Absence Of Divalent Cations Research Articles

Membrane attachment of Slr0006 in Synechocystis sp. PCC 6803 is determined by divalent ions

Slr0006 is one of the Synechocystis sp. PCC 6803 proteins strongly induced under carbon limiting conditions. Slr0006 has no predicted transmembrane helices or signal peptide sequence, yet it was exclusively recovered in the membrane fraction of Synechocystis, when the cells were broken in isolation buffers which contain divalent cations and are generally used for photosynthesis studies. Even subsequent washing of the membranes with high salt or various detergents did not release Slr0006, indicating strong binding of the Slr0006 protein to the membranes. Further, DNAse or RNAse treatment did not disturb the tight binding of Slr0006 protein to the membranes. Nevertheless, when the cells were broken in the absence of divalent cations, Slr0006 remained completely soluble. Binding of the Slr0006 to the membrane could not be properly reconstituted if the cations were added after breaking the cells in the absence of divalent ions. This unusual phenomenon has to be considered in identification and localization of other yet uncharacterized cyanobacterial proteins.

Physical Properties Affecting Cochleate Formation and Morphology Using Antimicrobial Oligo-acyl-lysyl Peptide Mimetics and Mixtures Mimicking the Composition of Bacterial Membranes in the Absence of Divalent Cations

Several cationic antimicrobial oligo-acyl-lysyl (OAK) peptide mimetics can form cochleate structures, that is, elongated multilayered cylindrical structures, with lipid mixtures mimicking the composition of bacterial cytoplasmic membranes. These cochleate structures do not require divalent cations for their assembly. In the present work, we use light microscopy to screen for cochleate formation in several OAK-lipid systems and freeze-fracture electron microscopy to assess their morphological features and size. We identify several factors that facilitate a structural change in these assemblies. Dehydration of the membrane interface and a high melting temperature are features of the lipids that enhance cochleate formation in OAK-based lipid systems. In addition, we observed that there is a specific length of the hydrocarbon linker in the OAK of 8-9 carbon atoms that provides optimal formation of these structures. The biophysical properties established in this study will allow for a better understanding of their role and suitability for biological studies.

Atp 4- Activates P2x Receptors

P2X receptors are non-selective cation channels that are involved in many important physiological processes, including neurotransmission and inflammation. Extracellular ATP released from synaptic vesicles or damaged tissues has been demonstrated to be the ligand for activating P2X receptors. However, it is not clear if ATP activates P2X receptors in its free anionic form as ATP4-, or in complex with divalent cations such as magnesium (MgATP)2-. Here we examined if ATP can open the channels in the absence of divalent cations, and tested the effects of different concentrations of Mg2+ on the dose response curve for channel activation by ATP. The results show that all seven subtypes of P2X receptors can be activated by ATP4-, and on all but one subtype of P2X receptor, Mg2+ has a modulatory effect. In the one exception, we found that Mg2+ competes with P2X receptors for ATP4-, suggesting that (MgATP)2- is not the agonist. In summary, our results suggest that free ATP, but not magnesium bound ATP, is the ligand for opening P2X receptors.

Cochleate Assemblies with Antimicrobial Oaks and Lipid Mixtures in Absence of Divalent Cations: Biophysical Properties and Strategies to Overcome Drug Resistance

We have recently shown that the cationic antimicrobial oligo-acyl-lysyl (OAK), C12K-7α8, can form cochleate structures with lipid mixtures mimicking the composition of bacterial cytoplasmic membranes. These assemblies can deliver drugs to bacteria, enabling them to overcome multidrug resistance and to effectively protect neutropenic mice from lethal infection upon single-dose systemic treatment (Livne et al., FASEB J, in press). The OAK-based lipid systems form cochleate structures with the novel feature that divalent cations are not required for their assembly, thus freeing the requirement for PS as well. We demonstrate that cochleate structures can assemble with a variety of OAKs analogs and several lipid mixtures, but with varying efficacy. We identify the biophysical characteristics that lead to cochleate formation in these OAK-based lipid systems and the requirements for enhancement of their biological activity. The novel fact that lipophilic OAKs can drive cochleate formation in the absence of Ca++ with a variety of phospholipid mixtures results in structures which can be exploited for drug delivery and permits their synergistic use with antibiotics to increase efficacy and reduce toxicity. Knowledge of the biophysical properties of these assemblies allowed us to optimize their effectiveness in combating bacterial multidrug resistance.

Structural Stability of Liposomes Modulated by Divalent Cations and an Amyloid

Liposomes, owing to their unique biocompatibility and biosimilarity, are increasingly used as drug delivery systems and biomembrane models. Divalent cations and proteins have been found to affect the physical-chemical properties of lipid bilayers, but the exact molecular mechanisms and the implications of such interactions, particularly in the case of liposomes, are largely unknown. In the present work we investigated the effect of divalent cations and amyloid on surface-adsorbed liposomes. Extruded DPPC vesicles with an average diameter of 110 nm were sedimented onto mica surface, and imaged with non-contact-mode atomic force microscopy in aqueous buffer. Preparing the vesicles in buffer containing 0.2, 0.4, 0.8, 20, 40, 80 mM MgCl2 or CaCl2 preserved their integrity. Furthermore, the mere addition of MgCl2 or CaCl2 at the above concentrations to the external medium also resulted in preserved vesicles. Slightly flattened liposomes with heights ranging between 30 and 80 nm were observed on the surface, but ruptured, completely flattened vesicles were unseen. In the absence of divalent cations we observed the coalescence of flat, micrometer-size patches of lipid bilayers. If divalent cations were removed from the external medium by size exclusion chromatography, vesicle-integrity dramatically decreased. To test the effect of amyloid on liposome structure, transthyretin sample containing a variety of aggregates were added to surface-adsorbed vesicles. Upon the addition of transthyretin, liposomes immediately ruptured and flattened. Our results demonstrate that surface-adsorbed liposomes stabilized with divalent cations represent a unique experimental system that may provide novel insights into the mechanisms of membrane-protein interactions.

NMR Solution Structure and Biophysical Characterization of Vibrio harveyi Acyl Carrier Protein A75H: EFFECTS OF DIVALENT METAL IONS

Bacterial acyl carrier protein (ACP) is a highly anionic, 9 kDa protein that functions as a cofactor protein in fatty acid biosynthesis. Escherichia coli ACP is folded at neutral pH and in the absence of divalent cations, while Vibrio harveyi ACP, which is very similar at 86% sequence identity, is unfolded under the same conditions. V. harveyi ACP adopts a folded conformation upon the addition of divalent cations such as Ca(2+) and Mg(2+) and a mutant, A75H, was previously identified that restores the folded conformation at pH 7 in the absence of divalent cations. In this study we sought to understand the unique folding behavior of V. harveyi ACP using NMR spectroscopy and biophysical methods. The NMR solution structure of V. harveyi ACP A75H displays the canonical ACP structure with four helices surrounding a hydrophobic core, with a narrow pocket closed off from the solvent to house the acyl chain. His-75, which is charged at neutral pH, participates in a stacking interaction with Tyr-71 in the far C-terminal end of helix IV. pH titrations and the electrostatic profile of ACP suggest that V. harveyi ACP is destabilized by anionic charge repulsion around helix II that can be partially neutralized by His-75 and is further reduced by divalent cation binding. This is supported by differential scanning calorimetry data which indicate that calcium binding further increases the melting temperature of V. harveyi ACP A75H by ∼20 °C. Divalent cation binding does not alter ACP dynamics on the ps-ns timescale as determined by (15)N NMR relaxation experiments, however, it clearly stabilizes the protein fold as observed by hydrogen-deuterium exchange studies. Finally, we demonstrate that the E. coli ACP H75A mutant is similarly unfolded as wild-type V. harveyi ACP, further stressing the importance of this particular residue for proper protein folding.

Technology Focus: EOR Performance and Modeling (January 2010)

Technology Focus The demand for oil continues to grow, and it is forecasted that oil will continue to dominate the world share of energy supply for the next 3 decades. Accordingly, new discoveries tend to decrease on an annual base, and enhanced oil recovery (EOR) has the potential to reclassify unrecoverable and contingent reserves. EOR is viable because there are many available mature technologies, its implementation usually does not require drilling and completion cost, and most of the existing infrastructure can be used. When I selected the EOR papers to highlight this year, I classified the papers into the categories of gas injection, chemical methods, thermal methods, conformance control, microbial EOR, carbonate-reservoir EOR, reservoir-problem-identification technologies, and novel EOR methods including low-salinity waterflooding, imbibition EOR, alkaline/surfactant/gas injection, and CO2-miscible combined with alkaline/surfactant/polymer injection. Not surprisingly, CO2 EOR and polymer floods have drawn great interest this last year because CO2 sequestration has been a great interest to the community and polymer flooding is a proven cost-effective chemical-EOR method. The published polymer papers cover mainly the topics of thermal-stability evaluation of different polymers, novel polymer-flooding modeling, polymer-rheology database, and the formation effects on polymer properties. The CO2 publications discussed mainly the feasibility-evaluation results of CO2-EOR projects in different oil fields worldwide. An EOR project is not just a matter of injecting materials, with many factors affecting whether the project is successful. The design and implementation of an EOR project require a systematic integration from a multidisciplinary team. It is critically important to identify the problems of mature oil fields for a successful EOR project, and, thus, the papers to identify channels in mature oil fields were included in this EOR feature. EOR Performance and Modeling additional reading available at OnePetro: www.onepetro.org SPE 121460 • “Stability of Partially Hydrolyzed Polyacrylamides at Elevated Temperatures in the Absence of Divalent Cations” by R.S. Seright, SPE, New Mexico Institute of Mining and Technology, et al. SPE 123971 • “Streamline-Based Simulation of Non-Newtonian-Polymer Flooding” by Abdulkareem M. AlSofi, SPE, Imperial College—London, et al. SPE 123930 • “Identification and Characterization of High-Conductive Layers in Waterfloods” by C.S. Kabir, SPE, Hess, et al. SPE 124930 • “Mechanistic Modeling of Emulsion Formation and Heat Transfer During the Steam-Assisted Gravity-Drainage (SAGD) Process” by Prince N. Azom, University of Texas at Austin, et al. SPE 124946 • “Pore-Level Mechanics of Forced and Spontaneous Imbibition of Aqueous Surfactant Solutions in Fractured Porous Media” by Amar J. Alshehri, SPE, Stanford University, et al.

Stability of Partially Hydrolyzed Polyacrylamides at Elevated Temperatures in the Absence of Divalent Cations

Summary At elevated temperatures in aqueous solution, partially hydrolyzed polyacrylamides (HPAMs) experience hydrolysis of amide side groups. However, in the absence of dissolved oxygen and divalent cations, the polymer backbone can remain stable so that HPAM solutions were projected to maintain at least half their original viscosity for more than 8 years at 100°C and for approximately 2 years at 120°C. Within our experimental error, HPAM stability was the same with and without oil (decane). An acrylamide-AMPS copolymer [with 25% 2-acrylamido-2-methylpropane sulphonic acid (AMPS)] showed similar stability to that for HPAM. Stability results were similar in brines with 0.3% NaCl, 3% NaCl, or 0.2% NaCl plus 0.1% NaHCO3. At temperatures of 160°C and greater, the polymers were more stable in brine with 2% NaCl plus 1% NaHCO3 than in the other brines. Even though no chemical oxygen scavengers or antioxidants were used in our study, we observed the highest level of thermal stability reported to date for these polymers. Our results provide considerable hope for the use of HPAM polymers in enhanced oil recovery (EOR) at temperatures up to 120°C if contact with dissolved oxygen and divalent cations can be minimized. Calculations performed considering oxygen reaction with oil and pyrite revealed that dissolved oxygen will be removed quickly from injected waters and will not propagate very far into porous reservoir rock. These findings have two positive implications with respect to polymer floods in high-temperature reservoirs. First, dissolved oxygen that entered the reservoir before polymer injection will have been consumed and will not aggravate polymer degradation. Second, if an oxygen leak (in the surface facilities or piping) develops during the course of polymer injection, that oxygen will not compromise the stability of the polymer that was injected before the leak developed or the polymer that is injected after the leak is fixed. Of course, the polymer that is injected while the leak is active will be susceptible to oxidative degradation. Maintaining dissolved oxygen at undetectable levels is necessary to maximize polymer stability. This can be accomplished readily without the use of chemical oxygen scavengers or antioxidants.

Purification and biochemical characterization of a D-galactose binding lectin from Japanese sea hare (Aplysia kurodai) eggs

A lectin was purified from Japanese sea hare Aplysia kurodai by lactosyl-agarose affinity chromatography. The molecular mass of the lectin was determined to be 56 and 32 kDa by SDS-PAGE under non-reducing and reducing conditions, respectively. It was found to agglutinate trypsinized and glutaraldehyde-fixed rabbit and human erythrocytes in the absence of divalent cations. The lectin exhibited stable thermo-tolerance as it retained hemagglutinating activity for 1 h even at 80 degrees C and showed stability at pH 10. By contrast, it was very sensitive at pH less than 5 and in the presence of the sulfhydryl-group preserving reagent, beta-mercaptoethanol. The hemagglutinating activity by the lectin was specifically inhibited by D-galactose, galacturonic acid, methyl-alpha- and methyl-beta-D-galactopyranoside, lactose, melibiose, and asialofetuin. The association rate constant (k(ass)) and dissociation rate constant (k(diss)) were determined for the lectin to be 4.3 x 10(5) M(-1) x sec(-1) and 2.2 x 10(-3) sec(-1), respectively, using a surface plasmon resonance biosensor. The lectin moderately inhibited cell proliferation in the P388 cell line dose dependently. Interestingly, lectin-treated cells did not show a fragmented DNA ladder as is caused by apoptosis, suggesting that the cell proliferation inhibition was caused by another unknown mechanism.

Properties of Orai1 mediated store‐operated current depend on the expression levels of STIM1 and Orai1 proteins

Two cellular proteins, stromal interaction molecule 1 (STIM1) and Orai1, are recently discovered essential components of the Ca2+ release activated Ca2+ (CRAC) channel. Orai1 polypeptides form the pore of the CRAC channel, while STIM1 plays the role of the endoplasmic reticulum Ca2+ sensor required for activation of CRAC current (I(CRAC)) by store depletion. It is not known, however, if the role of STIM1 is limited exclusively to Ca2+ sensing, or whether interaction between Orai1 and STIM1, either direct or indirect, also defines the properties of I(CRAC). In this study we investigated how the relative expression levels of ectopic Orai1 and STIM1 affect the properties of I(CRAC). The results show that cells expressing low Orai1 : STIM1 ratios produce I(CRAC) with strong fast Ca2+-dependent inactivation, while cells expressing high Orai1 : STIM1 ratios produce I(CRAC) with strong activation at negative potentials. Moreover, the expression ratio of Orai1 and STIM1 affects Ca2+, Ba2+ and Sr2+ conductance, but has no effect on the current in the absence of divalent cations. The results suggest that several key properties of Ca2+ channels formed by Orai1 depend on its interaction with STIM1, and that the stoichiometry of this interaction may vary depending on the relative expression levels of these proteins.

Harmony and Discord in Endothelial Calcium Entry

To The Editor: This letter addresses the recent report by Abdullaev et al.1 In 2006, the seminal work of Feske and colleagues2 identified membrane proteins that were named Orais, after the Greek keepers of Heaven’s gate: Harmony (Orai1), Justice (Orai2), and Peace (Orai3). The proteins emerged through a study of severe combined immune deficiency (SCID), which is caused by a defect in Ca2+ entry of T lymphocytes. A mutation in the Orai1 gene underlies the defect. There has been particular interest in Orais because of their relationship with the widely occurring store-operated Ca2+-entry (SOCE) phenomena, which reflect Ca2+-permeable plasma membrane mechanisms that respond to the depletion status of endoplasmic reticular Ca2+ stores. In lymphocytes, the store-operated mechanism is commonly observed as a Ca2+-selective and inwardly rectifying current, I CRAC. The mechanism is defective in SCID and rescued by expression of wild-type Orai1.2 In predicted structure Orais belong to the tetraspanin protein family3 rather than resembling known ion channels but mutations in Orais modify ion-selectivity, suggesting Orais generate ion permeation.4 The search by Abdullaev et al1 for I CRAC in cultured endothelial cells adds to an expanding view that the importance of Orai1 and I CRAC is not restricted to the immune system. A striking feature of I CRAC is its small amplitude, well below the size of most other whole-cell currents and close to the resolving power of whole-cell patch clamp. Abdullaev et al1 are to be commended for persisting when the current turned out to be at least 5 times smaller than the current of immune cells. Unfortunately, the current could only be convincingly shown in the absence of divalent cations, leaving open the possibility that Ca2+ shuts it down and, strictly, …

Structural and functional studies on a mesophilic stationary phase survival protein (Sur E) from Salmonella typhimurium

SurE, the stationary-phase survival protein of Salmonella typhimurium, forms part of a stress survival operon regulated by the stationary-phase RNA polymerase alternative sigma factor. SurE is known to improve bacterial viability during stress conditions. It functions as a phosphatase specific to nucleoside monophosphates. In the present study we reported the X-ray crystal structure of SurE from Salmonella typhimurium. The protein crystallized in two forms: orthorhombic F222; and monoclinic C2. The two structures were determined to resolutions of 1.7 and 2.7 A, respectively. The protein exists as a domain-swapped dimer. The residue D230 is involved in several interactions that are probably crucial for domain swapping. A divalent metal ion is found at the active site of the enzyme, which is consistent with the divalent metal ion-dependent activity of the enzyme. Interactions of the conserved DD motif present at the N-terminus with the phosphate and the Mg(2+) present in the active site suggest that these residues play an important role in enzyme activity. The divalent metal ion specificity and the kinetic constants of SurE were determined using the generic phosphatase substrate para-nitrophenyl phosphate. The enzyme was inactive in the absence of divalent cations and was most active in the presence of Mg(2+). Thermal denaturation studies showed that S. typhimurium SurE is much less stable than its homologues and an attempt was made to understand the molecular basis of the lower thermal stability based on solvation free-energy. This is the first detailed crystal structure analysis of SurE from a mesophilic organism.

Cap accessibility correlates with the initiation efficiency of alfalfa mosaic virus RNAs.

The rate of cap removal from the alfalfa mosaic virus (A1MV) RNAs with tobacco acid pyrophosphatase (TAP) depends on the RNA species. At 37 degrees C and in the absence of divalent cation, RNA 3 reacts more slowly than the other three, which are decapped at similar rates. In the presence of magnesium, at 25 degrees C, TAP also discriminates against RNA 1. Thus the order of reactivity with TAP largely mimics the hierarchy of initiation efficiencies of the A1MV RNAs (Godefroy-Colburn et al., preceding paper in this journal). Our interpretation of these findings is that cap accessibility is what limits the rate of reaction with initiation factors as well as with TAP. In this hypothesis, translational discrimination between naturally capped messages would be related to the rate of 'breathing' of their 5' ends.

Hydrolysis of Phosphatidylinositol 4,5-Bisphosphate Mediates Calcium-induced Inactivation of TRPV6 Channels

TRPV6 is a member of the transient receptor potential superfamily of ion channels that facilitates Ca(2+) absorption in the intestines. These channels display high selectivity for Ca(2+), but in the absence of divalent cations they also conduct monovalent ions. TRPV6 channels have been shown to be inactivated by increased cytoplasmic Ca(2+) concentrations. Here we studied the mechanism of this Ca(2+)-induced inactivation. Monovalent currents through TRPV6 substantially decreased after a 40-s application of Ca(2+), but not Ba(2+). We also show that Ca(2+), but not Ba(2+), influx via TRPV6 induces depletion of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2) or PIP(2)) and the formation of inositol 1,4,5-trisphosphate. Dialysis of DiC(8) PI(4,5)P(2) through the patch pipette inhibited Ca(2+)-dependent inactivation of TRPV6 currents in whole-cell patch clamp experiments. PI(4,5)P(2) also activated TRPV6 currents in excised patches. PI(4)P, the precursor of PI(4,5)P(2), neither activated TRPV6 in excised patches nor had any effect on Ca(2+)-induced inactivation in whole-cell experiments. Conversion of PI(4,5)P(2) to PI(4)P by a rapamycin-inducible PI(4,5)P(2) 5-phosphatase inhibited TRPV6 currents in whole-cell experiments. Inhibiting phosphatidylinositol 4 kinases with wortmannin decreased TRPV6 currents and Ca(2+) entry into TRPV6-expressing cells. We propose that Ca(2+) influx through TRPV6 activates phospholipase C and the resulting depletion of PI(4,5)P(2) contributes to the inactivation of TRPV6.

Isolation, purification, characterization and glycan-binding profile of a d-galactoside specific lectin from the marine sponge, Halichondria okadai

A lectin recognizing both Galβ1-3GlcNAc and Galβ1-4GlcNAc was purified from the demosponge Halichondria okadai by lactosyl-agarose affinity chromatography. The molecular mass of the lectin was determined to be 30 kDa by SDS-PAGE under reducing and non-reducing conditions and 60 kDa by gel permeation chromatography. The p I value of the lectin was 6.7. It was found to agglutinate trypsinized and glutaraldehyde-fixed rabbit and human erythrocytes in the presence and absence of divalent cations. The hemagglutinating activity by the lectin was inhibited by d-galactose, methyl- d-galactopyranoside, N-acetyl- d-galactosamine, methyl- N-acetyl- d-galactosaminide, lactose, melibiose, and asialofetuin. The K d of the lectin against p-nitrophenyl-β-lactoside was determined to be 2.76 × 10 − 5 M and its glycan-binding profile given by frontal affinity chromatography was shown to be similar to many other known galectins. Partial primary structure analysis of 7 peptides by cleavage with lysyl endopeptidase indicated that one of the peptides showed significant similarity with galectin purified from the sponge Geodia cydonium.

Novel Conantokins from Conus parius Venom Are Specific Antagonists of N-Methyl-D-aspartate Receptors

We report the discovery and characterization of three conantokin peptides from the venom of Conus parius. Each peptide (conantokin-Pr1, -Pr2, and -Pr3) contains 19 amino acids with three gamma-carboxyglutamate (Gla) residues, a post-translationally modified amino acid characteristic of conantokins. The new peptides contain several amino acid residues that differ from previous conantokin consensus sequences. Notably, the new conantokins lack Gla at the 3rd position from the N terminus, where the Gla residue is replaced by either aspartate or by another post-translationally modified residue, 4-trans-hydroxyproline. Conantokin-Pr3 is the first conantokin peptide to have three different post-translational modifications. Conantokins-Pr1 and -Pr2 adopt alpha-helical conformations in the presence of divalent cations (Mg2+ and Ca2+) but are generally unstructured in the absence of divalent cations. Conantokin-Pr3 adopts an alpha-helical conformation even in the absence of divalent cations. Like other conantokins, the new peptides induced sleep in young mice and hyperactivity in older mice upon intracranial injection. Electrophysiological assays confirmed that conantokins-Pr1, -Pr2, and -Pr3 are N-methyl-d-aspartate (NMDA) receptor antagonists, with highest potency for NR2B-containing NMDA receptors. Conantokin-Pr3 demonstrated approximately 10-fold selectivity for NR2B-containing NMDA receptors. However, conantokin-Pr2 showed minimal differences in potency between NR2B and NR2D. Conantokins-Pr1, -Pr2, and -Pr3 all demonstrated high specificity of block for NMDA receptors, when tested against various ligand-gated ion channels. Conus parius conantokins allow for a better definition of structural and functional features of conantokins as ligands targeting NMDA receptors.

Polymorphic Assemblies and Crystalline Arrays of Lens Tetraspanin MP20

Members of the tetraspanin superfamily function as transmembrane scaffold proteins that mediate the assembly of membrane proteins into specific signaling complexes. Tetraspanins also interact with each other and concentrate membrane proteins into tetraspanin-enriched microdomains (TEMs). Here we report that lens-specific tetraspanin MP20 can form multiple types of higher-order assemblies and we present crystalline arrays of MP20. When isolated in the absence of divalent cations, MP20 is solubilized predominantly in tetrameric form, whereas the presence of divalent cations during solubilization promotes the association of MP20 tetramers into higher-order species. This effect only occurs when divalent cations are present during solubilization but not when divalent cations are added to solubilized tetrameric MP20, suggesting that other factors may also be involved. When purified MP20 tetramers are reconstituted with native lens lipids in the presence of magnesium, MP20 forms two-dimensional (2D) crystals. A projection map at 18 Å resolution calculated from negatively stained 2D crystals showed that the building block of the crystal is an octamer consisting of two tetramers related to each other by 2-fold symmetry. In addition to 2D crystals, reconstitution of MP20 with native lipids also produced a variety of large protein–lipid complexes, and we present three-dimensional (3D) reconstructions of the four most abundant of these complexes in negative stain. The various complexes formed by MP20 most likely reflect the many ways in which tetraspanins can interact with each other to allow formation of TEMs.

SECRETED PHOSPHOPROTEIN 1 ISOLATED FROM UTERINE SECRETIONS PROMOTES INTEGRIN-MEDIATED ADHESION OF PORCINE TROPHECTODERM CELLS

In domestic pigs, over two-thirds of reproductive losses occur during the first 25 days of gestation, a period during which rapid conceptus development, trophoblast adhesion and implantation are initiated. Successful conceptus implantation requires pregnancy-specific alterations in extracellular matrix (ECM) at the conceptus-maternal interface. The pig luminal epithelium (LE) immediately adjacent to the implanting conceptus secretes SPP1 (also known as osteopontin), an ECM molecule involved in cell-cell and cell-ECM communication that is believed to mediate conceptus-uterus interactions. We hypothesized that SPP1 expressed by the uterine LE bridges to integrin receptors on conceptus trophectoderm to stimulate adhesion at the conceptus-maternal interface. The objective of this study was to determine if SPP1 isolated from uterine secretions (uterine milk) supports trophectoderm cell adhesion in vitro. To accomplish this, we employed a unilaterally pregnant sheep model, where the ovary ipsilateral to the right uterine horn was removed and a double ligature placed at the base of the right uterine horn at the bifurcation. At the following estrus, sheep were mated to generate gravid and nongravid uterine horns, hysterectomized on day 120 and the uterine milk within nongravid horns collected. SPP1 was purified from uterine milk using a DEAE-Sephacel column (ion exchange chromatography) followed by dual phenyl-Sepharose columns (hydrophobic chromatography). A 45 kDa fragment, previously identified at the apical surface of the sheep LE during pregnancy, and a 25 kDa fragment were isolated. The spontaneously immortalized pig trophectoderm cell line pTr2 was allowed to attach to serial dilutions (20 μg/ml to 40 ng/ml) of the following proteins: bovine milk SPP1 (bSPP1; purified in the manner described above), ovine uterine milk SPP1 (oSPP1), recombinant rat SPP1 containing the RGD cellbinding sequence (rSPP1-RGD), recombinant rat SPP1 containing an RAD sequence in place of the RGD sequence (rSPP1-RAD), full-length human fibronectin (FN; positive control) and BSA (negative control). Dose-dependent cell adhesion was observed for bSPP1, oSPP1 and rSPP1- RGD, all of which supported higher levels of adhesion than FN at concentrations ranging from 0.15 and 1.25 μg/ml. Only at concentrations of 2.5 μg/ml and higher did FN support greater pTr2 adhesion than the various SPP1 proteins. Cell adhesion was not supported by rSPP1-RAD over BSA controls at any concentration, strongly suggesting that adhesion to the various SPP1 proteins and FN occurred through RGD-binding integrin receptors. Integrins require the presence of divalent cations to allow ligand binding. This property was used to further confirm that pTr2 cell adhesion to SPP1 was integrin-mediated. Cells were allowed to attach in the presence of varying levels of divalent cations: 1) no cations; 2) 1 mM Ca2+ + 2 mM Mg2+; 3) 2 mM Ca2+ + 1 mM Mg2+; or 4) 0.1 mM Mn2+. No attachment was observed in the absence of divalent cations, confirming that porcine trophectoderm attachment to SPP1 is integrin-mediated. This study is the first to demonstrate that a native form of uterine SPP1, secreted by the uterine glandular epithelium and present at the conceptus-maternal interface, can promote dose- and cation-dependent integrin-mediated adhesion of pig trophectoderm cells. (Supported by NRI USDA Grant 2006-35203-17199). (platform)

Ionic Requirements for Membrane-Glass Adhesion and Giga Seal Formation in Patch-Clamp Recording

Patch-clamp recording has revolutionized the study of ion channels, transporters, and the electrical activity of small cells. Vital to this method is formation of a tight seal between glass recording pipette and cell membrane. To better understand seal formation and improve practical application of this technique, we examine the effects of divalent ions, protons, ionic strength, and membrane proteins on adhesion of membrane to glass and on seal resistance using both patch-clamp recording and atomic force microscopy. We find that H +, Ca 2+, and Mg 2+ increase adhesion force between glass and membrane (lipid and cellular), decrease the time required to form a tight seal, and increase seal resistance. In the absence of H + (10 −10 M) and divalent cations (<10 −8 M), adhesion forces are greatly reduced and tight seals are not formed. H + (10 −7 M) promotes seal formation in the absence of divalent cations. A positive correlation between adhesion force and seal formation indicates that high resistance seals are associated with increased adhesion between membrane and glass. A similar ionic dependence of the adhesion of lipid membranes and cell membranes to glass indicates that lipid membranes without proteins are sufficient for the action of ions on adhesion.

Insight into nucleotide and Ca2+binding to carp ?-actin

Nucleotides and Ca2+ binding to a-actin prepared from ordinary skeletal muscle of carp Cyprinus carpio was studied. When bound Ca2+ was removed with ethylenediaminetetraacetic acid, carp a-actin denatured more rapidly than chicken a-actin. Kinetic studies of the denaturation process showed that in the absence of divalent cations, the binding constants of ATP to carp and chicken actin were 5.0 ¥ 10/M and 1.2 ¥ 10/M, respectively. Competitive binding of Ca2+ between actin and 8-amino-2-[(2-amino-5-methylphenoxy)methyl]-6-methoxyquinoline-N,N,N′,N′-tetraacetic acid (Quin 2) showed that affinity of Ca2+ for carp actin was also lower than that for chicken actin by a factor of 1.6. These results indicated that carp actin could relatively easily denature due to the low affinities of these ligands. Enthalpy changes upon ATP binding to carp and chicken actin were -65 kJ/mol and -110 kJ/mol, respectively. Thermodynamic analyses of our results revealed that the entropy change associated with ATP binding to carp actin was significantly smaller than that to chicken actin, suggesting that structural stabilization upon ATP binding was less effective in carp actin.