Maleate Buffer Research Articles

Effect of buffer capacity on dissolution and supersaturation profiles of pioglitazone hydrochloride

The purpose of the present study was to investigate the effect of buffer capacity (β) on the dissolution, supersaturation, and precipitation (DSP) profiles of a salt form drug. The DSP of pioglitazone HCl (PIO–HCl, pKa = 5.8, intrinsic solubility = 0.4 μg/mL) was investigated under a non-sink condition (30 mg in 500 mL) in phosphate and maleate buffers with various β (2–32 mM/ΔpH) at pH 6.5. Precipitation from the bulk solution was investigated by a pH-shift method (pH 3.0 to 6.5). The slurry pH was measured to assess the solid surface pH. As β was increased, the degree of supersaturation in the non-sink dissolution test decreased from 25 to less than 3. The PIO-HCl particles transformed to the free base (PIO-FB) while retaining the outer shape of the initial particles. In the pH-shift precipitation test, β did not affect the precipitation of PIO-FB, and the shape of precipitant was different from that in the dissolution tests. The pH of the PIO-HCl slurry increased from 0.9 to 1.6 as β increased. In conclusion, buffer capacity had a marked impact on the PIO-HCl DSP profile. PIO-HCl transformed to PIO-FB before dissolving into the bulk phase.

Papillary Muscle Related Biomechanical Properties of Mitral Valve Chordae Tendineae

. Introduction Mitral valve is a complex structure including the annulus, the anterior leaflet, the posterior leaflet, the papillary muscles (PM), and the chordae tendineae connecting the leaflets and PM. The mechanical properties of the chordae play an important role in the normal functioning of the mitral valve: the chordae assists in maintaining the opening and closing configuration of the valve during cardiac cycle. Failure of certain chordae may lead to failure of the mitral valve and in severe cases, will lead to heart disease and mortality. In some cases, the ruptured chord need to be corrected by repair or replacement. Therefore, there has been high interest in the analysis of the function, mechanical properties and shape features of the mitral apparatus to improve the surgical effect. Chordae can be distinguished by leaflet location insertion as primary and secondary chordae. These finger-like chords connect the mitral valve to either anterolateral papillary muscle (APM) or posteromedial papillary muscle (PPM). The PPM has a higher risk to necrose and rupture in myocardial ischemia and infarction in clinic, but it is underlying mechanism is still unknown. Previous studies have shown importance in maintaining the asymmetric structure and realistic material property of the mitral valve for physiological load condition, but simplified the chord as symmetric structure, which is not true. In this study, the porcine heart chords were classified and measured according to the attached PMs. The uniaxial tensile test was utilized to analyze the biomechanical properties of the papillary muscle related chordae and histology observation was carried out for microstructure analysis. This study aims to analyze the anatomical and mechanical property differences in chords based on PMs which may help to understand the mitral valve function and to optimize the design of the artificial implantation or repair. Methods and materials Studies have shown that porcine valve was identified as an appropriate model for further investigation of the mitral valve system when considering the rarity of human valve. A total of 16 fresh porcine hearts were collected, infused in 4°C PBS buffer and infiltrated in physiological saline during the experiment, 9 of which were used for tensile testing, 6 for histological section staining, and 1 for TEM scanning. Based on insert position to PM (APM or PPM) and leaflets (primary on free edge and secondary on belly), the chord were divided into the APM primary chord, APM secondary chord, and PPM primary chord, and PPM secondary chord. The chords were separated from the valve, and the chordae diameter and length were measured via microscope, Markers were added at the target area of ​​the chord for strain measurement. The sample was then fixed on an Instron1000 uniaxial tensile test machine with sandpaper. Before the experiment, the specimens were preload from 0N to2N until the displacement curves were substantially coincident, and then tensile test. The sensor is used to record the stress change, and the CCD camera synchronously collects the displacement footage of the markers on target area until the chord sample is broken or slipped from tensile test machine. The MATLAB code was used to perform imaging processing and to obtain the stress-strain curve. Histological samples were fixed in 4 % glutaraldehyde in PO4 buffer (pH 7.4) for 5 hours, dehydrated with graduated concentrations of ethanol and embedded in paraffin. Radial sections were cut to 5 µm thick, masson stained and photos were then taken with microscope (ZEISS Stemi 2000-C) and camera (ZEISS AxioCam ICc5). The cross-sectional area ratio of collagen fibers and the amount of micro-vessel were observed. The microstructure sample were processed for transmission electron microscopy (TEM) observation. They were trimmed and fixed with 2.5 % glutaraldehyde, post-fixed with 1 % Osmium Tetroxide for 1 hour, 1 % uranyl acetate in Maleate buffer for 1 hour, and dehydrated with ethanol and propylene oxide, and then embedded in Epon. The difference in the configuration of collagen fibers was observed by TEM scanning. Result and conclusion There was no significant difference in the number of chord on each PM, and the PPM chord was longer. The Green strain-Cauchy stress curve showed that the Tangent Modulus (TM) of the PPM secondary chord was larger than that of the APM secondary chord. The number of blood vessels on APM primary chord was more than that of PPM primary chord, there was no significant difference in the area ratio of collagen fiber on chord of each PM. In our research, the Ogden nonlinear strain energy function was used to fit the experimental stress-strain data and to obtain material parameters. It provides a theoretical basis for the subsequent dynamic simulation using finite element method.

Development of a Substrate Identification Method for Human Scp1 Phosphatase Using Phosphorylation Mimic Phage Display

Protein phosphorylation is strictly regulated by protein kinases and protein phosphatases, and disordered regulation of protein phosphorylation often causes serious diseases, such as cancer. Protein phosphatases are divided into two major groups: tyrosine (Tyr) phosphatases and serine/threonine (Ser/Thr) phosphatases. Substrate trapping mutants are frequently used to characterize Tyr phosphatases and identify their substrates; however, a rapid and simple method to identify substrates for Ser/Thr phosphatases has yet to be developed. Recently it has reported that AlF4 -/AlF3 and BeF3 - form a complex with Mg2+ in the catalytic center of FCP/SCP phosphatases, and that the Mg2+-AlF4 -/AlF3 complex mimics the transition state of the hydrolysis step, while the Mg2+-BeF3 - complex mimics the aspartylphosphate intermediate. The main objective of this study was to develop a novel methodology, termed Phosphorylation Mimic Phage Display (PMPD), to identify substrates for Ser/Thr phosphatase Scp1 using peptide phage display libraries with Mg2+ and AlF4 -. Recombinant protein of human full-length Scp1 (rScp1) expressed in E. coli system was purified by Co2+ chelated affinity column, and then confirmed by SDS-PAGE and Western-blot analysis. The Ph.D.-C7C or M12 Phage Display Libraries (New England BioLabs, Beverly, MA) were screened using purified rScp1 immobilized on ELISA plate. Then, the plate was blocked with 0.5% (w/v) BSA in maleate buffer at 4°C for 3 h, before adding approximately 1×1010 plaque-forming units (pfu) of the phages in maleate blocking AlF4 - buffer to each well. After incubating, the wells were washed with maleate AlF4 - buffer to remove unbound phages. Then, phages were eluted with Mg2+ and AlF4 - free maleate buffer or with excess rScp1. After the third round of screening, the isolated phages were sequenced and subjected to binding analyses. After panning by PMPD method, 46 and 60 clones were isolated from the Ph.D. C7C and Ph.D. 12 phage libraries, respectively, as Mg2+ or/and AlF4 - -dependent binding clones. The binding analyses showed that M12-1 and Dep-3 specifically bind to Scp1 in an AlF4 --dependent manner. Notably, the Dep-3 peptide contained a Thr-Pro-Met-Ser sequence, which is similar to the Ser2-Pro3-Thr4-Ser5 (Ser/Thr-Pro-partially hydrophobic residue-Ser) sequence found in CTD, which is an endogenous substrate for Scp1. Binding analyses also showed that both BP-14 and M12-6a bound to Scp1 in a Mg2+-dependent manner. BP-14 peptide contained Ser- Thr-Tyr and Pro-Phe-Glu sequences, which are similar to the Ser-Thr-Trp and Ile-Phe-Glu sequences found in M12-6a, suggesting that one or both of these tripeptides may be the binding motif(s) recognized by Scp1. We developed a substrate identification method for the Ser/Thr phosphatase Scp1 using a novel phage display method with AlF4 -. Dep-3 showed a core sequence similar to that of the CTD of RNA polymerase II, an endogenous Scp1 substrate, suggesting that this method is applicable for identifying novel Scp1 substrate candidates. This method will also be applicable for other FCP/SCP-type phosphatases, allowing us to better understand the substrate recognition mechanisms of Ser/Thr phosphatases.

Kinetic modelling of ascorbic and dehydroascorbic acids concentrations in a model solution at different temperatures and oxygen contents

The degradation kinetics of vitamin C (ascorbic and dehydroascorbic acids, AA and DHA) were determined under controlled conditions of temperature (50–90 °C) and oxygen concentrations in the gas phase (10–30% mol/mol) using a specific reactor. The degradation of vitamin C in malate buffer (20 mM, pH 3.8), mimetic of an apple puree, was assessed by sampling at regular intervals and spectrophotometric quantification of AA and DHA levels at 243 nm. The results showed that AA degradation increased with temperature and oxygen concentration, while DHA exhibited the behaviour of an intermediate species, appearing then disappearing. A kinetic model was successfully developed to simulate the experimental data by two first order consecutive reactions. The first one represented AA degradation as a function of temperature and concentration in dissolved oxygen, and the second reflected DHA degradation as a function of temperature only, both adequately following Arrhenius' law.

Efficiency of three buffers for extracting β-glucosidase enzyme in different soil orders: Evaluating the role of soil organic matter

The objective of this research was to evaluate extraction methods for β-glucosidases comparing three buffer solutions (MUB, acetate, and maleate) at different incubation times (0.5 h to 10 h) and in three different soil orders (Mollisols, Andisols and Ultisols). Seven acidic soils were evaluated, showing differences in pH, OM, and clay contents. To evaluate the effect of OM as enzymes source, one soil of each order was treated to partially remove its OM and then the enzyme assay was performed. When using MUB and maleate buffers the highest (32 and 31 µg- p NP g-soil -1 h -1 in average, respectively) were found, and the latter was significantly (p < 0.050) correlated with the soil clay content. The activity obtained with acetate buffer was much lower (38.2 µg- p NP g-soil -1 h -1 in average). The use of MUB buffer with 1 h of incubation is suggested as extraction method, showing good reproducibility and allowing to express higher enzyme potential for soil comparisons. For the Andisol and Ultisol, the enzyme activity significantly decreased with the OM removal (%) indicating that OM is the major source of the measured β-glucosidase activity, while a different trend was observed for the Mollisol, in which the mineral fraction (mainly 2:1 type clay) appears to be involved in the increased enzyme activity displayed after the initial OM removal.

Spectrophotometric method for fast quantification of ascorbic acid and dehydroascorbic acid in simple matrix for kinetics measurements

A simple, rapid and reliable method was developed for quantifying ascorbic (AA) and dehydroascorbic (DHAA) acids and validated in 20mM malate buffer (pH 3.8). It consists in a spectrophotometric measurement of AA, either directly on the solution added with metaphosphoric acid or after reduction of DHAA into AA by dithiothreitol. This method was developed with real time measurement of reactions kinetics in bulk reactors in mind, and was checked in terms of linearity, limits of detection and quantification, fidelity and accuracy. The linearity was found satisfactory on the range of 0–6.95mM with limits of detection and quantification of 0.236mM and 0.467mM, respectively. The method was found acceptable in terms of fidelity and accuracy with a coefficient of variation for repeatability and reproducibility below 6% for AA and below 15% for DHAA, and with a recovery range of 97–102% for AA and 88–112% for DHAA.

Matching phosphate and maleate buffer systems for dissolution of weak acids: Equivalence in terms of buffer capacity of bulk solution or surface pH?

The development of in vitro dissolution tests able to anticipate the in vivo fate of drug products has challenged pharmaceutical scientists over time, especially in the case of ionizable compounds. In the seminal model proposed by Mooney et al. thirty-five years ago, the pH at the solid–liquid interface (pH0) was identified as a key parameter in predicting dissolution rate. In the current work it is demonstrated that the in vitro dissolution of the weak acid ibuprofen in maleate and phosphate buffer systems is a function of the pH0, which in turn is affected by properties of the drug and the medium. The reported pH0 for ibuprofen dissolution in bicarbonate buffer, the predominant buffer species in the human small intestine under fasting conditions, can be achieved by reducing the phosphate buffer concentration to 5.0mM or the maleate buffer concentration to 2.2mM. Using this approach to identify the appropriate buffer/buffer capacity combination for in vitro experiments in FaSSIF-type media, it would be possible to increase the physiological relevance of this important biopharmaceutics tool. However, the necessity of monitoring and adjusting the bulk pH during the experiments carried out in 5.0mM phosphate or 2.2mM maleate buffers must also be taken into consideration.

Determination of arginine catabolism by salivary pellet

To determine the formation of ammonium from arginine by oral bacteria residing in saliva and dental plaque, an arginolytic activity assay based on the work described by Nascimento et al. [2] was developed. Following the original methodology, insufficient ammonium production could be determined.To improve the method for our research goal, the following modifications were made to the original protocols:•The following changes were made to the arginine catabolism assay resulting in a 1000-fold increase in sensitivity: (i) the salivary pellet was washed and concentrated five times resulting in the removal of low density compounds interfering with the assay, (ii) the pH of the Tris–maleate buffer was increased from 6.0 to 7.5 resulting in a better conversion of arginine to ammonium and (iii) the incubation time was increased to 3h to ensure that non-responders and salivary pellets low in cell numbers could yield detectable levels of ammonium.•Removal of a centrifuge step from the protein determination resulted in a higher protein yield improving the accuracy of the assay.•Changing from the use of the toxic, environmentally hazardous, mercury containing Nessler's reagent to a colorimetric enzyme assay achieved a safer and greener determination of ammonium concentration.

Thermophysical Properties of Carboxylic and Amino Acid Buffers at Subzero Temperatures: Relevance to Frozen State Stabilization

Macromolecules and other thermolabile biologicals are often buffered and stored in frozen or dried (freeze-dried) state. Crystallization of buffer components in frozen aqueous solutions and the consequent pH shifts were studied in carboxylic (succinic, malic, citric, tartaric acid) and amino acid (glycine, histidine) buffers. Aqueous buffer solutions were cooled from room temperature (RT) to -25 °C and the pH of the solution was measured as a function of temperature. The thermal behavior of frozen solutions was investigated by differential scanning calorimetry (DSC), and the crystallized phases were identified by X-ray diffractometry (XRD). Based on the solubility of the neutral species of each buffer system over a range of temperatures, it was possible to estimate its degree of supersaturation at the subambient temperature of interest. This enabled us to predict its crystallization propensity in frozen systems. The experimental and the predicted rank orderings were in excellent agreement. The malate buffer system was robust with no evidence of buffer component crystallization and hence negligible pH shift. In the citrate and tartrate systems, at initial pH < pK(a)(2), only the most acidic buffer component (neutral form) crystallized on cooling, causing an increase in the freeze-concentrate pH. In glycine buffer solutions, when the initial pH was ∼3 units < isoelectric pH (pI = 5.9), β-glycine crystallization caused a small decrease in pH, while a similar effect but in the opposite direction was observed when the initial pH was ∼3 units > pI. In the histidine buffer system, depending on the initial pH, either histidine or histidine HCl crystallized.

Predicting the Crystallization Propensity of Carboxylic Acid Buffers in Frozen Systems—Relevance to Freeze-Drying

Selective crystallization of buffer components in frozen solutions is known to cause pronounced pH shifts. Our objective was to study the crystallization behavior and the consequent pH shift in frozen aqueous carboxylic acid buffers. Aqueous carboxylic acid buffers were cooled to -25°C and the pH of the solution was measured as a function of temperature. The thermal behavior of solutions during freezing and thawing was investigated by differential scanning calorimetry. The crystallized phases in frozen solution were identified by X-ray diffractometry. The malate buffer system was robust with no evidence of buffer component crystallization and hence negligible pH shift. In the citrate and tartarate systems, at initial pH <pKa2 , only the most acidic buffer component (neutral form) crystallized on cooling, causing an increase in the freeze-concentrate pH. Carboxylic acid buffers were rank ordered based on their propensity to crystallize in frozen solutions. From the aqueous solubility values of these carboxylic acids, which have been reported over a range of temperatures, it was also possible to estimate the degree of supersaturation at the subambient temperature of interest. This enabled us to predict their crystallization propensity in frozen systems. The experimental and the predicted rank orderings were in excellent agreement.

Efficient production of cellulase in the culture of Acremonium cellulolyticus using untreated waste paper sludge

Cellulase was produced by Acremonium cellulolyticus using untreated waste paper sludge (PS) as the carbon source. The clay present in PS did not show any inhibitory effect on cellulase production but did alter the pH during fermentation. On the flask scale, the maleate buffer concentration and pH were key factors that affected the efficiency of cellulase production from PS cellulose. Optimum cellulase production in a 3-L fermentor of working volume 1.5 L was achieved by controlling the pH value at 6.0 using 2 M NaOH and 2 M maleic acid, and the productivity reached 8.18 FPU/mL. When 40.89 g/L PS cellulose, 2.2 g/L (NH(4) )(2) SO(4) , and 4.4 g/L urea were added to a 48-h culture, the cellulase activity was 9.31 FPU/mL at the flask scale and 10.96 FPU/mL in the 3-L fermentor. These values are ∼80% of those obtained when pure cellulose is used as the carbon source. The method developed here presents a new route for the utilization of PS.

Cytochrome P450 2B1 Mediates Complement-dependent Sublytic Injury in a Model of Membranous Nephropathy

Membranous nephropathy is a disease that affects the filtering units of the kidney, the glomeruli, and results in proteinuria accompanied by loss of kidney function. Passive Heymann nephritis is an experimental model that mimics membranous nephropathy in humans, wherein the glomerular epithelial cell (GEC) injury induced by complement C5b-9 leads to proteinuria. We examined the role of cytochrome P450 2B1 (CYP2B1) in this complement-mediated sublytic injury. Overexpression of CYP2B1 in GECs significantly increased the formation of reactive oxygen species, cytotoxicity, and collapse of the actin cytoskeleton following treatment with anti-tubular brush-border antiserum (anti-Fx1A). In contrast, silencing of CYP2B1 markedly attenuated anti-Fx1A-induced reactive oxygen species generation and cytotoxicity with preservation of the actin cytoskeleton. Gelsolin, which maintains an organized actin cytoskeleton, was significantly decreased by complement C5b-9-mediated injury but was preserved in CYP2B1-silenced cells. In rats injected with anti-Fx1A, the cytochrome P450 inhibitor cimetidine blocked an increase in catalytic iron and ROS generation, reduced the formation of malondialdehyde adducts, maintained a normal distribution of nephrin in the glomeruli, and provided significant protection at the onset of proteinuria. Thus, GEC CYP2B1 contributes to complement C5b-9-mediated injury and plays an important role in the pathogenesis of passive Heymann nephritis.

Brush border enzymes and absorptive capacity in extrahepatic portal venous obstruction in children

Portal hypertension may affect intestinal functions, brush border enzymes and absorption parameters. Information about these in extrahepatic portal venous obstruction (EHPVO) in children is limited and poorly reported. We therefore studied the brush border enzymes and absorptive capacity in EHPVO in children. The study was conducted on 52 children of EHPVO. The diagnosis of EHPVO was made on the basis of clinical presentation and ultrasound examination. All patients underwent upper gastrointestinal endoscopy. Endoscopic tissue biopsies from duodenum were taken on aluminum foils and kept immediately at -20°C for estimation of brush border enzymes. Tissue biopsies were homogenized in sodium maleate buffer, 0.1M pH 6.0, by a homogenizer and processed for the enzymes: lactase, maltase, and sucrase. Enzyme levels were compared to normal healthy controls (n=20). d-Xylose test, stool acid steatocrit for fat excretion in stools and stool alpha-1 antitrypsin were done to know about the absorptive parameters. Enzyme levels of lactase (6.21±5.67IU/mg) and sucrase (37.07±21.06IU/mg) in EHPVO group were significantly lower as compared to lactase (23.32±10.48IU/mg) and sucrase (95.96±46.55IU/mg) in normal healthy controls. Maltase levels were lower, but difference was not statistically significant in EHPVO group (56.90±28.65IU/mg) as compared to normal controls (63.28±22.88IU/mg). There was no significant difference of urinary d-xylose and stool fat in patients with normal or short stature EHPVO patients. EHPVO leads to decrease in levels of brush border enzymes in small bowel but their absorption capacity remains normal.

Efficient cellulase-catalyzed saccharification of untreated paper sludge targeting for biorefinery

This aim of this study was to optimize the conditions for efficient saccharification of paper sludge (PS) considering PS cellulose concentration, buffer capacity, and cellulase activity from Acremonium cellulolyticus. PS is a cellulosic biomass, the residue of paper making industry, composed of 24.5% cellulose, 10.5% clay, and 65% water. Clay present in PS did not show any inhibitory effect on the saccharification process but influenced the buffer pH. Maleate buffer in the pH range 5.2–5.6 was suitable for the saccharification of PS cellulose. The maximum reducing sugar concentration that could be obtained from 75.6 kg m −3 of PS cellulose was predicted to be 37.8 kg m −3. This was experimentally confirmed to be 38.4 kg m −3 under the optimal saccharification conditions at pH 5.2 (1.06 kmol m −3 Maleate buffer) using Cellulase 20,000 FPU m −3. Almost 100% saccharification yield (SY) was obtained at a low PS cellulose concentration of 15 kg m −3, but this value decreased when the cellulose content increased. When PS was hydrolyzed with commercial cellulase GC220, the SY and glucose content were 54% and 60% of SY, respectively. The corresponding values with Cellulosin T2 were 63% and 75% of SY, respectively. A. cellulolyticus yielded SY and glucose content values almost 10% higher than those obtained with other cellulases.

What is modulating solubility in simulated intestinal fluids?

The aim of this study was to understand which parameters are responsible for the selective modulation of compounds solubility in simulated intestinal fluids. The solubility of 25 chemically diverse reference compounds was measured in simulated intestinal fluid (FaSSIF-V2) and in aqueous phosphate and maleate buffers. Electrostatic interactions between compounds and the bio-relevant medium components seem to explain the different solubility behavior observed for acids and bases. The solubility of ionized acids is not increased in FaSSIF-V2 probably due to electrostatic repulsions with the media components. Lipophilicity plays an important role but mainly for charged bases with a log P > 4 (or log D 6.5 > 1.9). When the aqueous solubility is mainly driven by lipophilicity, the FaSSIF-V2 components seem to improve the solubility of basic compounds to a greater extent than for compounds whose solubility is limited by crystal packing. These results suggest that ionization, lipophilicity and crystal packing play important but peculiar roles in controlling solubility in FaSSIF-V2 compared to that in aqueous buffer and this information could be useful to guide medicinal chemists and formulation scientists.

Molecular Interplay between Endostatin, Integrins, and Heparan Sulfate

Endostatin is an endogenous inhibitor of angiogenesis. Although several endothelial cell surface molecules have been reported to interact with endostatin, its molecular mechanism of action is not fully elucidated. We used surface plasmon resonance assays to characterize interactions between endostatin, integrins, and heparin/heparan sulfate. alpha5beta1 and alphavbeta3 integrins form stable complexes with immobilized endostatin (KD=approximately 1.8x10(-8) M, two-state model). Two arginine residues (Arg27 and Arg139) are crucial for the binding of endostatin to integrins and to heparin/heparan sulfate, suggesting that endostatin would not bind simultaneously to integrins and to heparan sulfate. Experimental data and molecular modeling support endostatin binding to the headpiece of the alphavbeta3 integrin at the interface between the beta-propeller domain of the alphav subunit and the betaA domain of the beta3 subunit. In addition, we report that alpha5beta1 and alphavbeta3 integrins bind to heparin/heparan sulfate. The ectodomain of the alpha5beta1 integrin binds to haparin with high affinity (KD=15.5 nM). The direct binding between integrins and heparin/heparan sulfate might explain why both heparan sulfate and alpha5beta1 integrin are required for the localization of endostatin in endothelial cell lipid rafts.

Regulation of Podosome Formation in Macrophages by a Splice Variant of the Sodium Channel SCN8A

Voltage-gated sodium channels initiate electrical signaling in excitable cells such as muscle and neurons. They also are expressed in non-excitable cells such as macrophages and neoplastic cells. Previously, in macrophages, we demonstrated expression of SCN8A, the gene that encodes the channel NaV1.6, and intracellular localization of NaV1.6 to regions near F-actin bundles, particularly at areas of cell attachment. Here we show that a splice variant of NaV1.6 regulates cellular invasion through its effects on podosome and invadopodia formation in macrophages and melanoma cells. cDNA sequence analysis of SCN8A from THP-1 cells, a human monocyte-macrophage cell line, confirmed the expression of a full-length splice variant that lacks exon 18. Immunoelectron microscopy demonstrated NaV1.6-positive staining within the electron dense podosome rosette structure. Pharmacologic antagonism with tetrodotoxin (TTX) in differentiated THP-1 cells or absence of functional NaV1.6 through a naturally occurring mutation (med) in mouse peritoneal macrophages inhibited podosome formation. Agonist-mediated activation of the channel with veratridine caused release of sodium from cationic vesicular compartments, uptake by mitochondria, and mitochondrial calcium release through the Na/Ca exchanger. Invasion by differentiated THP-1 and HTB-66 cells, an invasive melanoma cell line, through extracellular matrix was inhibited by TTX. THP-1 invasion also was inhibited by small hairpin RNA knockdown of SCN8A. These results demonstrate that a variant of NaV1.6 participates in the control of podosome and invadopodia formation and suggest that intracellular sodium release mediated by NaV1.6 may regulate cellular invasion of macrophages and melanoma cells.

Bone Morphogenetic Protein 1 Prodomain Specifically Binds and Regulates Signaling by Bone Morphogenetic Proteins 2 and 4

Highly purified fractions of bone extracts capable of inducing ectopic bone formation have been reported to contain peptides corresponding to the mature active regions of the TGF-beta-like bone morphogenetic proteins (BMPs) 2-7, and to the prodomain region of the metalloproteinase BMP1. Co-purification of BMPs 2-7 with BMP1 prodomain sequences through the multiple biochemical steps used in these previous reports has suggested the possibility of interactions between the BMP1 prodomain and BMPs 2-7. Here we demonstrate that the BMP1 prodomain binds BMPs 2 and 4 with high specificity and with a KD of approximately 11 nM, in the physiological range. It is further demonstrated that the BMP1 prodomain is capable of modulating signaling by BMPs 2 and 4 in vitro and in vivo, and that endogenous BMP1 prodomain-BMP4 complexes exist in cell culture media and in tissues.

Exovesicles from Human Activated Dendritic Cells Fuse with Resting Dendritic Cells, Allowing Them to Present Alloantigens

Dendritic cells (DCs) can release microvesicles, but the latter's numbers, size, and fate are unclear. Fluorescently labeled DCs were visualized by laser-scanning microscopy. Using a Surpass algorithm, we were able to identify and quantify per cell several hundred microvesicles released from the surface of stimulated DCs. We show that most of these microvesicles are not of endocytic origin but result from budding of the plasma membrane, hence their name, exovesicle. Using a double vital staining, we show that exovesicles isolated from activated DCs can fuse with the membrane of resting DCs, thereby allowing them to present alloantigens to lymphocytes. We concluded that, within a few hours from their release, exovesicles may amplify local or distant adaptive immunological response.

PPARβ/δ Regulates Paneth Cell Differentiation Via Controlling the Hedgehog Signaling Pathway

All 4 differentiated epithelial cell types found in the intestinal epithelium derive from the intestinal epithelial stem cells present in the crypt unit, in a process whose molecular clues are intensely scrutinized. Peroxisome proliferator-activated receptor beta (PPARbeta) is a nuclear hormone receptor activated by fatty acids and is highly expressed in the digestive tract. However, its function in intestinal epithelium homeostasis is understood poorly. To assess the role of PPARbeta in the small intestinal epithelium, we combined various cellular and molecular approaches in wild-type and PPARbeta-mutant mice. We show that the expression of PPARbeta is particularly remarkable at the bottom of the crypt of the small intestine where Paneth cells reside. These cells, which have an important role in the innate immunity, are strikingly affected in PPARbeta-null mice. We then show that Indian hedgehog (Ihh) is a signal sent by mature Paneth cells to their precursors, negatively regulating their differentiation. Importantly, PPARbeta acts on Paneth cell homeostasis by down-regulating the expression of Ihh, an effect that can be mimicked by cyclopamine, a known inhibitor of the hedgehog signaling pathway. We unraveled the Ihh-dependent regulatory loop that controls mature Paneth cell homeostasis and its modulation by PPARbeta. PPARbeta currently is being assessed as a drug target for metabolic diseases; these results reveal some important clues with respect to the signals controlling epithelial cell fate in the small intestine.