Detecting endotoxin with a flow cytometry-based magnetic aptasensor

Endotoxin, which is also known as lipopolysaccharide (LPS), is a marker for intruding gram-negative pathogens. It is essential to detect endotoxin quickly and sensitively in a complex milieu. A new flow cytometry (FCM)-based magnetic aptasensor assay that employs two endotoxin-binding aptamers and magnetic beads has been developed to detect endotoxin. The endotoxin-conjugated sandwich complex on magnetic beads was observed by scanning confocal laser microscopy. The resulting magnetic aptasensor rapidly detected (<1 min) endotoxin within a broad dynamic detection range of 10⁻⁸ to 10⁰ mg/ml in the presence of bovine serum albumin (BSA), RNA, sucrose, and glucose, which are most likely to coexist with endotoxin in the majority of biological liquids. Only 2 μl of magnetic aptasensor was required to quantify the endotoxin solution. Furthermore, the magnetic aptasensor could be regenerated seven times and still presented an outstanding response to the endotoxin solution. Therefore, the magnetic aptasensor exhibited high sensitivity, selectivity, and reproducibility, thereby serving as a powerful tool for the quality control and high-throughput detection of endotoxin in the food and pharmaceutical industries.     

Sensitive detection of C-reactive protein in serum by immunoprecipitation–microchip capillary gel electrophoresis

Sepsis represents a significant cause of mortality in intensive care units. Early diagnosis of sepsis is essential to increase the survival rate of patients. Among others, C-reactive protein (CRP) is commonly used as a sepsis marker. In this work we introduce immune precipitation combined with microchip capillary gel electrophoresis (IP–MCGE) for the detection and quantification of CRP in serum samples. First high-abundance proteins (HSA, IgG) are removed from serum samples using affinity spin cartridges, and then the remaining proteins are labeled with a fluorescence dye and incubated with an anti-CRP antibody, and the antigen/antibody complex is precipitated with protein G-coated magnetic beads. After precipitation the complex is eluted from the beads and loaded onto the MCGE system. CRP could be reliably detected and quantified, with a detection limit of 25 ng/μl in serum samples and 126 pg/μl in matrix-free samples. The overall sensitivity (LOQ = 75 ng/μl, R² = 0.9668) of the method is lower than that of some specially developed methods (e.g., immune radiometric assay) but is comparable to those of clinically accepted ELISA methods. The straightforward sample preparation (not prone to mistakes), reduced sample and reagent volumes (including the antibodies), and high throughput (10 samples/3 h) are advantages and therefore IP–MCGE bears potential for point-of-care diagnosis.     

Accelerated dereplication of crude extracts using HPLC-PDA-MS-SPE-NMR: Quinolinone alkaloids of Haplophyllum acutifolium

Direct hyphenation of analytical-scale high-performance liquid chromatography, photo-diode array detection, mass spectrometry, solid-phase extraction and nuclear magnetic resonance spectroscopy (HPLC-PDA-MS-SPE-NMR) has been used for accelerated dereplication of crude extract of Haplophyllum acutifolium (syn. Haplophyllum perforatum). This technique allowed fast on-line identification of six quinolinone alkaloids, named haplacutine A-F, as well as of acutine, haplamine, eudesmine, and 2-nonylquinolin-4(1H)-one. Acutine and haplacutine E, isolated by preparative-scale HPLC, showed moderate antiplasmodial activity with IC₅₀ values of 2.17 ± 0.22 μM and 3.79 ± 0.24 μM, respectively (chloroquine-sensitive Plasmodium falciparum 3D7 strain).     

Multicomponent mesofluidic system for the detection of veterinary drug residues based on competitive immunoassay

An automated multicomponent mesofluidic system (MCMS) based on biorecognitions carried out on meso-scale glass beads in polydimethylsiloxane (PDMS) channels was developed. The constructed MCMS consisted of five modules: a bead introduction module, a bioreaction module, a solution handling module, a liquid driving module, and a signal collection module. The integration of these modules enables the assay to be automated and reduces it to a one-step protocol. The MCMS has successfully been applied toward the detection of veterinary drug residues in animal-derived foods. The drug antigen-coated beads (?250 μm) were arrayed in the PDMS channels (?300 μm). The competitive immunoassay was then carried out on the surface of the glass beads. After washing, the Cy3-labeled secondary antibody was introduced to probe the antigen-antibody complex anchored to the beads. The fluorescence intensity of each bead was measured and used to determine the residual drug concentration. The MCMS is highly sensitive, with its detection limits ranging from 0.02 (salbutamol) to 3.5 μg/L (sulfamethazine), and has a short assay time of 45 min or less. The experimental results demonstrate that the MCMS proves to be an economic, efficient, and sensitive platform for multicomponent detection of compound residues for contamination in foods or the environment.     

Nanomolar detection of hydrogen peroxide at a new polynuclear cluster of tin pentacyanonitrosylferrate nanoparticle-modified carbon ceramic electrode

This work describes a new electrochemical sensor for hydrogen peroxide based on tin pentacyanonitrosylferrate (SnPCNF)-modified carbon ceramic electrode (CCE). The modified electrode was constructed by using a sol-gel technique involving two steps: construction of CCE containing metallic tin (Sn) powder and then electrochemical creation of SnPCNF film on the surface of CCE. The modified electrode was characterized by energy-dispersive X-ray, Fourier transform infrared, scanning electron microscopy, and cyclic voltammetry (CV) techniques. The charge transfer coefficient (α) and charge transfer rate constant (k_s) for the modifying film were calculated. The electrocatalytic activity of the modified electrode toward the reduction of hydrogen peroxide was studied by CV and chronoamperometry. A linear calibration curve was obtained over the hydrogen peroxide concentration range of 0.5 to 69.4 μM using a hydrodynamic amperometric technique. The limit of detection (for a signal-to-noise ratio of 3) and sensitivity were found to be 92 nM and 0.89 μA/μM, respectively. Furthermore, the diffusion coefficient of hydrogen peroxide (D) and catalytic rate constant (k_cat) were calculated.     

Analysis of the antiviral drugs acyclovir and valacyclovir-hydrochloride in tsetse flies (Glossina pallidipes) using LC-MSMS

A new simple, sensitive and precise liquid chromatography-tandem mass spectrometry method has been developed and validated for the determination of valacyclovir-HCl and acyclovir in tsetse flies (Glossina pallipides). Tsetse flies were extracted by ultrasonication with acidified methanol/acetonitrile, centrifuged and cleaned up by solid phase dispersion using MgSO₄ and MSPD C₁₈ material. Samples were analysed using a Waters Alliance 2695 series HPLC with a C₁₈ Gemini analytical column (150 mm × 4.6 mm × 5 μm) and a guard cartridge column connected to a Waters Quattro-Micro triple-quadrupole mass spectrometer. The isocratic mobile phase consisted of methanol:acetonitrile:water (60:30:10, v/v/v) plus formic acid (0.1%) at a flow rate of 0.25 ml/min. The precursor > product ion transition for valacyclovir (m/z 325.1 > 152) and acyclovir (m/z 226.1 > 151.9) were monitored in positive electrospray multiple reaction monitoring mode. The method was validated at fortification levels of 0.5, 1 and 2 μg/g. The range of calibration for both drugs was 0.45-4.5 μg/g. The overall accuracy of the method was 92% for valacyclovir and 95% for acyclovir with corresponding within-laboratory reproducibilities of 4.4 and 3.4%, respectively. Mean recoveries were above 80% for both drugs and repeatability ranged from 0.7 to 6.1%. For both drugs the limits of detection and quantification were 0.0625 and 0.2 μg/g, respectively. The method was applied in experiments on the mass rearing of tsetse flies for sterile insect technique (SIT) applications, in which the flies were fed with blood meals containing acyclovir or valcyclovir-HCl prior to analysis to assess effects on Glossina pallidipes Salivary Gland Hypertrophy syndrome.     

Synthesis, crystal structure and magnetic properties of a polynuclear Cu(II) complex: catena-poly[aqua(di-2-pyridylamine)copper(II)(μ-formato-O,O′)nitrate]

The synthesis, X-ray structure, spectroscopic and magnetic properties of a zig-zag formato-bridged chain complex with the formula [Cu(dpyam)(μ-O₂CH)(OH₂)]_n(NO₃)_n (1) (in which dpyam = di-2-pyridylamine) is described.The geometry of the copper(II) ion is distorted square pyramidal with a basal plane consisting of two nitrogen atoms of the dpyam ligand (Cu-N distances 1.987(3) and 2.010(3) Å) and two oxygen atoms of two different formato ligands (Cu-O distances 1.974(2) and 1.975(2) Å). A coordinated water molecule occupies the axial position at a distance of 2.222(3) Å. The copper atoms are bridged unsymmetrically by a formato anion in a syn-anti arrangement, resulting in a polymeric zig-zag chain structure.The magnetic susceptibility measurements (5-280 K) agree with a very weak ferromagnetic chain interaction between the Cu centres with a J value of 0.75 cm⁻¹.     

Low-lying electronic states of the ferrous high-spin (S=2) heme in deoxy-Mb and deoxy-Hb studied by highly-sensitive multi-frequency EPR

The low-lying electronic states of the ferrous high-spin heme in deoxy-myoglobin (deoxy-Mb) and deoxy-hemoglobin (deoxy-Hb) were probed by multi-frequency electron paramagnetic resonance (MFEPR) spectroscopy. An unexpected broad EPR signal was measured at the zero magnetic field using cavity resonators at 34-122 GHz that could not be simulated using any parameter sets for the S = 2 spin Hamiltonian assuming spin quintet states in the ⁵B₂ ground state. Furthermore, we have observed novel, broad EPR signals measured at 70-220 GHz and 1.5 K using a single pass transmission probe. These signals are attributed to the ferrous high-spin heme in deoxy-Mb and deoxy-Hb. The resonant peaks shifted to a higher magnetic field with increasing frequency. The energy level separation between the ground singlet and the first excited state at the zero magnetic field was directly estimated to be 3.5 cm^− 1 for deoxy-Hb. For deoxy-Mb, the first two excited singlet states are separated by 3.3 cm^− 1 and 6.5 cm^− 1, respectively, from the ground state. The energy gap at the zero magnetic field is directly derived from our MFEPR for deoxy-Mb and deoxy-Hb and strongly supports the theoretical analyses based on the Mössbauer and magnetic circular dichroism experiments.     

Determination of sitagliptinin human plasma using protein precipitation and tandem mass spectrometry

A simple offline LC–MS/MS method for the quantification of sitagliptin in human plasma is described. Samples are prepared using protein precipitation. Filtration of the supernatants through a Hybrid-SPE-PPT plate was found to be necessary to reduce ionization suppression caused by co-elution of phospholipids with sitagliptin. The sitagliptin and its stable isotope labeled internal standard (IS) were chromatographed under hydrophilic interaction chromatography conditions on a Waters Atlantis HILIC Silica column (2.1 mm × 50 mm, 3 μm) using a mobile phase of ACN/H₂O (80/20, v/v) containing 10 mM NH₄Ac (pH 4.7). The sample drying after protein precipitation due to high organic content in the sample is not necessary, because HILIC column was used. The analytes were detected with a tandem mass spectrometer employing a turbo ion spray (TIS) interface in positive ionization mode. The multiple reaction monitoring (MRM) transitions were m/z 408 → 235 for sitagliptin and m/z 412 → 239 for IS. The lower limit of quantitation (LLOQ) for this method is 1 ng/mL when 100 μL of plasma is processed. The linear calibration range is 1–1000 ng/mL for sitagliptin. Intra-day precision and accuracy were assessed based on the analysis of six sets of calibration standards prepared in six lots of human control plasma. Intra-day precision (RSD%, n = 6) ranged from 1.2% to 6.1% and the intra-day accuracy ranged from 97.6% to 103% of nominal values.     

Determination of the major constituents in fruit of Arctium lappa L. by matrix solid-phase dispersion extraction coupled with HPLC separation and fluorescence detection

The arctiin and arctigenin in the fruit of Arctium lappa L. were extracted by matrix solid-phase dispersion (MSPD) and determined by high-performance liquid chromatography (HPLC) with fluorescence detection. The experimental conditions for the MSPD were optimized. Silica gel was selected as dispersion adsorbent and methanol as elution solvent. The calibration curve showed good relationship (r > 0.9998) in the concentration range of 0.010–5.0 μg mL⁻¹ for arctiin and 0.025–7.5 μg mL⁻¹ for arctigenin. The recoveries were between 74.4% and 100%. The proposed method consumed less sample, time and solvent compared with conventional methods, including ultrasonic and Soxhlet extraction.     

Dinuclear copper(II) chloro complex of the ligand 2,3,5,6-tetra(2-pyridyl)pyrazine

Crystallographic and magnetic studies have been performed on the complex, [{CuCl}₂(μ-tppz)][PF₆]₂, where tppz is 2,3,5,6-tetra-2-pyridinylpyrazine. The crystal structure revealed an infinite, ionic chain wherein Cu(II) ions are respectively above and below the plane of the pyrazine moiety of the bridging tppz ligand with the pyridine moieties moving out of the pyrazine plane in order to coordinate to Cu(II). Each chloride ligand bonds equatorially to Cu(II) in a [{CuCl}₂(μ-tppz)]²⁺ ion and axially to a neighboring [{CuCl}₂(μ-tppz)]²⁺ ion so as to form a one-dimensional chain in the solid state. The temperature-dependent magnetic susceptibilitity could be satisfactorily fitted by using a modified Bleaney-Bowers expression (for H = −JS_a · S_bJ = −5.6 cm⁻¹ and g = 2.16) where the exchange interaction is suggested to involve the orbitals of the tppz ligand.     

Long-range nonanomalous diffusion of quantum dot-labeled aquaporin-1 water channels in the cell plasma membrane

Aquaporin-1 (AQP1) is an integral membrane protein that facilitates osmotic water transport across cell plasma membranes in epithelia and endothelia. AQP1 has no known specific interactions with cytoplasmic or membrane proteins, but its recovery in a detergent-insoluble membrane fraction has suggested possible raft association. We tracked the membrane diffusion of AQP1 molecules labeled with quantum dots at an engineered external epitope at frame rates up to 91 Hz and over times up to 6 min. In transfected COS-7 cells, >75% of AQP1 molecules diffused freely over ∼7 μm in 5 min, with diffusion coefficient, D_1-3 ∼ 9 × 10⁻¹⁰ cm²/s. In MDCK cells, ∼60% of AQP1 diffused freely, with D_1-3 ∼ 3 × 10⁻¹⁰ cm²/s. The determinants of AQP1 diffusion were investigated by measurements of AQP1 diffusion following skeletal disruption (latrunculin B), lipid/raft perturbations (cyclodextrin and sphingomyelinase), and bleb formation. We found that cytoskeletal disruption had no effect on AQP1 diffusion in the plasma membrane, but that diffusion was increased greater than fourfold in protein de-enriched blebs. Cholesterol depletion in MDCK cells greatly restricted AQP1 diffusion, consistent with the formation of a network of solid-like barriers in the membrane. These results establish the nature and determinants of AQP1 diffusion in cell plasma membranes and demonstrate long-range nonanomalous diffusion of AQP1, challenging the prevailing view of universally anomalous diffusion of integral membrane proteins, and providing evidence against the accumulation of AQP1 in lipid rafts.     

Synthesis, structure and redox properties of an unexpected trinuclear copper(II) complex with aspartame: [Cu(apm)2Cu(μ-N,O:O′-apm)2(H2O)Cu(apm)2(H2O)] · 5H2O

Structural, magnetic and spectroscopic data of a new trinuclear copper(II) complex with the ligand aspartame (apm) are described. [Cu(apm)₂Cu(μ-N,O:O′-apm)₂(H₂O)Cu(apm)₂(H₂O)] · 5H₂O crystallizes in the triclinic system, space group P1 (#1) with a = 7.3300(1) Å, b = 15.6840(1) Å, c = 21.5280(1) Å, α = 93.02(1)°, β = 93.21(1)°, γ = 92.66(1)° and Z = 1. Aspartame coordinates to Cu(II) through the carboxylate and β-amino groups. The carboxylate groups of the two central ligands act as bidentate bridges in a syn-anti conformation while the carboxylate groups of the four peripheral ligands are monodentate in a syn conformation. The central copper ion is in a distorted square pyramidal geometry with the apical position being occupied by one oxygen atom of the water molecule. The two terminal copper(II) atoms are coordinated to the ligands in the same position but their coordination sphere differs from each other due to the fact that one copper atom has a water molecule in an apical position leading to an octahedral coordination sphere while the other copper atom is exclusively coordinated to aspartame ligands forming a distorted square pyramidal coordination sphere. Thermal analysis is consistent with the X-ray structure. EPR spectra and CV curves indicate a rupture of the trinuclear framework when this complex is dissolved in ethanol or DMF, forming a mononuclear species, with a tetragonal structure.     

Detection of single quantum dots in model organisms with sheet illumination microscopy

Single-molecule detection and tracking is important for observing biomolecule interactions in the microenvironment. Here we report selective plane illumination microscopy (SPIM) with single-molecule detection in living organisms, which enables fast imaging and single-molecule tracking and optical penetration beyond 300 μm. We detected single nanocrystals in Drosophila larvae and zebrafish embryo. We also report our first tracking of single quantum dots during zebrafish development, which displays a transition from flow to confined motion prior to the blastula stage. The new SPIM setup represents a new technique, which enables fast single-molecule imaging and tracking in living systems.     

Role of exchange and dipolar interactions in the radical pair model of the avian magnetic compass

It is not yet understood how migratory birds sense the Earth's magnetic field as a source of compass information. One suggestion is that the magnetoreceptor involves a photochemical reaction whose product yields are sensitive to external magnetic fields. Specifically, a flavin-tryptophan radical pair is supposedly formed by photoinduced sequential electron transfer along a chain of three tryptophan residues in a cryptochrome flavoprotein immobilized in the retina. The electron Zeeman interaction with the Earth's magnetic field (∼50 μT), modulated by anisotropic magnetic interactions within the radicals, causes the product yields to depend on the orientation of the receptor. According to well-established theory, the radicals would need to be separated by >3.5 nm in order that interradical spin-spin interactions are weak enough to permit a ∼50 μT field to have a significant effect. Using quantum mechanical simulations, it is shown here that substantial changes in product yields can nevertheless be expected at the much smaller separation of 2.0 ± 0.2 nm where the effects of exchange and dipolar interactions partially cancel. The terminal flavin-tryptophan radical pair in cryptochrome has a separation of ∼1.9 nm and is thus ideally placed to act as a magnetoreceptor for the compass mechanism.     

Local spin dynamics in magnetic molecular chains studied by NMR and μSR

We present NMR and μ⁺SR study of spin dynamics in one-dimensional and quasi-one-dimensional molecular magnets of recent synthesis. In particular, we focus on the so called Gd(hfac)₃NIT-R and CoPhOMe magnetic chains families. For Gd-R helimagnets we show some differences between “weakly frustrated systems” and “fully frustrated systems”. The different behaviour is due to the different radical inserted in the chains (R = Me, Ph for “weakly frustrated systems” and R = iPr, Et for “fully frustrated systems”). The existence of different phase transitions, particularly to 3D long-range magnetic order in Gd-Ph and to chiral order in Gd-iPr, is remarked together with a comparison between results obtained from macroscopic and microscopic investigating techniques. As regards CoPhOMe slowly relaxing chain, the ¹H NMR measurements confirm the freezing of the spins at low temperature, which prevents the 3D long-range order, and display the presence of two relaxation mechanisms related to distinct contributions to the local spin relaxation.     

Development of cloxacillin loaded multiple-unit alginate-based floating system by emulsion-gelation method

This work investigates the development, optimization and in vitro evaluation of liquid paraffin-entrapped multiple-unit alginate-based floating system containing cloxacillin by emulsion-gelation method for gastro retentive delivery. The effect of process variables like drug to polymer ratio by weight, and liquid paraffin to water ratio by volume on various physicochemical properties in case of liquid paraffin-entrapped calcium alginate beads containing cloxacillin applicable to drug entrapment efficiency, density and drug release was optimized using 3² factorial design and analyzed using response surface methodology. The observed (actual values) responses were coincided well with the predicted values, given by the optimization technique. The optimized beads showed drug entrapment efficiency of 64.63 ± 0.78%, density of 0.90 ± 0.05 g/cm³, and drug release of 56.72 ± 0.85% in simulated gastric fluid (pH 1.2) after 8 h with floating lag time of 8.45 min and floated well over 12 h in simulated gastric fluid (pH 1.2). The average size of all dried beads ranged from 1.73 ± 0.04 to 1.97 ± 0.08 mm. The beads were characterized by SEM and FTIR for surface morphology and excipients-drug interaction analysis, respectively. All these beads showed prolonged sustained release of cloxacillin over 8 h in simulated gastric fluid (pH 1.2). The cloxacillin release profile from liquid paraffin beads followed Korsmeyer-Peppas model over a period of 8 h with anomalous (non-Fickian) diffusion mechanism for drug release.     

Horizontal or vertical photobioreactors? How to improve microalgae photosynthetic efficiency

The productivity of a vertical outdoor photobioreactor was quantitatively assessed and compared to a horizontal reactor. Daily light cycles in southern Spain were simulated and applied to grow the microalgae Chlorella sorokiniana in a flat panel photobioreactor.The maximal irradiance around noon differs from 400 μmol photons m⁻² s⁻¹ in the vertical position to 1800 μmol photons m⁻² s⁻¹ in the horizontal position. The highest volumetric productivity was achieved in the simulated horizontal position, 4 g kg culture⁻¹ d⁻¹. The highest photosynthetic efficiency was found for the vertical simulation, 1.3 g of biomass produced per mol of PAR photons supplied, which compares favorably to the horizontal position (0.85 g mol⁻¹) and to the theoretical maximal yield (1.8 g mol⁻¹). These results prove that productivity per unit of ground area could be greatly enhanced by placing the photobioreactors vertically.     

Development of pH-sensitive tamarind seed polysaccharide-alginate composite beads for controlled diclofenac sodium delivery using response surface methodology

The present study deals with the development of novel pH-sensitive tamarind seed polysaccharide (TSP)-alginate composite beads for controlled diclofenac sodium delivery using response surface methodology by full 3² factorial design. The effect of polymer-blend ratio (sodium alginate:TSP) and cross-linker (CaCl₂) concentration on the drug encapsulation efficiency (DEE, %) and drug release from diclofenac sodium loaded TSP-alginate composite beads prepared by ionotropic gelation was optimized. The observed responses were coincided well with the predicted values by the experimental design. The DEE (%) of these beads containing diclofenac sodium was within the range between 72.23 ± 2.14 and 97.32 ± 4.03% with sustained in vitro drug release (69.08 ± 2.36-96.07 ± 3.54% in 10 h). The in vitro drug release from TSP-alginate composite beads containing diclofenac sodium was followed by controlled-release pattern (zero-order kinetics) with case-II transport mechanism. Particle size range of these beads was 0.71 ± 0.03-1.33 ± 0.04 mm. The swelling and degradation of the developed beads were influenced by different pH of the test medium. The FTIR and NMR analyses confirmed the compatibility of the diclofenac sodium with TSP and sodium alginate used to prepare the diclofenac sodium loaded TSP-alginate composite beads. The newly developed TSP-alginate composite beads are suitable for controlled delivery of diclofenac sodium for prolonged period.     

Role of Protein Kinase C in Endothelin Converting Enzyme-1 trafficking and shedding from endothelial cells

This study aimed to determine the consequences of Protein Kinase C (PKC) mediated Endothelin Converting Enzyme-1 (ECE-1) phosphorylation and its relationship to ECE-1 expression and shedding. The proteins on the surface of EA.hy926 cells were labelled with EZ-Link NHS-SS-Biotin both prior to (control) and following stimulation by 2 μM phorbol 12-myristate 13-acetate (PMA) which activates PKC. The biotinylated proteins were isolated using neutravidin beads, resolved by gel electrophoresis and analysed by western blotting using anti-ECE-1 antibodies. Significant increase in ECE-1 expression at the cell surface was observed following stimulation by PMA, compared to unstimulated control cells (170 ± 32.3% of control, n = 5). The ECE-1 activity (expressed as μM substrate cleaved/min) was determined by monitoring the cleavage of a quenched fluorescent substrate. The specificity of cleavage was confirmed using the ECE-1 inhibitor CGS35066. The stimulation of cells by PMA (1 μM, 6 h) significantly increased the ECE-1 activity (0.28 ± 0.02; n = 3) compared to the control (0.07 ± 0.02; n = 3). This increase was prevented by prior incubation with the PKC inhibitor bisindolymaleimide (BIM; 2 μM for 1 h; 0.10 ± 0.01; n = 3). Treatment with PMA also increased the activity of ECE-1 in the media (0.18 ± 0.01; n = 3) compared to control (0.08 ± 0.01; n = 3). In addition, this study confirmed by western immunoblotting that only the extracellular region of ECE-1 is released from the cell surface. These data indicate for the first time that PKC activation induces the trafficking and shedding of ECE to and from the cell surface, respectively.