Isotonic Buffer Research Articles

Mitochondria isolated in nearly isotonic KCl buffer: Focus on cardiolipin and organelle morphology

Rat liver mitochondria were isolated in parallel in two different isolation buffers: a standard buffer containing mannitol/sucrose and a nearly physiological KCl based solution. The two different organelle preparations were comparatively characterized by respiratory activity, heme content, microsomal and Golgi contamination, electron microscopy and lipid analyses. The substitution of saccharides with KCl in the isolation buffer does not induce the formation of mitoplasts or disruption of mitochondria. Mitochondria isolated in KCl buffer are coupled and able to maintain a stable transmembrane charge separation. A number of biochemical and functional differences between the two organelle preparations are described; in particular KCl mitochondria exhibit lower cardiolipin content and smaller intracristal compartments in comparison with the standard mitochondrial preparation.

Large T Antigen Promotes JC Virus Replication in G2-arrested Cells by Inducing ATM- and ATR-mediated G2 Checkpoint Signaling

Large T antigen (TAg) of the human polyomavirus JC virus (JCV) possesses DNA binding and helicase activities, which, together with various cellular proteins, are required for replication of the viral genome. We now show that JCV-infected cells expressing TAg accumulate in the G(2) phase of the cell cycle as a result of the activation of ATM- and ATR-mediated G(2) checkpoint pathways. Transient transfection of cells with a TAg expression vector also induced G(2) checkpoint signaling and G(2) arrest. Analysis of TAg mutants with different subnuclear localizations suggested that the association of TAg with cellular DNA contributes to the induction of G(2) arrest. Abrogation of G(2) arrest by inhibition of ATM and ATR, Chk1, and Wee1 suppressed JCV genome replication. In addition, abrogation of the G(2)-M transition by Cdc2 depletion disabled Wee1 depletion-induced suppression of JCV genome replication, suggesting that JCV replication is facilitated by G(2) arrest resulting from G(2) checkpoint signaling. Moreover, inhibition of ATM and ATR by caffeine suppressed JCV production. The observation that oligodendrocytes productively infected with JCV in vivo also undergo G(2) arrest suggests that G(2) checkpoint inhibitors such as caffeine are potential therapeutic agents for JCV infection.

Transdermal drug delivery of labetalol hydrochloride: Feasibility and effect of penetration enhancers

Objectives:The objective of this study is to investigate the feasibility of transdermal drug delivery of Labetalol Hydrochloride (LHCl) and to study the effect of different penetration enhancers on the skin permeability of LHCl.Methods:The permeability experiments were conducted using a horizontal glass diffusion cell with a diffusional area of 2.37 cm-2 on albino rat skin. The effect of various penetration enhancers namely turpentine oil, dimethyl formamide (DMF), menthol, dimethyl sulfoxide, pine oil, and 2-pyrollidone, and the effect of the concentration of drug and enhancer in the donor phase on the skin permeability of LHCl was studied.Results:The apparent partition coefficient of the drug was found to be 6.95, suggesting it to be a lipophilic drug. The preliminary skin permeation studies revealed that the permeation of LHCL through albino rat skin was moderate (Kp = 6.490 × 10-2 cm hr-1) from isotonic phosphate buffer of pH 7.4. An appreciable increase in the LHCl permeability coefficient was observed on using a co-solvent (ethanol 95%) with the penetration enhancers in the donor phase. DMSO (10% v/v) was found to be the most effective enhancer for Labetalol hydrochloride (Enhancement Factor = 1.165). An increase in the concentration of drug and enhancer in the donor cell accentuated the permeability coefficient of LHCl.Conclusions:It was concluded that LHCl could be delivered via the dermal route with the use of 10% DMSO as the penetration enhancer.

Lysophospholipids enhance taurine release from rat retinal vascular endothelial cells under hypoosmotic stress

Sphingosine 1-phosphate (S1P) and lysophosphatidic acid (LPA) are simple bioactive lysophospholipids which exhibit an effect on blood vessels via their G protein-coupled receptors. The purpose of this study was (i) to clarify the impact of S1P and LPA on the release of taurine as an organic osmolyte from rat retinal vascular endothelial cells (RVECs) under hypoosmotic stress and (ii) to quantify the gene expression levels of S1P and LPA receptors in RVECs. When cultured RVECs (TR-iBRB2 cells) that had been preloaded with [ 3H]taurine were exposed to hypotonic buffer (230 mOsm) for 1 to 10 min, [ 3H]taurine release from the cells was several times greater than that using an isotonic buffer (300 mOsm). S1P and LPA significantly enhanced the [ 3H]taurine release under hypotonic conditions in a time- and concentration-dependent manner, whereas S1P and LPA had no significant effect under isotonic conditions. Quantitative real-time PCR revealed that freshly isolated RVECs predominantly express mRNAs for S1P 1, S1P 4 and LPA 4. The S1P-enhanced [ 3H]taurine release under hypoosmotic conditions was significantly inhibited by an S1P 1 receptor antagonist. Inhibitor of the small GTPase Rho, C3 exotoxin, attenuated S1P- and LPA-enhanced [ 3H]taurine release. These results suggest that S1P and LPA play a novel role in the regulation of osmolyte efflux from RVECs in response to hypoosmotic stress via the activation of their specific receptors.

Phosphorylation of α6-Tubulin by Protein Kinase Cα Activates Motility of Human Breast Cells

Engineered overexpression of protein kinase Calpha (PKCalpha) was previously shown to endow nonmotile MCF-10A human breast cells with aggressive motility. A traceable mutant of PKCalpha (Abeyweera, T. P., and Rotenberg, S. A. (2007) Biochemistry 46, 2364-2370) revealed that alpha6-tubulin is phosphorylated in cells expressing traceable PKCalpha and in vitro by wild type PKCalpha. Gain-of-function, single site mutations (Ser-->Asp) were constructed at each PKC consensus site in alpha6-tubulin (Ser158, Ser165, Ser241, and Thr337) to simulate phosphorylation. Following expression of each construct in MCF-10A cells, motility assays identified Ser165 as the only site in alpha6-tubulin whose pseudophosphorylation reproduced the motile behavior engendered by PKCalpha. Expression of a phosphorylation-resistant mutant (S165N-alpha6-tubulin) resulted in suppression of MCF-10A cell motility stimulated either by expression of PKCalpha or by treatment with PKCalpha-selective activator diacylglycerol-lactone. MCF-10A cells treated with diacylglycerol-lactone showed strong phosphorylation of endogenous alpha-tubulin that could be blocked when S165N-alpha6-tubulin was expressed. The S165N mutant also inhibited intrinsically motile human breast tumor cells that express high endogenous PKCalpha levels (MDA-MB-231 cells) or lack PKCalpha and other conventional isoforms (MDA-MB-468 cells). Comparison of Myc-tagged wild type alpha6-tubulin and S165N-alpha6-tubulin expressed in MDA-MB-468 cells demonstrated that Ser165 is also a major site of phosphorylation for endogenously active, nonconventional PKC isoforms. PKC-stimulated motility of MCF-10A cells was nocodazole-sensitive, thereby implicating microtubule elongation in the mechanism. These findings support a model in which PKC phosphorylates alpha-tubulin at Ser165, leading to microtubule elongation and motility.

Radioactive Labeling of Defined HPMA-Based Polymeric Structures Using [18F]FETos for In Vivo Imaging by Positron Emission Tomography

During the last decades polymer-based nanomedicine has turned out to be a promising tool in modern pharmaceutics. The following article describes the synthesis of well-defined random and block copolymers by RAFT polymerization with potential medical application. The polymers have been labeled with the positron-emitting nuclide fluorine-18. The polymeric structures are based on the biocompatible N-(2-hydroxypropyl)-methacrylamide (HPMA). To achieve these structures, functional reactive ester polymers with a molecular weight within the range of 25,000-110,000 g/mol were aminolyzed by 2-hydroxypropylamine and tyramine (3%) to form (18)F-labelable HPMA-polymer precursors. The labeling procedure of the phenolic tyramine moieties via the secondary labeling synthon 2-[(18)F]fluoroethyl-1-tosylate ([(18)F]FETos) provided radiochemical fluoroalkylation yields of ∼80% for block copolymers and >50% for random polymer architectures within a synthesis time of 10 min and a reaction temperature of 120 °C. Total synthesis time including synthon synthesis, (18)F-labeling, and final purification via size exclusion chromatography took less than 90 min and yielded stable (18)F-labeled HPMA structures in isotonic buffer solution. Any decomposition could be detected within 2 h. To determine the in vivo fate of (18)F-labeled HPMA polymers, preliminary small animal positron emission tomography (PET) experiments were performed in healthy rats, demonstrating the renal clearance of low molecular weight polymers. Furthermore, low metabolism rates could be detected in urine as well as in the blood. Thus, we expect this new strategy for radioactive labeling of polymers as a promising approach for in vivo PET studies.

Development of a novel HPLC‐MS/MS method for the determination of aconitine and its application to in vitro and rat microdialysis samples

A sensitive and selective LC-MS/MS method was developed and validated for the determination of aconitine in microdialysate and rat plasma. Extraction of plasma sample was conducted by use of 1% trichloracetic acid and acetonitrile solution with 10 ng/mL internal standard (propafenone) spiked. Microdialysates were analyzed without sample purification. After sample preparation, 2 microL were injected and separated with an isocratic mobile phase consisting of acetonitrile:0.1% formic acid (60:40, v/v) at a flow rate of 0.3 mL/min. The Agilent G6410A triple quadrupole LC/MS system was operated under the multiple-reaction monitoring mode (MRM) using the electrospray ionization technique in positive mode. Overall, the assay exhibited good precision and accuracy. The diffusion properties of aconitine investigated in in vitro microdialysis experiments revealed unfavourable concentration dependence avertable by keeping a constant pH 5.77 using isotonic phosphate buffer solution as perfusate. The mean relative recoveries were 48.23% [coefficient of variation (CV 4.47%)] and 55.38% (CV 2.89%) for retrodialysis and recovery experiments, respectively. The in vivo recovery of aconitine was 34.48% (CV 3.05%) and was stable over the 6 h study period. Following characterization of aconitine both in vitro and in vivo microdialysis, the developed setting is suitable for application in pharmacokinetics and pharmacodynamics studies.

Effect of phosphate salts concentrations, supporting electrolytes, and calcium phosphate salt precipitation on the pH of phosphate buffer solutions

A monobasic and a dibasic phosphate salt are usually added to water to form a standard phosphate buffer solution. The solution pH depends primarily on the concentration ratio R of the two salts, and secondarily on their concentrations and on the concentrations of the supporting electrolytes such as NaCl or KCl. Various ideal and non-ideal solution thermodynamic models are presented. The ideal solution model predicts that the pH should depend only on R, and underestimates the pH at physiological osmolarities by 0.3–0.5 pH units at both 298 K and 310 K, depending on the phosphate, NaCl, and KCl concentrations. Model predictions using the non-ideal extended Debye–Hückel (D–H) equation agree with the data up to ca. ±0.1 pH units at 298 K and 310 K. The pH predictions for the standard isotonic phosphate buffer saline, containing 2 mM of monobasic and 8 M of dibasic sodium phosphate, 137 mM of NaCl, and 2.7 mM of KCl agree well with the data. When CaCl 2 is added, up to 3 mM, a phosphate salt often precipitates, affecting the free Ca 2+ ion concentration, the phosphate ion concentrations, and the pH. The conditions for the formation at equilibrium of hydroxyapatite (Ca 5(PO 4) 3(OH)), or dicalcium phosphate dehydrate (CaHPO 4·2H 2O), or tricalcium phosphate (Ca 3(PO 4) 2), are predicted. The results are important in determining and predicting electrolyte concentrations for achieving desirable pH values while avoiding precipitation for various biological or biomedical applications.

Cell size dynamics and viability of cells exposed to hypotonic treatment and electroporation for electrofusion optimization

Background. Various electrofusion parameters have to be adjusted to obtain the optimal electrofusion ef- ficiency. Based on published data, good electrofusion conditions can be achieved with the hypotonic treat- ment. However, the duration of the hypotonic treatment before electroporation and buffer hypoosmolarity have to be adjusted in order to cause cell swelling, to avoid regulatory volume decrease and to preserve cell viability. The aims of our study were to determine cell size dynamics and viability of four different cell lines in hypotonic buffer and to study the influence of the electroporation on the selected cell line in hypotonic buffer. Materials and methods. Cell size dynamics of different cell lines exposed to hypotonic buffer and elec- troporation were analyzed by time-resolved cell size measurements. The viability of hypotonically treated or/and electroporated cells was determined 24 h after the experiment by a modified crystal violet (CV) viability assay. Results. In our experimental conditions the hypotonic treatment at 100 mOsm was efficient for CHO, V79 and B16-F1 cell lines. The optimal duration of the treatment was between two and five minutes. On the other hand the same hypotonic treatment did not cause cell swelling of NS1 cells. Cell swelling was also observed after electroporation of B16-F1 in isotonic buffer and it was amplified when hypotonic buffer was used. In addition, the regulatory volume decrease was successfully inhibited with electroporation. Conclusions. Cell size dynamics in hypotonic conditions should be studied for each cell line since they differ in their sensitivity to the hypotonic treatment. The inhibition of cell regulatory volume decrease by electroporation may be beneficial in achieving higher electrofusion efficiency. The hypotonic treatment in itself did not significantly affect the cell viability; however, electric field parameters for electroporation should be carefully selected taking into account the hypotonically induced volume increase of cells.

Ketorolac Ester Enhancer-prodrugs: Preparation and Evaluation of Their Physicochemical Properties

Six ester analogues of Ketorolac were synthesized as potential enhancer prodrugs for transdermal delivery. Solubility of these esters was determined in 10% propylene glycol (PG)/isotonic phosphate buffer (IPB) at room temperature while lipophilicity was obtained as partition coefficients (log P) and capacity factors (k') using HPLC. Stability of the prodrugs in skin extract and in plasma was investigated at . The lipophilicity of the potential prodrugs increased in proportion to their alkyl chain length. Good linear relationship between partition coefficients (log P) and capacity factors (log k') was observed (). All of the analogues were fairly stable but slowly degraded in IPB over a 12 hour period. However, their stability in skin extract and in plasma varied with most compounds gradually decomposing over a 12 hour period. Although unsaturation of the alkyl ester chain did not alter the over all lipophilicity of the compound, the half-life was significantly affected. In plasma, degradation of the esters was slower than in the skin extract, which is a desirable trait for enhancer-prodrugs. However, the overall hydrolysis in the skin extract needs to be facilitated for the development of an effective enhancer prodrug. The analogue with the shortest half life in the skin extract was the unsaturated C-12 analogue of 0.96 hr.

ClC-3 and IClswell are Required for Normal Neutrophil Chemotaxis and Shape Change

Polymorphonuclear leukocytes undergo directed movement to sites of infection, a complex process known as chemotaxis. Extension of the polymorphonuclear leukocyte (PMN) leading edge toward a chemoattractant in association with uropod retraction must involve a coordinated increase/decrease in membrane, redistribution of cell volume, or both. Deficits in PMN phagocytosis and trans-endothelial migration, both highly motile PMN functions, suggested that the anion transporters, ClC-3 and ICl(swell), are involved in cell motility and shape change ( Moreland, J. G., Davis, A. P., Bailey, G., Nauseef, W. M., and Lamb, F. S. (2006) J. Biol. Chem. 281, 12277-12288 ). We hypothesized that ClC-3 and ICl(swell) are required for normal PMN chemotaxis through regulation of cell volume and shape change. Using complementary chemotaxis assays, EZ-TAXIScantrade mark and dynamic imaging analysis software, we analyzed the directed cell movement and morphology of PMNs lacking normal anion transporter function. Murine Clcn3(-/-) PMNs and human PMNs treated with anion transporter inhibitors demonstrated impaired chemotaxis in response to formyl peptide. This included decreased cell velocity and failure to undergo normal cycles of elongation and retraction. Impaired chemotaxis was not due to a diminished number of formyl peptide receptors in either murine or human PMNs, as measured by flow cytometry. Murine Clcn3(-/-) and Clcn3(+/+) PMNs demonstrated a similar regulatory volume decrease, indicating that the ICl(swell) response to hypotonic challenge was intact in these cells. We further demonstrated that ICl(swell) is essential for shape change during human PMN chemotaxis. We speculate that ClC-3 and ICl(swell) have unique roles in regulation of PMN chemotaxis; ICl(swell) through direct effects on PMN volume and ClC-3 through regulation of ICl(swell).

A Trypanosoma cruzi Phosphatidylinositol 3-Kinase (TcVps34) Is Involved in Osmoregulation and Receptor-mediated Endocytosis

Trypanosoma cruzi, the etiological agent of Chagas disease, has the ability to respond to a variety of environmental changes during its life cycle both in the insect vector and in the vertebrate host. Because regulation of transcription initiation seems to be nonfunctional in this parasite, it is important to investigate other regulatory mechanisms of adaptation. Regulatory mechanisms at the level of signal transduction pathways involving phosphoinositides are good candidates for this purpose. Here we report the identification of the first phosphatidylinositol 3-kinase (PI3K) in T. cruzi, with similarity with its yeast counterpart, Vps34p. TcVps34 specifically phosphorylates phosphatidylinositol to produce phosphatidylinositol 3-phosphate, thus confirming that it belongs to class III PI3K family. Overexpression of TcVps34 resulted in morphological and functional alterations related to vesicular trafficking. Although inhibition of TcVps34 with specific PI3K inhibitors, such as wortmannin and LY294,000, resulted in reduced regulatory volume decrease after hyposmotic stress, cells overexpressing this enzyme were resistant to these inhibitors. Furthermore, these cells were able to recover their original volume faster than wild type cells when they were submitted to severe hyposmotic stress. In addition, in TcVps34-overexpressing cells, the activities of vacuolar-H+-ATPase and vacuolar H+-pyrophosphatase were altered, suggesting defects in the acidification of intracellular compartments. Furthermore, receptor-mediated endocytosis was partially blocked although fluid phase endocytosis was not affected, confirming a function for TcVps34 in membrane trafficking. Taken together, these results strongly support that TcVps34 plays a prominent role in vital processes for T. cruzi survival such as osmoregulation, acidification, and vesicular trafficking.

Direct Interaction of Nuclear Liver X Receptor-β with ABCA1 Modulates Cholesterol Efflux

Cholesterol is an essential component of eukaryotic cells; at the same time, however, hyperaccumulation of cholesterol is harmful. Therefore, the ABCA1 gene, the product of which mediates secretion of cholesterol, is highly regulated at both the transcriptional and post-transcriptional levels. The transcription of ABCA1 is regulated by intracellular oxysterol concentration via the nuclear liver X receptor (LXR)/retinoid X receptor (RXR); once synthesized, ABCA1 protein turns over rapidly with a half-life of 1-2 h. Here, we show that the LXRbeta/RXR complex binds directly to ABCA1 on the plasma membrane of macrophages and modulates cholesterol secretion. When cholesterol does not accumulate, ABCA1-LXRbeta/RXR localizes on the plasma membrane, but is inert. When cholesterol accumulates, oxysterols bind to LXRbeta, and the LXRbeta/RXR complex dissociates from ABCA1, restoring ABCA1 activity and allowing apoA-I-dependent cholesterol secretion. LXRbeta can exert an immediate post-translational response, as well as a rather slow transcriptional response, to changes in cellular cholesterol accumulation. Thus, we provide the first demonstration that protein-protein interaction suppresses ABCA1 function. Furthermore, we show that LXRbeta is involved in both the transcriptional and post-transcriptional regulation of the ABCA1 transporter.

Ca2+ Signaling Occurs via Second Messenger Release from Intraorganelle Synthesis Sites

SummaryCyclic ADP-ribose is an important Ca2+-mobilizing cytosolic messenger synthesized from β-NAD+ by ADP-ribosyl cyclases (ARCs). However, the focus upon ectocellular mammalian ARCs (CD38 and CD157) has led to confusion as to how extracellular enzymes generate intracellular messengers in response to stimuli. We have cloned and characterized three ARCs in the sea urchin egg and found that endogenous ARCβ and ARCγ are intracellular and located within the lumen of acidic, exocytotic vesicles, where they are optimally active. Intraorganelle ARCs are shielded from cytosolic substrate and targets by the organelle membrane, but this barrier is circumvented by nucleotide transport. We show that a β-NAD+ transporter provides ARC substrate that is converted luminally to cADPR, which, in turn, is shuttled out to the cytosol via a separate cADPR transporter. Moreover, nucleotide transport is integral to ARC activity physiologically because three transport inhibitors all inhibited the fertilization-induced Ca2+ wave that is dependent upon cADPR. This represents a novel signaling mechanism whereby an extracellular stimulus increases the concentration of a second messenger by promoting messenger transport from intraorganelle synthesis sites to the cytosol.

Effect of buffer composition and preparation protocol on the dispersion stability and interfacial behavior of aqueous DPPC dispersions

The effect of the buffer composition and the preparation protocol on the dynamic surface tension (DST) and vesicle sizes of aqueous dipalmitoylphosphatidylcholine (DPPC) dispersions was studied. Four isotonic buffers were used in preparing DPPC dispersions at physiological conditions for possible biological applications: (1) a standard PBS solution; (2) the above PBS with 1 mM CaCl 2; (3) PBS with one tenth the previous standard phosphate salt concentrations and 2.5 mM CaCl 2; and (4) 150 mM NaCl with 2.5 mM CaCl 2 and 10 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid). Two protocols, with a new method and an old method (Bangham method), were used in preparing the DPPC dispersions. The DPPC dispersions prepared with the new method contained mostly vesicles and were quite stable at 25 or 37 °C. Dynamic light scattering (DLS) and spectroturbidimetry (ST) results showed that the DPPC vesicle sizes in buffer (4) were much smaller than those in the other buffers. When the DPPC dispersions were prepared with the new method, the diameter of the DPPC particles was smaller than those with the old method. The DPPC vesicles with the new method were more stable than those with the other method. The DPPC dispersions of 1000 ppm at 37 °C with the new method produced, at pulsating area conditions at 20 cycles per minute, low tension minima ( γ min), lower than 10 mN/m, in buffers (1), (2), and (4). With buffer (4) the DSTs were lower and were achieved faster than with the other buffers. A minimum concentration of 1000 or 250 ppm DPPC was needed to produce DSTs lower than 10 mN/m within 10 min or less, with buffer (2) or (4), respectively. IRRAS results suggest that DPPC in buffer (2) or (4) forms a close-packed monolayer at the interface. These results have implications for designing efficient protocols of lipid dispersion preparation and lung surfactant replacement formulations in treating respiratory disease.

The Nucleoside Diphosphate Kinase D (NM23-H4) Binds the Inner Mitochondrial Membrane with High Affinity to Cardiolipin and Couples Nucleotide Transfer with Respiration

Nucleoside diphosphate kinase (NDPK/Nm23), responsible for intracellular di- and triphosphonucleoside homeostasis, plays multiple roles in cellular energetics, signaling, proliferation, differentiation and tumor invasion. The only human NDPK with a mitochondrial targeting sequence is NDPK-D, the NME4 gene product, which is a peripheral protein of mitochondrial membranes. Subfractionation of rat liver and HEK 293 cell mitochondria revealed that NDPK-D is essentially bound to the inner membrane. Surface plasmon resonance analysis of the interaction using recombinant NDPK-D and model liposomes showed that NDPK-D interacts electrostatically with anionic phospholipids, with highest affinity observed for cardiolipin. Mutation of the central arginine (Arg-90) in a surface-exposed basic RRK motif unique to NDPK-D strongly reduced interaction with anionic phospholipids. Due to its symmetrical hexameric structure, NDPK-D was able to cross-link anionic phospholipid-containing liposomes, suggesting that NDPK-D could promote intermembrane contacts. Latency assays with isolated mitochondria and antibody binding to mitoplasts indicated a dual orientation for NDPK-D. In HeLa cells, stable expression of wild type but not of the R90D mutant led to membrane-bound enzyme in vivo. Respiration was significantly stimulated by the NDPK substrate TDP in mitochondria containing wild-type NDPK-D, but not in those expressing the R90D mutant, which is catalytically equally active. This indicates local ADP regeneration in the mitochondrial intermembrane space and a tight functional coupling of NDPK-D with oxidative phosphorylation that depends on its membrane-bound state.

Characterization of Binding Affinity of CJ-023,423 for Human Prostanoid EP4 Receptor

In order to characterize the receptor binding pharmacology of CJ-023,423, a potent and selective EP₄ antagonist, we performed a radioligand receptor binding assay under various assay conditions. An acidic (pH 6) and hypotonic buffer is a conventional, well-known buffer for prostaglandin E₂ receptor binding assays. CJ-023,423 showed moderate binding affinity for human EP₄ receptor under conventional buffer conditions. However, its binding affinity was greatly increased under neutral (pH 7.4) and isotonic buffer conditions. In this report, the binding mechanism between CJ-023,423 and human EP₄ receptor is discussed based on the binding affinities determined under various assay conditions.

Preparation and characterization of pH- and temperature-sensitive pullulan microspheres for controlled release of drugs

Most part of pH- and temperature-sensitive microspheres used for the controlled delivery of drugs are not biodegradable. Therefore, the aim of this work is to prepare pH- and temperature-sensitive microspheres from biodegradable and biocompatible natural polymers. Pullulan microspheres were prepared by suspension cross-linking with epichlorohydrin of an aqueous solution of the polymer. In order to confer them temperature sensitivity, poly( N-isopropylacrylamide- co-acrylamide) was grafted onto pullulan microspheres. Then, the pH-sensitive units (–COOH) were introduced by reaction between the remaining –OH groups of the pullulan with succinic anhydride. The grafted pullulan microspheres are more hydrophilic than pullulan microspheres, their swelling degree as well as water regain increase significantly. The thermo-sensitivity of the carboxylated microspheres depends to the number and the ionization form (–COOH/–COO −) of carboxylic groups. At a low exchange capacity (0.35 meq/g), microspheres are thermo-sensitive both in the protonated and deprotonated form of –COOH groups. At a higher exchange capacity (2.25 meq/g), microspheres are almost unswellable in the protonated form and swell extensively in the ionized form (up to 28 times than their dried form) loosing in a great extent the thermo-sensitive properties. In isotonic phosphate buffer pH = 7.4, both thermo-sensitive and pH/thermo-sensitive microspheres possess a phase transition temperature close to that of the human body temperature. Loading and release profiles of lysozyme, taken as a molecular model system, were investigated.

A Role for the Receptor for Advanced Glycation End Products in Idiopathic Pulmonary Fibrosis

Idiopathic pulmonary fibrosis (IPF) is a severely debilitating disease associated with a dismal prognosis. There are currently no effective therapies for IPF, thus the identification of novel therapeutic targets is greatly needed. The receptor for advanced glycation end products (RAGE) is a member of the immunoglobulin superfamily of cell surface receptors whose activation has been linked to various pathologies. In healthy adult animals, RAGE is expressed at the highest levels in the lung compared to other tissues. To investigate the hypothesis that RAGE is involved in IPF pathogenesis, we have examined its expression in two mouse models of pulmonary fibrosis and in human tissue from IPF patients. In each instance we observed a depletion of membrane RAGE and its soluble (decoy) isoform, sRAGE, in fibrotic lungs. In contrast to other diseases in which RAGE signaling promotes pathology, immunohistochemical and hydroxyproline quantification studies on aged RAGE-null mice indicate that these mice spontaneously develop pulmonary fibrosis-like alterations. Furthermore, when subjected to a model of pulmonary fibrosis, RAGE-null mice developed more severe fibrosis, as measured by hydroxyproline assay and histological scoring, than wild-type controls. Combined with data from other studies on mouse models of pulmonary fibrosis and human IPF tissues indicate that loss of RAGE contributes to IPF pathogenesis.

Regulation of Phosphorylation of Thr-308 of Akt, Cell Proliferation, and Survival by the B55α Regulatory Subunit Targeting of the Protein Phosphatase 2A Holoenzyme to Akt

Akt is a protein serine/threonine kinase that is involved in the regulation of diverse cellular processes. Phosphorylation of Akt at regulatory residues Thr-308 and Ser-473 leads to its full activation. The protein phosphatase 2A (PP2A) has long been known to negatively regulate Akt activity. The PP2A holoenzyme consists of the structural subunit (A), catalytic subunit (C), and a variable regulatory subunit (B). Here we report the identification of the specific B regulatory subunit that targets the PP2A holoenzyme to Akt. We found endogenous association of PP2A AB55C holoenzymes with Akt by co-immunoprecipitation analyses in pro-lymphoid FL5.12 cells. Akt was shown to associate with ectopically expressed B55alpha subunit in NIH3T3 cells. The direct interaction between B55alpha subunit and Akt was confirmed using in vitro pulldown analyses. Intriguingly, we found that overexpression of B55alpha subunit significantly impaired phosphorylation at Thr-308, but to a lesser extent at Ser-473 of Akt in both FL5.12 and NIH3T3 cells. Concomitantly, phosphorylation of a subset of Akt substrates, including FoxO3a, was substantially decreased by B55alpha overexpression in these cells. Silencing of B55alpha expression markedly increased phosphorylation at Thr-308 but not at Ser-473 in both FL5.12 cells and NIH3T3 cells. Consistently, PP2A AB55alphaC holoenzymes preferentially dephosphorylated phospho-Thr-308 rather than phospho-Ser-473 in in vitro dephosphorylation assays. Furthermore, B55alpha overexpression retarded proliferation of NIH3T3 cells, and knockdown of B55alpha expression increased survival of FL5.12 cells upon interleukin-3 deprivation. Together, our data demonstrate that B55alpha-dependent targeting of the PP2A holoenzyme to Akt selectively regulates Akt phosphorylation at Thr-308 to regulate cell proliferation and survival.