Decrease In Electrophoretic Mobility Research Articles

Fullerene C60 functionalized Sm2O3@Eu2O3 bimetallic oxides core shell nanocomposites for zeta potential and energy storage applications

Nanocomposites of Fullerene C60 (FC60) with lanthanide oxides show better energy storage properties compared with any of them alone. In the present research, we have synthesized FC60 (2, 5 and 10 wt%) anchored on Sm2O3@Eu2O3 bimetallic oxides by simple chemical precipitation method. The FC60:Sm2O3@Eu2O3 core shell nanocomposites (NCs) were characterized by XRD (X-ray diffraction), SEM (scanning electron microscopy), EDX (energy dispersive X-ray), UV–visible, XPS (X-ray photoelectron spectroscopy), and BET (Brunauer–Emmett–Teller) analytical methods. XRD studies revealed mixed phase of monoclinic and orthorhombic crystal structure with irregularly scattered small plate/flake like morphology agglomerated and formed as large grains. Electrochemical supercapacitive behaviour of Sm2O3@Eu2O3 bimetallic oxides showed specific capacitance (SCs) of 665.075F/g in 3 M KOH as electrolyte. The SCs of FC60 (5 wt% and 10 wt%):Sm2O3@Eu2O3 electrode material in 3 M KOH solution was found to be 795.92F/g and 1120F/g at a current density of 1 A/g (scan rate of 10 mV/s). FC60 (10 wt%):Sm2O3@Eu2O3 NCs showed excellent capacitive retention of 90.8 % after 10,000 cycles. The Zeta potential (ζ) studies of FC60:Sm2O3@Eu2O3 NCs showed decrease in the mean Zeta potential (ζ) from 34.23 mV to 11.97 mV with decrease in electrophoretic mobility (2.667 to 0.933 (μm cm/Vs)) attributed to the cationic nature of the FC60:Sm2O3@Eu2O3 NCs. The FC60:Sm2O3@Eu2O3 NCs with high cycle stability and charge density can be a better candidate for high energy storage devices, supercapacitor, dielectric related applications.

Interaction between gold nanoparticles and blood proteins

Introduction Metallic nanoparticles constitute promising biosensing systems, due to their high affinity to biomolecules such as proteins, which form protein coronas of distinct compositions on their surface [1]. Gold nanoparticles (AuNP) are particularly interesting given their relatively easy, quick and inexpensive synthesis, low toxicity and ease of functionalization with bifunctional molecules. Usually, these molecules have thiol groups bound to the AuNP surface and bio-friendly chemical groups at the opposite end, allowing for controlled protein adsorption. Such functionalised AuNPs may be used as probing agents for a patient’s droplet of blood and the health state can be based on the composition of AuNP-adsorbed protein corona. It is thus important to understand the behaviour of each plasma protein in the corona, divided into a tightly-bound inmost monolayer (hard corona) and looser outer layers (soft corona), removable through centrifugation [2]. Materials and methods AuNP synthesis was performed according to a modified Turkevich method and AuNP diameter and concentration was determined by UV-Vis spectroscopy. AuNPs were functionalised with 11-mercaptoundecanoic acid and further conjugated with bovine or human serum albumin or fibrinogen. These single protein conjugates were evaluated for hydrodynamic diameter changes after centrifugation by dynamic light scattering (DLS). Agarose gel electrophoresis (AGE) allowed to determine electrophoretic mobility and concentration-dependent conjugation efficiency. Analysis of AGE profiles was by the open source electrophoresis gel image processing software eReuss. Results DLS showed a decrease in hydrodynamic diameter for centrifuged conjugates of 40 nm gold nanoparticles, with too high polydispersity indexes for the 13 nm ones, suggesting aggregation. AGE revealed a decrease in electrophoretic mobility as the protein-to-AuNP ratio increases, data fitted to a Langmuir adsorption model. Protein and AuNP concentrations during incubation affect the electrophoretic mobility profile. In fact, depending on the protein-to-AuNP ratio, migration is proportional to the colloidal suspension volume in which conjugation occurred. Discussion and conclusions Although centrifugation can induce AuNP aggregation, it appears to affect the protein corona. A decrease in hydrodynamic diameter as determined by DLS appears in centrifuged samples comparatively to its uncentrifuged counterparts, which corroborates the soft corona being more loosely bound. Moreover, AGE suggests that for equal protein:AuNP ratios, the volume of sample is determinant for the conjugation process.

Capillary Zone Electrophoresis-Tandem Mass Spectrometry for Large-Scale Phosphoproteomics with the Production of over 11,000 Phosphopeptides from the Colon Carcinoma HCT116 Cell Line.

Phosphoproteomics requires better separation of phosphopeptides to boost the coverage of the phosphoproteome. We argue that an alternative separation method that produces orthogonal phosphopeptide separation to the widely used LC needs to be considered. Capillary zone electrophoresis (CZE) is one important alternative because CZE and LC are orthogonal for phosphopeptide separation and because the migration time of peptides in CZE can be accurately predicted. In this work, we coupled strong cation exchange (SCX)-reversed-phase LC (RPLC) to CZE-MS/MS for large-scale phosphoproteomics of the colon carcinoma HCT116 cell line. The CZE-MS/MS-based platform identified 11,555 phosphopeptides. The phosphopeptide data set is at least 100% larger than that from previous CZE-MS/MS studies and will be a valuable resource for building a model for predicting the migration time of phosphopeptides in CZE. Phosphopeptides migrate significantly slower than corresponding unphosphopeptides under acidic conditions of CZE separations and in a normal polarity. According to our modeling data, phosphorylation decreases peptide's charge roughly by one charge unit, resulting in dramatic decrease in electrophoretic mobility. Preliminary investigations demonstrate that electrophoretic mobility of phosphopeptides containing one phosphoryl group can be predicted with the same accuracy as for nonmodified peptides ( R2 ≈ 0.99). The CZE-MS/MS and LC-MS/MS were complementary in large-scale phosphopeptide identifications and produced different phosphosite motifs from the HCT116 cell line. The data highlight the value of CZE-MS/MS for phosphoproteomics as a complementary separation approach for not only improving the phosphoproteome coverage but also providing more insight into the phosphosite motifs.

Influence of Analyte Concentration on Stability Constant Values Determined by Capillary Electrophoresis.

The influence of analyte concentration when compared with the concentration of a charged ligand in background electrolyte (BGE) on the measured values of electrophoretic mobilities and stability constants (association, binding or formation constants) is studied using capillary electrophoresis (CE) and a dynamic mathematical simulator of CE. The study is performed using labile complexes (with fast kinetics) of iron (III) and 5-sulfosalicylate ions (ISC) as an example. It is shown that because the ligand concentration in the analyte zone is not equal to that in BGE, considerable changes in the migration times and electrophoretic mobilities are observed, resulting in systematic errors in the stability constant values. Of crucial significance is the slope of the dependence of the electrophoretic mobility decrease on the ligand equilibrium concentration. Without prior information on this dependence to accurately evaluate the stability constants for similar systems, the total ligand concentration must be at least >50-100 times higher than the total concentration of analyte. Experimental ISC peak fronting and the difference between the direction of the experimental pH dependence of the electrophoretic mobility decrease and the mathematical simulation allow assuming the presence of capillary wall interaction.

The role of membrane lipids in forming the electrokinetic properties of red blood cells in solid tumors and psoriasis

Aim. Analysis of the electrophoretic mobility of red blood cells in patients with the diseases caused by increased cell proliferation activity, and its relationship with the lipid spectrum of erythrocyte membranes.
 Methods. The blood of 42 patients with malignant tumors of epithelial tissues (colon, kidney, prostate, urine bladder and cervical cancer) and 16 patients with skin diseases was studied. The control group consisted of 20 healthy individuals. Electrophoretic mobility of red blood cells was assessed by microelectrophoresis. Lipid fractions levels in erythrocytes was determined by one-dimensional thin layer chromatography.
 Results. Over than two-fold decrease of electrophoretic mobility of erythrocytes was observed in patients with malignant tumors of epithelial tissues and skin diseases associated with cell proliferation processes (psoriasis) - by 59 and 60%, respectively In inflammatory skin diseases (microbial eczema), the decrease was less pronounced (30%). The contents of charged phospholipid in erythrocyte membranes in case of cancer and psoriasis has changed in a similar way: a significant reduction in sphingomyelin level in the absence of significant differences in phosphatidylethanolamine and phosphatidylcholine levels. Multidirectional statistically significant changes in the contents of lysophosphatidylcholine were revealed depending on the stage of cancer, their level was decreased two-fold in case of psoriasis. The changes of cholesterol, diacylglycerols, free fatty acids levels in the membranes of red blood cells in case of cancer and psoriasis were divergent.
 Conclusion. In diseases caused by cell proliferation activation, electrophoretic mobility of erythrocytes is reduced by more than 50%. Kinetic characteristic of erythrocytes may be used for detecting the conditions of pathologic cell proliferation, including malignant tumors, since they alter in cases of phospholipid membranes lipid spectrum disorders.

Surface electric charge of thylakoid membranes from genetically modified tobacco plants under freezing stress

In the present study we characterize for the first time electrokinetic and light scattering properties of thylakoids from freezing-tolerant tobacco plants, transformed to accumulate osmoprotectants (proline: AtP5Cs, VacP5Cs; fructan: SacB; glycine betaine: codA). Tobacco plants of wild type (WT) and transformed variants were cultivated at 2°C (cold acclimated) and −2°C (freezing stressed). “Lower salt” thylakoids (I=0.0006) of WT and SacB plants exhibited a decrease in electrophoretic mobility (EPM) after (2°C) treatment. AtP5Cs thylakoids (22°C) show a substantial increase in negative electrical charge (σ) upon illumination. We observed that “low salt”SacB thylakoids at 22°C and 2°C increased the σ on their membrane surfaces during the process of acclimation. WT (22°C) and AtP5Cs thylakoids (2°C) in “low salt” media (I=0.0156) showed a substantial increase in surface electrical charge upon illumination. Cold acclimation on WT and freezing stress on transformed plants resulted in a decrease in aggregation of thylakoids at both ionic strengths.There was a large enhancement in the relaxation capacity of reverse photosynthetic reactions in codA and SacB tobacco after freezing stress. Maximal intensity of the delayed light emission following low temperature stimuli was decreased, revealing a path for tobacco transformants to improve their cold stress tolerance.Here, we suggest the EPM value as an indicator for stability of thylakoids undergone genetic transformation.

Extracting Information from the Ionic Strength Dependence of Electrophoretic Mobility by Use of the Slope Plot

The effective mobility (μ(ep)) is the main parameter characterizing the electrophoretic behavior of a given solute. It is well-known that μ(ep) is a decreasing function of the ionic strength for all solutes. Nevertheless, the decrease depends strongly on the nature of the solute (small ions, polyelectrolyte, nanoparticles). Different electrophoretic models from the literature can describe this ionic strength dependence. However, the complexity of the ionic strength dependence with the solute characteristics and the variety of analytical expressions of the different existing models make the phenomenological ionic strength dependence difficult to comprehend. In this work, the ionic strength dependence of the effective mobility was systematically investigated on a set of different solutes [small mono- and multicharged ions, polyelectrolytes, and organic/inorganic (nano)particles]. The phenomenological decrease of electrophoretic mobility with ionic strength was experimentally described by calculating the relative electrophoretic mobility decrease per ionic strength decade (S) in the range of 0.005-0.1 M ionic strength. Interestingly, the "slope plot" displaying S as a function of the solute electrophoretic mobility at 5 mM ionic strength allows for defining different zones that are characteristic of the solute nature. This new representative approach should greatly help experimentalists to better understand the ionic strength dependence of analyte and may contribute to the characterization of unknown analytes via their ionic strength dependence of electrophoretic mobility.

Electrophoretic mobility of partially denatured DNA in a gel: Qualitative and semiquantitative differences between bubbles and split ends

Partially melted DNA is known to exhibit an abrupt decrease of electrophoretic mobility in a gel. Although this is the main phenomenon exploited in TGGE/DGGE (temperature gradient gel electrophoresis/denaturing gradient gel electrophoresis), not much is known about the physical processes responsible for the blocking. While there is a commonly used formula for the reduced mobility based on the theory of branched polymers, it does not discriminate between denatured domains bounded on one (split end) or two sides (bubble). To better understand how the blocking occurs in both of these cases, a coarse-grained model of DNA gel electrophoresis is simulated using Langevin Dynamics. The simulations reveal that the low-field mobility is much more sensitive to denatured domains located at the ends of a DNA fragment. A denatured domain occurring at the center of a fragment indeed reduces the mobility, but at a much lower rate.

Field- and concentration-dependence of electrostatics in non-polar colloidal asphaltene suspensions

Electrostatic stabilization has recently been found to be an important factor in non-polar colloidal asphaltene suspensions. However, the nature of charging in asphaltene systems may be quite different than in other non-polar colloid systems. For instance, the origin of charging in asphaltene colloids arises from both positive and negative charges native to the asphaltenes. In part due to this bimodality of surface charge, some dispersants are shown to stabilize asphaltene colloids at concentrations below their cmc. This effect does not arise from ionic effects of the dispersant, but rather from preferential dispersant adsorption onto charged sites on the colloidal asphaltene surface. Due to the long-range nature of electrostatics in non-polar systems, electrophoretic mobility can depend on both electric-field and particle volume fraction. We investigate the field- and concentration- dependence of electrophoretic mobility in non-polar colloidal asphaltene suspensions. Our results suggest that colloidal asphaltene suspensions stabilized by non-ionic dispersants can exhibit large screening lengths even in the presence of dispersant micelles. Due in part to the large screening length, a decrease in electrophoretic mobility can occur even at low colloidal particle concentrations.

Thermal stability of porcine pepsin influenced by Al(III) ion: DSC study

Differential scanning calorimetry (DCS) has been used to determine thermodynamic profile of pepsin and the in vitro effect of Al(III) ions. Thermograms of pepsin unfolding in the presence and absence of aluminum were used to determine the binding constant, KL, in the pepsin-aluminium model system. The thermodynamic parameters were derived from DSC profiles at different ligand concentrations (1, 5 and 10 mM). The temperatures of thermal transitions (Tm), calorimetric (ΔHcal) and van’t Hoff enthalpy (ΔHVH), Gibbs free energy, Δ(ΔG), of Al(III) binding to pepsin, as well as an average number of ligands bound to the native protein, were obtained from DSC profiles too. Temperature-dependent changes in the protein structure were also monitored by native PAGE electrophoresis. Increasing the temperature causes the decrease in electrophoretic mobility. Increase in concentration of Al(III) decelerate the migration of pepsin samples on concentration dependent manner. Analysis showed that ligand binding increases thermal stability of protein.

The Structural Basis for Substrate Recognition by Mammalian Polynucleotide Kinase 3′ Phosphatase

Mammalian polynucleotide kinase 3' phosphatase (PNK) plays a key role in the repair of DNA damage, functioning as part of both the nonhomologous end-joining (NHEJ) and base excision repair (BER) pathways. Through its two catalytic activities, PNK ensures that DNA termini are compatible with extension and ligation by either removing 3'-phosphates from, or by phosphorylating 5'-hydroxyl groups on, the ribose sugar of the DNA backbone. We have now determined crystal structures of murine PNK with DNA molecules bound to both of its active sites. The structure of ssDNA engaged with the 3'-phosphatase domain suggests a mechanism of substrate interaction that assists DNA end seeking. The structure of dsDNA bound to the 5'-kinase domain reveals a mechanism of DNA bending that facilitates recognition of DNA ends in the context of single-strand and double-strand breaks and suggests a close functional cooperation in substrate recognition between the kinase and phosphatase active sites.

DNA as molecular target of analogous palladium and platinum anti- Trypanosoma cruzi compounds: A comparative study

In the search for drugs with anti-trypanosome activity, we had previously synthesized two series of platinum and palladium analogous compounds of the formula [M IICl 2(HL)], where HL were bioactive 5-nitrofuryl or 5-nitroacroleine thiosemicarbazone derivatives. In this work, we thoroughly characterized [M IICl 2(HL)] complexes interaction with DNA by using different techniques: gel electrophoresis, DNA viscosity measurements, circular dichroism (CD) and atomic force microscopy (AFM). Electrophoresis results showed that all complexes induced a withdrawal of DNA superhelicity demonstrated by a decrease in electrophoretic mobility of supercoiled DNA form. This effect on migration was dependent on dose but also on the nature of both the metal and the ligand. In general, the effect produced by palladium complexes was significantly more intense than that observed for the corresponding platinum analogs. Differences between palladium and platinum complexes were also observed in CD experiments. While palladium complexes induce evident calf thymus (CT)-DNA profile changes compatible with B-DNA to Z-DNA conformational transition, no clear effect was observed for platinum ones. Additionally, AFM studies showed that changes in the shape of plasmid DNA, like supercoiling, kinks and thickness increase resulted more intense for the former. In addition, either Pd or Pt complexes increased the viscosity of CT DNA solutions in a concentration dependent manner. Although the nature of DNA interaction of both series of analogous palladium and platinum complexes seemed to be similar, an explanation for the observed differential intensity of the effect could be related to the known kinetic stability differences between palladium and platinum compounds.

Electrophoretic mobilities of dissolved polyelectrolyte charging agent and suspended non-colloidal titanium during electrophoretic deposition

Coarse (≤20 μm) titanium particles were deposited on low-carbon steel substrates by cathodic electrophoretic deposition (EPD) with ethanol as suspension medium and poly(diallyldimethylammonium chloride) (PDADMAC) as polymeric charging agent. Preliminary data on the electrophoretic mobilities and electrical conductivities on the suspensions of these soft particles as well as the solutions themselves as a function of PDADMAC level were used as the basis for the investigation of the EPD parameters in terms of the deposition yield as a function of five experimental parameters: (a) PDADMAC addition level, (b) solids loading, (c) deposition time, (d) applied voltage, and (e) electrode separation. These data were supported by particle sizing by laser diffraction and deposit surface morphology by scanning electron microscopy (SEM). The preceding data demonstrated that Ti particles of ∼1–12 μm size, electrosterically modified by the PDADMAC charging agent, acted effectively as colloidal particles during EPD. Owing to the non-colloidal nature of the particles and the stabilization of the Ti particles by electrosteric forces, the relevance of the zeta potential is questionable, so the more fundamental parameter of electrophoretic mobility was used. A key finding from the present work is the importance of assessing the electrophoretic mobilities of both the suspensions and solutions since the latter, which normally is overlooked, plays a critical role in the ability to interpret the results meaningfully. Further, algebraic uncoupling of these data plus determination of the deposit yield as a function of charging agent addition allow discrimination between the three main mechanistic stages of the electrokinetics of the process, which are: (1) surface saturation; (2) compression of the diffuse layer, growth of polymer-rich layer, and/or competition between the mobility of Ti and PDADMAC; and (3) little or no decrease in electrophoretic mobility of Ti, establishment of polymer-rich layer, and/or dominance of the mobility of the PDADMAC over that of Ti.

Preparation and Biophysical Characterization of Pluronic F127-Dendrimer Conjugate as a Delivery Agent of Antisense Oligonucleotides

A series of pluronic F127 conjugated PAMAM dendrimer generation 5 were prepared to evaluate the potential use of dendrimer-pluronic conjugates as a delivery agent of antisense oligonucleotides. The structural features of the conjugates were identified by FT-IR and 1H-NMR spectra. The prepared dendrimer generation 5-pluronic F127 conjugates showed particle size in the range of 50 to 130 nm with reduced zeta potential. The electrophoretic property of conjugates was further confirmed by capillary zone electrophoresis (CZE), demonstrating the decrease in electrophoretic mobility of conjugates as the number of pluronic F127 on surface of dendrimer increases. The number of free amino groups on a pluronic F127 conjugated dendrimer was quantitatively determined by fluorescamine assay, measuring the fluorescence intensity of amino groups reactive to fluorescamine on dendrimer generation 5-pluronic F127 conjugates. In MTT assay, dendrimer G5-pluronic F127 conjugates were safe on gingival fibroblast cells and acute toxicity was reduced as the mole ratio of dendrimer to pluronic increased. Complex formation of dendrimer G5-pluronic F127 conjugates with oligonucleotides was dose dependent.

Effect of Glutamate on the Electrical Properties of Cationic Solid Lipid Nanoparticles Containing Stearylamine and Dioctadecyldimethyl Ammonium Bromide

Electrical properties, including electrophoretic mobility, zeta potential, total surface charge density, and surface charge density resulting from primary amino groups, of cationic solid lipid nanoparticles (CSLNs) were investigated in the present study. Cationic lipids including stearylamine (SA) and dioctadecyldimethyl ammonium bromide (DODAB) were covered on the external cores of CSLNs. The influences of glutamate concentration in the medium, composition of cationic lipids, and surfactant species were especially analyzed. The results indicated that an increase in the mole ratio of SA in the cationic lipid caused an increase in the average diameter of CSLNs. Also, the average diameter of Span 20-stabilized CSLNs was larger than that of Tween 80-stabilized CSLNs. The electrostatic traits of CSLNs were reduced as the mole ratio of SA increased, and the electricity of Span 20-stabilized CSLNs was weaker than that of Tween 80-stabilized CSLNs. An increase in the glutamate concentration in the medium led to a decrease in electrophoretic mobility, zeta potential, and total surface charge density of CSLNs. As the glutamate concentration increased, surface charge density resulting from primary amino groups increased, and that from quaternary amino groups decreased as a result of the adsorption of negatively charged glutamate on CSLN surfaces. Ohshima's soft particle theory was adopted to describe the electrical behavior of CSLNs, and the deviations of zeta potential predicted by the Smoluchowski, Happel, and Kuwabara models were normally greater than 10%.

Activation of Cytosolic Phospholipase A2α in Resident Peritoneal Macrophages by Listeria monocytogenes Involves Listeriolysin O and TLR2

Eicosanoid production by macrophages is an early response to microbial infection that promotes acute inflammation. The intracellular pathogen Listeria monocytogenes stimulates arachidonic acid release and eicosanoid production from resident mouse peritoneal macrophages through activation of group IVA cytosolic phospholipase A2 (cPLA2alpha). The ability of wild type L. monocytogenes (WTLM) to stimulate arachidonic acid release is partially dependent on the virulence factor listeriolysin O; however, WTLM and L. monocytogenes lacking listeriolysin O (DeltahlyLM) induce similar levels of cyclooxygenase 2. Arachidonic acid release requires activation of MAPKs by WTLM and DeltahlyLM. The attenuated release of arachidonic acid that is observed in TLR2-/- and MyD88-/- macrophages infected with WTLM and DeltahlyLM correlates with diminished MAPK activation. WTLM but not DeltahlyLM increases intracellular calcium, which is implicated in regulation of cPLA2alpha. Prostaglandin E2, prostaglandin I2, and leukotriene C4 are produced by cPLA2alpha+/+ but not cPLA2alpha-/- macrophages in response to WTLM and DeltahlyLM. Tumor necrosis factor (TNF)-alpha production is significantly lower in cPLA2alpha+/+ than in cPLA2alpha-/- macrophages infected with WTLM and DeltahlyLM. Treatment of infected cPLA2alpha+/+ macrophages with the cyclooxygenase inhibitor indomethacin increases TNFalpha production to the level produced by cPLA2alpha-/- macrophages implicating prostaglandins in TNFalpha down-regulation. Therefore activation of cPLA2alpha in macrophages may impact immune responses to L. monocytogenes.

The influence of Al3+ ion on porcine pepsin activity in vitro

The in vitro effect of Al3+ ions in the concentration range 1.7·10− 6 M–8.7·10− 3 M on pepsin activity at pH 2, via kinetic parameters and its electrophoretic mobility was evaluated. Kinetic study demonstrated the existence of an activation effect of Al3+ at pH 2 on pepsin molecule. Kinetic analysis with respect to concentrations of haemoglobin showed that Al3+ ions increase the maximal velocity (Vmax) and kcat values rather than apparent affinity for substrate (KS) implying the non-competitive nature of activation which indicated that aluminium was a non-essential activator of partial non-competitive type. The values of the equilibrium constants KS and KmA for dissociation of corresponding complexes were evaluated as 0.904 ± 0.083 mM and 8.56 ± 0.51 μM, respectively. Dissociation constant KA, of activator from enzyme-activator complex calculated via kinetic and direct measurement of Al3+ binding data, as well as activation constant A50, the activator concentration that gives a rate equal to half at a saturating concentration of activator, were found to be 8.82 ± 0.90 μM, 8.39 ± 0.76 μM, and 8.05 ± 0.48 μM respectively. Native PAGE electrophoresis shows the decrease in electrophoretic mobility of pepsin and confirms modification of the electric charge and conformational changes of pepsin caused by bound Al3+ on the pepsin molecule. Al3+ induced conformational changes of pepsin were verified by UV-VIS and IR spectra. Moreover, the absence of conformational changes in the haemoglobin molecule in the presence of Al3+ ions confirms that the obtained activation is a consequence of conformational changes caused only in the pepsin molecule.

Site-specific Phosphorylation and Caspase Cleavage Differentially Impact Tau-Microtubule Interactions and Tau Aggregation

The microtubule-associated protein tau is hyperphosphorylated and forms neurofibrillary tangles in Alzheimer disease. Additionally caspase-cleaved tau is present in Alzheimer disease brains co-localized with fibrillar tau pathologies. To further understand the role of site-specific phosphorylation and caspase cleavage of tau in regulating its function, constructs of full-length tau (T4) or tau truncated at Asp421 (T4C3) to mimic caspase-3 cleavage with and without site-directed mutations that mimic phosphorylation at Thr231/Ser235, Ser396/Ser404, or at all four sites (Thr231/Ser235/Ser396/Ser404) were made and expressed in cells. Pseudophosphorylation of T4, but not T4C3, at either Thr231/Ser235 or Ser396/Ser404 increased its phosphorylation at Ser262 and Ser199. Pseudophosphorylation at Thr231/Ser235 impaired the microtubule binding of both T4 and T4C3. In contrast, pseudophosphorylation at Ser396/Ser404 only affected microtubule binding of T4C3 but did make T4 less soluble and more aggregated, which is consistent with the previous finding (Abraha, A., Ghoshal, N., Gamblin, T. C., Cryns, V., Berry, R. W., Kuret, J., and Binder, L. I. (2000) J. Cell Sci. 113, 3737-3745) that pseudophosphorylation at Ser396/Ser404 enhances tau polymerization in vitro. In situ T4C3 was more prevalent in the cytoskeletal and microtubule-associated fractions compared with T4, whereas purified recombinant T4 bound microtubules with higher affinity than did T4C3 in an in vitro assay. These data indicate the importance of cellular factors in regulating tau-microtubule interactions and that, in the cells, phosphorylation of T4 might impair its microtubule binding ability more than caspase cleavage. Treatment of cells with nocodazole revealed that pseudophosphorylation of T4 at both Thr231/Ser235 and Ser396/Ser404 diminished the ability of tau to protect against microtubule depolymerization, whereas with T4C3 only pseudophosphorylation at Ser396/Ser404 attenuated the ability of tau to stabilize the microtubules. These results show that site-specific phosphorylation and caspase cleavage of tau differentially affect the ability of tau to bind and stabilize microtubules and facilitate tau self-association.

Phosphorylation of Frizzled-3

Wnts are secreted proteins important to many biological processes. frizzled genes encode a family of Wnt receptors that signal to the intracellular compartment through the cytosolic protein Disheveled. Limited information is available concerning the regulation of Frizzleds at a biochemical level. We report here that Xenopus Frizzled-3 is phosphorylated in a Disheveled-dependent manner that appears to require the DEP domain of Disheveled. Phosphorylation of serine 576 causes a decrease in electrophoretic mobility and accounts for a significant fraction of receptor phosphorylation, although additional residues in the C-terminal tail are also phosphorylated. In addition, mutations that interfere with Frizzled-3 function also interfere with phosphorylation, but these inactive mutants can be phosphorylated when an active form of Frizzled-3 is co-expressed. Mutation of C-terminal serines including serine 576 significantly enhances Frizzled-3-mediated induction of neural crest markers, suggesting that C-terminal phosphorylation plays a role in down-regulating Frizzled signaling.

Interaction of DNA Fragmentation Factor (DFF) with DNA Reveals an Unprecedented Mechanism for Nuclease Inhibition and Suggests That DFF Can Be Activated in a DNA-bound State

DNA fragmentation factor (DFF) is a complex of the DNase DFF40 (CAD) and its chaperone/inhibitor DFF45 (ICAD-L) that can be activated during apoptosis to induce DNA fragmentation. Here, we demonstrate that DFF directly binds to DNA in vitro without promoting DNA cleavage. DNA binding by DFF is mediated by the nuclease subunit, which can also form stable DNA complexes after release from DFF. Recombinant and reconstituted DFF is catalytically inactive yet proficient in DNA binding, demonstrating that the nuclease subunit in DFF is inhibited in DNA cleavage but not in DNA binding, revealing an unprecedented mode of nuclease inhibition. Activation of DFF in the presence of naked DNA or isolated nuclei stimulates DNA degradation by released DFF40 (CAD). In transfected HeLa cells transiently expressed DFF associates with chromatin, suggesting that DFF could be activated during apoptosis in a DNA-bound state.