1-myristoyl)-glycerophosphorylethanolamine, methotrexate-gamma-glycerophosphorylethanolamine, methotrexate-gamma-phosphorylethanolamine, nor methotrexate-gamma-ethanolamine resemble methotrexate-gamma-DMPE sensitized liposomes or the free derivative in their ability to block tritiated deoxyuridine incorporation into DNA. When added extracellularly, these putative metabolites manifest a higher ID50 concentration and/or, unlike the liposomes or unincorporated methotrexate-gamma-DMPE, utilize the methotrexate transport system to enter cells. Additionally, we have synthesized methotrexate-gamma-dihexadecylphosphatidylethanolamine and methotrexate-gamma-hexadecylphosphorylethanolamine, analogs of methotrexate-gamma-DMPE that cannot be hydrolyzed by phospholipases A2, C and D; liposomes prepared with these derivatives are markedly less potent cytotoxic agents than methotrexate-gamma-DMPE sensitized liposomes. All together, these results are consistent with the conclusion that methotrexate-gamma-DMPE must undergo intracellular metabolism to exert optimal inhibition; they also bear on possible mechanisms by which methotrexate-gamma-DMPE may enter cells.     

Aromatic stacking in the sugar binding site of the lactose permease

Major determinants for substrate recognition by the lactose permease of Escherichia coli are at the interface between helices IV (Glu126, Ala122), V (Arg144, Cys148), and VIII (Glu269). We demonstrate here that Trp151, one turn of helix V removed from Cys148, also plays an important role in substrate binding probably by aromatic stacking with the galactopyranosyl ring. Mutants with Phe or Tyr in place of Trp151 catalyze active lactose transport with time courses nearly the same as wild type. In addition, apparent K(m) values for lactose transport in the Phe or Tyr mutants are only 6- or 3-fold higher than wild type, respectively, with a comparable V(max). Surprisingly, however, binding of high-affinity galactoside analogues is severely compromised in the mutants; the affinity of mutant Trp151-->Phe or Trp151-->Tyr is diminished by factors of at least 50 or 20, respectively. The results demonstrate that Trp151 is an important component of the binding site, probably orienting the galactopyranosyl ring so that important H-bond interactions with side chains in helices IV, V, and VIII can be realized. The results are discussed in the context of a current model for the binding site.     

Alkaline hemolysis fragility is dependent on cell shape: results from a morphology tracker.

BACKGROUND: The morphometric analysis of red blood cells (RBCs) is an important area of study and has been performed previously for fixed samples. We present a novel method for the analysis of morphologic changes of live erythrocytes as a function of time. We use this method to extract information on alkaline hemolysis fragility. Many other toxins lyse cells by membrane poration, which has been studied by averaging over cell populations. However, no quantitative data are available for changes in the morphology of individual cells during membrane poration-driven hemolysis or for the relation between cell shape and fragility. METHODS: Hydroxide, a porating agent, was generated in a microfluidic enclosure containing RBCs in suspension. Automatic cell recognition, tracking, and morphometric measurements were done by using a custom image analysis program. Cell area and circular shape factor (CSF) were measured over time for individual cells. Implementations were developed in MATLAB and on Kestrel, a parallel computer that affords higher speed that approaches real-time processing. RESULTS: The average CSF went through a first period of fast increase, corresponding to the conversion of discocytes to spherocytes under internal osmotic pressure, followed by another period of slow increase until the fast lysis event. For individual cells, the initial CSF was shown to be inversely correlated to cell lifetime (linear regression factor R=0.44), with discocytes surviving longer than spherocytes. The inflated cell surface area to volume ratio was also inversely correlated to lifetime (R=0.43) but not correlated to the CSF. Lifetime correlated best to the ratio of cell inflation volume (Vfinal-Vinitial) to surface area (R=0.65). CONCLUSIONS: RBCs inflate at a rate proportional to their surface area, in agreement with a constant flux model, and lyse after attaining a spherical morphology. Spherical RBCs display increased alkaline hemolysis fragility (shorter lifetimes), providing an explanation for the increased osmotic fragility of RBCs from patients who have spherocytosis.     

磷脂酰丝氨酸外翻和磷脂氧化在凋亡细胞被吞噬细胞清除过程中的协作效应

为探讨磷脂酰丝氨酸(phosphatidylserine,PS)外翻和磷脂氧化在凋亡细胞被吞噬细胞清除中的作用,用脂质体整合的方法将不同的磷脂整合到红细胞上或用N-乙酰马来酰胺(N-ethylmaleimide,NEM)预处理红细胞然后整合磷脂,制备含不同凋亡信号的红细胞模型,测定巨噬细胞对整合不同磷脂信号红细胞的结合率和吞噬率。结果表明,单独整合PS或用NEM处理造成PS外翻,可显著性提高巨噬细胞对红细胞的结合率,但对吞噬率没有影响;同时整合PS和氧化磷脂(氧化PS或氧化磷脂酰胆碱(phosphatidylcholine,PC)),或用NEM处理造成PS外翻后再整合氧化PS或氧化PC,不仅可显著提高巨噬细胞对红细胞的结合率,而且可显著性提高吞噬率。这些结果提示PS外翻可能参与了巨噬细胞对凋亡细胞的结合,而磷脂氧化可能启动了巨噬细胞对凋亡细胞的吞噬,二者协作才可能完成巨噬细胞对凋亡细胞的清除。