Ultra-deformable free fatty acid based nano-carriers for topical delivery of Luteolin: A potential paradigm for management of Methicillin-Resistant Staphylococcus aureus skin infections.

The incidences of antimicrobial resistance in particular, Methicillin-Resistant Staphylococcus aureus (MRSA) have increased during the last two decades. However, conventional dosage forms are unable to evade the barrier effect of the stratum corneum to permit deep penetration of the skin to resolve deep skin infections. There is, therefore, an urgent need for an advanced drug delivery system. Thus the study reported herein was aimed to fabricate a novasome-loaded luteolin (LUT) to improve its topical delivery and to enhance its antibacterial activity. The system was investigated for the impact of the type of surfactant, stearic acid concentration (g %), cholesterol amount (mg) and Brij 52 amount (mg) on the percent entrapment efficiency, particle size, poly-dispersity index and zeta potential. Statistical optimization of these factors was conducted using the Design-Expert® software. The optimum formulation was further in-vitro characterized by release study, differential scanning calorimetry, transmission electron microscope, x-ray diffraction and antibacterial activity. Formulation F2 composed of Span 60, 0.4 g % of stearic acid, 100 mg cholesterol and 30 mg Brij 52 was selected as the optimum formula based on the highest desirability value (0.634). F2 demonstrated enhanced antimicrobial activity with lower minimum inhibitory concentrations against a panel of MRSA clinical isolates when compared to LUT dispersion. Furthermore, the F2 formula exhibited higher anti-virulence activity by effectively inhibiting biofilm formation and suppressing α-hemolysin activity in MRSA isolates. It also demonstrated improved biosafety based on cytotoxicity assessment on human skin fibroblasts (HSF). Finally, when assessed in an in vivo skin infection mouse model, the F2 formula and commercially available fusidic acid preparation significantly reduced the microbial load of infected skin lesions compared to both the negative control and LUT dispersion-treated groups. Based on the aforementioned results, the validity of novasomes as a nano-carrier to boost in vitro and in vivo anti-MRSA activity of LUT could be affirmed.

Eradication of meticillin-resistant Staphylococcus aureus from human skin by the novel LL-37-derived peptide P10 in four pharmaceutical ointments.

Skin bacterial colonization/infection is a frequent cause of morbidity in patients with chronic wounds and allergic/inflammatory skin diseases. This study aimed to develop a novel approach to eradicate meticillin-resistant Staphylococcus aureus (MRSA) from human skin. To achieve this, the stability and antibacterial activity of the novel LL-37-derived peptide P10 in four ointments was compared. Results indicate that P10 is chemically stable and antibacterial in hypromellose gel and Softisan-containing cream, but not in Cetomacrogol cream (with or without Vaseline), at 4 °C for 16 months. Reduction in MRSA counts on Leiden human epidermal models (LEMs) by P10 in hypromellose gel was greater than that of the peptide in Cetomacrogol cream or phosphate buffered saline. P10 did not show adverse effects on LEMs irrespective of the ointment used, while Cetomacrogol with Vaseline and Softisan cream, but not hypromellose gel or Cetomacrogol cream, destroyed MRSA-colonized LEMs. Taking all this into account, P10 in hypromellose gel dose-dependently reduced MRSA colonizing the stratum corneum of the epidermis as well as biofilms of this bacterial strain on LEMs. Moreover, P10 dose-dependently reduced MRSA counts on ex-vivo human skin, with P10 in hypromellose gel being more effective than P10 in Cetomacrogol and Softisan creams. P10 in hypromellose gel is a strong candidate for eradication of MRSA from human skin.

Scrolls: novel microparticulate systems for enhanced delivery to/across the skin.

We describe the scroll system as a new microparticulate structured delivery system for enhanced delivery to/across the skin. The basic components of the scroll system are non-ionic surface active of the type of alkyl polyglycol ethers and a glycol. The unique structures are preserved with addition of various ingredients such as polymers, vegetable oils, pharmaceuticals, and permeation enhancers but are dismissed when amphiphile is withdrawn. The microparticles have a unique scroll structure with multiple "wrapping." Besides enabling superior permeation of drugs into/across the skin, the drugs delivered by scroll systems were more effective in vitro and in vivo compared to controls. Model drugs presented high entrapment capacity in scroll systems. The systems are stable over time and are safe for skin application. In order to form, they require a small number of ingredients, simple preparation method, and are environment friendly. The scroll systems may be new potential tools in the dermal/transdermal pharmaceutical and cosmetic industry.

High Efficacy but Low Potency of δ-Opioid Receptor-G Protein Coupling in Brij-58-Treated, Low-Density Plasma Membrane Fragments.

PRINCIPAL FINDINGS: HEK293 cells stably expressing PTX-insensitive δ-opioid receptor-Gi1α (C351I) fusion protein were homogenized, treated with low concentrations of non-ionic detergent Brij-58 at 0°C and fractionated by flotation in sucrose density gradient. In optimum range of detergent concentrations (0.025-0.05% w/v), Brij-58-treated, low-density membranes exhibited 2-3-fold higher efficacy of DADLE-stimulated, high-affinity [32P]GTPase and [35S]GTPγS binding than membranes of the same density prepared in the absence of detergent. The potency of agonist DADLE response was significantly decreased. At high detergent concentrations (>0.1%), the functional coupling between δ-opioid receptors and G proteins was completely diminished. The same detergent effects were measured in plasma membranes isolated from PTX-treated cells. Therefore, the effect of Brij-58 on δ-opioid receptor-G protein coupling was not restricted to the covalently bound Gi1α within δ-opioid receptor-Gi1α fusion protein, but it was also valid for PTX-sensitive G proteins of Gi/Go family endogenously expressed in HEK293 cells. Characterization of the direct effect of Brij-58 on the hydrophobic interior of isolated plasma membranes by steady-state anisotropy of diphenylhexatriene (DPH) fluorescence indicated a marked increase of membrane fluidity. The time-resolved analysis of decay of DPH fluorescence by the "wobble in cone" model of DPH motion in the membrane indicated that the exposure to the increasing concentrations of Brij-58 led to a decreased order and higher motional freedom of the dye. SUMMARY: Limited perturbation of plasma membrane integrity by low concentrations of non-ionic detergent Brij-58 results in alteration of δ-OR-G protein coupling. Maximum G protein-response to agonist stimulation (efficacy) is increased; affinity of response (potency) is decreased. The total degradation plasma membrane structure at high detergent concentrations results in diminution of functional coupling between δ-opioid receptors and G proteins.

ATP serves as an endogenous inhibitor of UDP-glucuronosyltransferase (UGT): a new insight into the latency of UGT.

We have suggested that adenine-related compounds are allosteric inhibitors of UGT in rat liver microsomes (RLM) treated with detergent. To clarify whether the same occurs with a pore-forming peptide, alamethicin, the effects of adenine-related compounds on 4-metylumbelliferone (4-MU) glucuronidation were examined using RLM and human liver microsomes (HLM). ATP inhibited 4-MU glucuronidation when polyoxyethylene cetyl alcohol ether (Brij-58)-treated RLM were used (IC(50) = approximately 70 µM). However, alamethicin-treated RLM exhibited a lower susceptibility (IC(50) = approximately 460 µM) than Brij-58-treated RLM. A similar phenomenon was observed when pooled HLM were used. Then, the endogenous ATP content of RLM was determined in the presence and absence of alamethicin or detergent, and although no ATP remained in the microsomal pellets after Brij-58 treatment, more than half of the microsomal ATP remained even after treatment with alamethicin. Furthermore, the V(max) in the absence of an adenine-related compound was approximately three times higher in Brij-58-treated than in alamethicin-treated RLM. The difference in the inhibitory potency observed was due to the difference in remaining endogenous ATP and the accessibility of exogenous ATP to the luminal side of the endoplasmic reticulum (ER), where the active site of UDP-glucuronosyltransferase (UGT) is located. Gefitinib (Iressa), a protein tyrosine kinase inhibitor, markedly inhibited human UGT1A9 activity. It is interesting to note that AMP antagonized Gefitinib-provoked inhibition of UGT1A9, and ATP exhibited an additive inhibitory effect at a lower concentration. Therefore, Gefitinib inhibits UGT1A9 at the common ATP-binding site shared with ATP and AMP. Releasing adenine nucleotide from the ER is suggested to be one of the mechanisms that explain the "latency" of UGT.

A bilateral comparison study of pimecrolimus cream 1% and a topical medical device cream in the treatment of patients with atopic dermatitis.

Corticosteroids are the mainstay of therapy for atopic dermatitis, but long-term use is associated with adverse effects. We sought to evaluate the clinical efficacy of two steroid-sparing creams for atopic dermatitis. Twenty patients were enrolled in an investigator-blinded, bilateral comparison study. Patients applied pimecrolimus cream twice daily to a target lesion on one side of the body and also applied a topical medical device cream three times daily on a symmetrical target lesion on the opposite side of the body for four weeks. Clinical assessments including Physician Global Assessment (PGA), Target Lesion Symptom Score (TLSS), subject self-assessment and digital photography were performed at the baseline, 2 week, and 4 week visits. Seventy-five percent of patients (pimecrolimus, 15 of 20; topical medical device, 15 of 20) were rated "clear" (0) or "almost clear" (1) by PGA for both medications after four weeks. Percent improvement of the PGA from randomization for pimecrolimus cream and the topical medical device cream were 72.50 and 71.67 respectively (P=0.9283). PGA scores decreased significantly from baseline for both treatments (P=0.004). Overall, there was no statistically significant difference between treatment groups for PGA scores throughout the study (P=0.8236). No cutaneous side effects were noted. Our study was limited by a small sample size and lack of double-blinding; however, both treatments were found to be safe and effective in treating atopic dermatitis over four weeks. Significant improvements were noted for all efficacy variables. In conclusion, a lipid-rich, non-steroidal, topical medical device cream was as effective in improving atopic dermatitis as pimecrolimus cream.

The effect of detergents on trimeric G-protein activity in isolated plasma membranes from rat brain cortex: correlation with studies of DPH and Laurdan fluorescence.

The effect of non-ionic detergents on baclofen (GABAB-R agonist)-stimulated G-protein activity was measured as a [(35)S]GTPgammaS binding assay in the plasma membranes (PM) isolated from the brain tissue. The effect was clearly biphasic--a decrease in the activity was followed by an activation maximum and finally, at high concentrations, drastic inhibition of the G-protein activity was noticed. Contrarily, specific radioligand binding to GABAB-receptor was inhibited in the whole range of detergent concentrations step by step, i.e. it was strictly monophasic. The magnitude of both detergent effects was decreased in the same order of potency: Brij58>Triton X-100>Digitonin. The identical order was found when comparing detergents ability to alter fluorescence anisotropy of the membrane probe 1,6-diphenyl-1,3,5-hexatriene (rDPH) incorporated into the hydrophobic PM interior. Decrease of rDPH, in the order of Brij58>Triton X-100>Digitonin, was reflected as decrease of the S-order parameter and rotation correlation time phi paralleled by an increase of diffusion wobbling constant Dw (analysis by time-resolved fluorescence according to "wobble-in-cone" model). The influence of the detergents on the membrane organization at the polar headgroup region was characterized by Laurdan generalized polarization (GP). As before, the effect of detergents on GP parameters proceeded in the order: Brij58>Triton X-100>Digitonin.

Fatty acyl-CoA as an endogenous activator of UDP-glucuronosyltransferases.

The acyl-CoA-dependent modulation of hepatic microsomal UDP-glucuronosyltransferase (UGT) function in rats was studied. Oleoyl- and palmitoyl-CoAs inhibited UGT activity toward 4-methylumbelliferone in the presence of Brij 58. However, acyl-CoAs enhanced UGT activity in untreated microsomes. A maximum activation of about 8-fold over the control was observed at 15 microM oleoyl-CoA, whereas 50 microM or more oleoyl-CoA had an inhibitory effect on UGT function. Medium- and long-chain acyl-CoAs also exhibited similar effects. On the basis of resistance to tryptic digestion of UGTs, oleoyl-CoA at 15 microM has no ability to change the permeability of the endoplasmic reticulum (ER) membrane, although perturbation of the membrane occurred with 50 microM oleoyl-CoA. N-Ethylmaleimide and 5,5'-dithiobis(2-nitrobenzoic acid) abolished the oleoyl-CoA (15 microM)-dependent activation of microsomal UGT. These results suggest that: (1) acyl-CoAs play a role as an endogenous activator of UGTs, and (2) a sulfhydryl group is required for the activation of UGT by physiological concentrations of acyl-CoAs.

Acute cholesterol depletion impairs functional expression of tissue factor in fibroblasts: modulation of tissue factor activity by membrane cholesterol.

Cholesterol, in addition to providing rigidity to the fluid membrane, plays a critical role in receptor function, endocytosis, recycling, and signal transduction. In the present study, we examined the effect of membrane cholesterol on functional expression of tissue factor (TF), a cellular receptor for clotting factor VIIa. Depletion of cholesterol in human fibroblasts (WI-38) with methyl-beta-cyclodextrin-reduced TF activity at the cell surface. Binding studies with radiolabeled VIIa and TF monoclonal antibody (mAB) revealed that reduced TF activity in cholesterol-depleted cells stems from the impairment of VIIa interaction with TF rather than the loss of TF receptors at the cell surface. Repletion of cholesterol-depleted cells with cholesterol restored TF function. Loss of caveolar structure on cholesterol removal is not responsible for reduced TF activity. Solubilization of cellular TF in different detergents indicated that a substantial portion of TF in fibroblasts is associated with noncaveolar lipid rafts. Cholesterol depletion studies showed that the TF association with these rafts is cholesterol dependent. Overall, the data presented herein suggest that membrane cholesterol functions as a positive regulator of TF function by maintaining TF receptors, probably in noncaveolar lipid rafts, in a high-affinity state for VIIa binding.

Enhanced macrocyclizing activity of the thioesterase from tyrocidine synthetase in presence of nonionic detergent.

Macrocyclization carried out by thioesterase domains of multimodular nonribosomal peptide synthetases (NRPSs) is a key step in the biosynthesis of many biologically active peptides. The thioesterase excised from tyrocidine synthetase is a versatile macrocyclization catalyst and a useful tool for chemoenzymatic synthesis of diverse cyclic peptides. However, its utility is limited by its short lifetime of catalytic activity as well as significant flux of the acyl-enzyme intermediate to hydrolysis. The addition of Brij 58, a nonionic detergent, above the critical micelle concentration, has dramatic effects on enzyme activity: catalytic activity is extended to >60 min and the rate of cyclization (but not hydrolysis) increases 6-fold, resulting in a net 150- to 300-fold increase in cyclic product yields. This enhanced activity allowed enzymatic macrocyclization of a solid phase library of tyrocidine decapeptides to identify acceptable substitutions at the Orn9 position which had previously been inaccessible for diversification.

Partitioning of the serotonin transporter into lipid microdomains modulates transport of serotonin.

The serotonin transporter (SERT) is an integral membrane protein responsible for the clearance of serotonin from the synaptic cleft following the release of the neurotransmitter. SERT plays a prominent role in the regulation of serotoninergic neurotransmission and is a molecular target for multiple antidepressants as well as substances of abuse. Here we show that SERT associates with lipid rafts in both heterologous expression systems and rat brain and that the inclusion of the transporter into lipid microdomains is critical for serotonin uptake activity. SERT is present in a subpopulation of lipid rafts, which is soluble in Triton X-100 but insoluble in other non-ionic detergents such as Brij 58. Disaggregation of lipid rafts upon depletion of cellular cholesterol results in a decrease of serotonin transport capacity (V(max)), due to the reduction of turnover number of serotonin transport. Our data suggest that the association of SERT with lipid rafts may represent a mechanism for regulating the transporter activity and, consequently, serotoninergic signaling in the central nervous system, through the modulation of the cholesterol content in the cell membrane. Furthermore, SERT-containing rafts are detected in both intracellular and cell surface fractions, suggesting that raft association may be important for trafficking and targeting of SERT.

Effect of membrane perturbants on the activity and phase distribution of inositol phosphorylceramide synthase; development of a novel assay.

The effect of 26 different membrane-perturbing agents on the activity and phase distribution of inositol phosphorylceramide synthase (IPC synthase) activity in crude Candida albicans membranes was investigated. The nonionic detergents Triton X-100, Nonidet P-40, Brij, Tween, and octylglucoside all inactivated the enzyme. However, at moderate concentrations, the activity of the Triton X-100- and octylglucoside-solubilized material could be partially restored by inclusion of 5 mM phosphatidylinositol (PI) in the solubilization buffer. The apparent molecular mass of IPC synthase activity solubilized in 2% Triton X-100 was between 1.5 x 10(6) and 20 x 10(6) Da, while under identical conditions, octylglucoside-solubilized activity remained associated with large presumably membrane-like structures. Increased detergent concentrations produced more drastic losses of enzymatic activity. The zwitterionic detergents Empigen BB, N-dodecyl-N,N-(dimethylammonio)butyrate (DDMAB), Zwittergent 3-10, and amidosulfobetaine (ASB)-16 all appeared capable of solubilizing IPC synthase. However, these agents also inactivated the enzyme essentially irreversibly. Solubilization with lysophospholipids again resulted in drastic losses of enzymatic activity that were not restored by the inclusion of PI. Lysophosphatidylinositol also appeared to compete, to some extent, with the donor substrate phosphatidylinositol. The sterol-containing agent digitonin completely inactivated IPC synthase. By contrast, sterol-based detergents such as 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate (CHAPSO), and taurodeoxycholate (tDOC) had little or no effect on the enzyme activity. The IPC synthase activity in C. albicans membranes remained largely intact and sedimentable at CHAPS concentrations (4%) where >90% of the phospholipids and 60% of the total proteins were extracted from the membranes. At 2.5% CHAPS, a concentration where approximately 50% of the protein and 80% of the phospholipids are solubilized, there was no detectable loss of enzyme activity, and it was found that the detergent-treated membranes had significantly improved properties compared to crude, untreated membranes as the source of IPC synthase activity. In contrast to assays utilizing intact membranes or Triton X-100 extracts, assays using CHAPS- or tDOC-washed membranes were found to be reproducible, completely dependent on added acceptor substrate (C(6)-7-nitro-2-1,3-benzoxadiazol-4-yl (NBD)-ceramide), and >95% dependent on added donor substrate (PI). Product formation was linear with respect to both enzyme concentration and time, and transfer efficiency was improved more than 20-fold as compared to assays using crude membranes. Determination of kinetic parameters for the two IPC synthase substrates using CHAPS-washed membranes resulted in K(m) values of 3.3 and 138.0 microM for C(6)-NBD-ceramide and PI, respectively. In addition, the donor substrate, PI, was found to be inhibitory at high concentrations with an apparent K(i) of 588.2 microM.

The effect of incubation conditions on the enzyme kinetics of udp-glucuronosyltransferases.

Traditionally, the Michaelis-Menten equation has been used to determine kinetic parameters for in vitro glucuronidation assays. Recently, estradiol-3-glucuronide formation was shown to exhibit non-Michaelis-Menten kinetics consistent with autoactivation. A concern with the observation of nontraditional kinetics is that they may result as an artifact of the incubation conditions. To examine this concern, the formation of estradiol-3-glucuronide was investigated using human liver microsomes prepared by two different methods, a range of assay conditions, and activation by alamethecin, sonication, or Brij 58 (polyoxyethylene monocetyl ether). Interestingly, holding the other assay components constant, estradiol-3-glucuronide formation was up to 2.5-fold greater using microsomes prepared in phosphate buffer compared with those prepared in sucrose. Incubations activated by alamethecin consistently exhibited the highest rates of estradiol glucuronidation versus the other activators. Furthermore, estradiol-3-glucuronidation exhibited autoactivation kinetics in all of the conditions investigated (n = 1.2-1.7). Nontraditional kinetics were also observed when other UGT1A1 substrates such as ethinylestradiol, buprenorphine, and anthraflavic acid were studied with both human liver microsomes and recombinant UGT1A1. Naphthol, propofol, morphine, and androstanediol were used as probe UGT substrates selective for UGT1A6, UGT1A9, UGT2B7, and UGT2B15, respectively. Of these substrates, only androstanediol exhibited nontraditional kinetics using human liver microsomes. In conclusion, the Hill and/or Michaelis-Menten equations should be used to fit kinetic data to obtain an accurate assessment of in vitro glucuronidation.

Resistance of cell membranes to different detergents.

Partial resistance of cell membranes to solubilization with mild detergents and the analysis of isolated detergent-resistant membranes (DRMs) have been used operationally to define membrane domains. Given the multitude of detergents used for this purpose, we sought to investigate whether extraction with different detergents might reflect the same underlying principle of domain formation. We therefore compared the protein and lipid content of DRMs prepared with a variety of detergents from two cell lines. We found that the detergents differ considerably in their ability to selectively solubilize membrane proteins and to enrich sphingolipids and cholesterol over glycerophospholipids as well as saturated over unsaturated phosphatidylcholine. In addition, we observed cell type-dependent variations of the molecular characteristics of DRMs and the effectiveness of particular detergents. These results make it unlikely that different detergents reflect the same aspects of membrane organization and underscore both the structural complexity of cell membranes and the need for more sophisticated analytical tools to understand their architecture.

Direct and indirect antibody-induced TX-100 resistance of cell surface antigens.

Translocation into detergent-insoluble microdomains (rafts) represents one of the earliest events in the process of cell activation, which follows the binding of surface receptor with natural ligand or mimicking antibody. In this study, the antibody-induced TX-100 resistance of surface antigens has been studied utilizing flow cytometry on TX-100 extracted cells. TX-100 resistance was evaluated by the ratio of antigen retained on the cells after detergent extraction compared with mAb-pretreated and untreated cells. All the antigens under study except CD98 demonstrated antibody-induced TX-100 resistance if the cells were treated with monoclonal antibodies and further cross-linked with secondary antibodies prior to lysis. CD20, CD5, and sIgM molecules were capable of transferring into TX-100-insoluble state in the absence of additional cross-linking. The experiments on modification of raft and cytoskeletal components of the cell, as well as the data on co-localization of TX-100-resistant antigens with raft and cytoskeletal markers strongly indicate that antibody-induced TX-100 resistance of antigens is mainly related to the translocation of antigens into lipid microdomains.

Evaluation of nonionic and zwitterionic detergents as membrane protein solubilizers in two-dimensional electrophoresis.

The solubilizing power of various nonionic and zwitterionic detergents as membrane protein solubilizers for two-dimensional electrophoresis was investigated. Human red blood cell ghosts and Arabidopsis thaliana leaf membrane proteins were used as model systems. Efficient detergents could be found in each class, i.e. with oligooxyethylene, sugar or sulfobetaine polar heads. Among the commercially available nonionic detergents, dodecyl maltoside and decaethylene glycol mono hexadecyl ether proved most efficient. They complement the more classical sulfobetaine detergents to widen the scope of useful detergents for the solubilization of membrane proteins in proteomics.

Change in creep behavior of plexiform bone with phosphate ion treatment.

The effect of phosphate ions on the mechanical properties of plexiform bone in tension was investigated with an in-vitro model. Bone samples were treated with saline and phosphate ion solutions for three days at 25 degrees C and 37 degrees C and tested in tension. The mechanical properties of the bone samples treated with phosphate were not different than controls (saline treated). Electro kinetic measurements on plexiform bone particles treated with phosphate ions at 37 degrees C showed that phosphate ions alter electro kinetic potentials of bone particles by interacting with bone mineral as compare to saline treated particles near physiological pH. Because of the limited diffusion properties of intact plexiform bone tissue, the tension experiments indicate that, the effect of phosphate ions on the bone mineral-matrix interface is negligible after three days treatment. On the other hand, electro kinetic measurements demonstrated that in a short period of treatment time, phosphate ions diffuse through organic matrix barrier and interact with bone mineral when plexiform bone is in the particle form. As a final experiments bone samples were tested at 37 degrees C in three point bending configuration for three days in saline and phosphate buffer solution. The maximum tension stress generated in bending samples was about 75 percent of the tension yield stress of the samples. The creep experiments showed that the bending rigidity of bone samples tested in phosphate solution reduced in time hence the creep deformation increased compare to control samples tested in saline. This observation is attributed to the acceleration of phosphate ion diffusion into the bending samples due to micro cracks accumulation in bone tissue during the creep experiments which facilitated the phosphate ion interaction with bone mineral.

T cell receptor can be recruited to a subset of plasma membrane rafts, independently of cell signaling and attendantly to raft clustering.

The constitutive/inducible association of the T cell receptor (TCR) with isolated detergent-resistant, lipid raft-derived membranes has been studied in Jurkat T lymphocytes. Membranes resistant to 1% Triton X-100 contained virtually no CD3epsilon, part of the TCR complex, irrespective of cell stimulation. On the other hand, membranes resistant either to a lower Triton X-100 concentration (i.e. 0.2%) or to the less hydrophobic detergent Brij 58 (1%) contained (i) a low CD3epsilon amount (approximate 2.7% of total) in resting cells and (ii) a several times higher amount of the TCR component, after T cell stimulation with either antigen-presenting cells or with phytohemagglutinin. It appeared that CD3/TCR was constitutively associated with and recruited to a raft-derived membrane subset because (i) all three membrane preparations contained a similar amount of the raft marker tyrosine kinase Lck but no detectable amounts of the conventional membrane markers, CD45 phosphatase and transferrin receptor; (ii) a larger amount of particulate membranes were resistant to solubilization with 0.2% Triton X-100 and Brij 58 than to solubilization with 1% Triton X-100; and (iii) higher cholesterol levels were present in membranes resistant to either the lower Triton X-100 concentration or to Brij 58, as compared with those resistant to 1% Triton X-100. The recruitment of CD3 to the raft-derived membrane subset appeared (i) to occur independently of cell signaling events, such as protein-tyrosine phosphorylation and Ca(2+) mobilization/influx, and (ii) to be associated with clustering of plasma membrane rafts induced by multiple cross-linking of either TCR or the raft component, ganglioside GM(1). We suggest that during T cell stimulation a lateral reorganization of rafts into polarized larger domains can determine the recruitment of TCR into these domains, which favors a polarization of the signaling cascade.

Hexokinase translocation during neutrophil activation, chemotaxis, and phagocytosis: disruption by cytochalasin D, dexamethasone, and indomethacin.

Neutrophils expend large amounts of energy to perform demanding cell functions. To better understand energy production and flow during cell activation, immunofluorescence microscopy was employed to determine the location of the key metabolic enzyme hexokinase during various conditions. Hexokinase is translocated from the neutrophil's cytosol to its periphery in response to N-formyl-methionyl-leucyl-phenylalanine (fMLP) and other activating stimuli, but not during exposure to the formyl peptide receptor antagonist N-tert-BOC-phe-leu-phe-leu-phe (Boc-PLPLP). Translocation was observed from 10(-6) to 10(-9)M fMLP. However, fMLP did not affect the intracellular distribution of lactate dehydrogenase. Hexokinase accumulated at the lamellipodium of cells exposured to an fMLP gradient whereas it localized to the phagosome after latex bead uptake. Thus, hexokinase is differentially translocated within cells depending upon the prevailing physiological conditions. Further studies noted that cytochalasin D, dexamethasone, and indomethacin blocked hexokinase translocation. Parallel regulation of reactive oxygen metabolite (ROM) production was shown. We speculate that hexokinase translocation participates in neutrophil activation.

Sequestration of epidermal growth factor receptors in non-caveolar lipid rafts inhibits ligand binding.

Cholesterol depletion has been shown to increase mitogen-activated protein kinase activation in response to stimulation with epidermal growth factor (EGF) (Furuchi, T., and Anderson, R. G. W. (1998) J. Biol. Chem. 273, 21099-21104). However, the underlying mechanisms are unknown. We show that cholesterol depletion increases EGF binding, whereas cholesterol loading lowers EGF binding. Based on binding analyses, we demonstrate that the observed changes in EGF binding are caused by alterations in the number of EGF receptors available for ligand binding, whereas the affinity of the receptor for EGF remains unaltered. We also show by immunofluorescence that in unstimulated cells the EGF receptor is localized in non-caveolar lipid rafts containing the ganglioside GM1 and that patching of these rafts by cholera toxin B-chain causes co-patching of EGF receptors. Experiments with solubilization in different detergents at 4 degrees C show that the association of the EGF receptor with these rafts is sensitive to Triton X-100 extraction but insensitive to extraction with another non-ionic detergent, Brij 58. Furthermore, experiments with cholesterol-depleted cells show that the association is cholesterol-dependent. We propose that non-caveolar lipid rafts function as negative regulators of EGF receptor signaling by sequestering a fraction of the EGF receptors in a state inaccessible for ligand binding.