The Balance of Fluid and Osmotic Pressures across Active Biological Membranes with Application to the Corneal Endothelium

The movement of fluid and solutes across biological membranes facilitates the transport of nutrients for living organisms and maintains the fluid and osmotic pressures in biological systems. Understanding the pressure balances across membranes is crucial for studying fluid and electrolyte homeostasis in living systems, and is an area of active research. In this study, a set of enhanced Kedem-Katchalsky (KK) equations is proposed to describe fluxes of water and solutes across biological membranes, and is applied to analyze the relationship between fluid and osmotic pressures, accounting for active transport mechanisms that propel substances against their concentration gradients and for fixed charges that alter ionic distributions in separated environments. The equilibrium analysis demonstrates that the proposed theory recovers the Donnan osmotic pressure and can predict the correct fluid pressure difference across membranes, a result which cannot be achieved by existing KK theories due to the neglect of fixed charges. The steady-state analysis on active membranes suggests a new pressure mechanism which balances the fluid pressure together with the osmotic pressure. The source of this pressure arises from active ionic fluxes and from interactions between solvent and solutes in membrane transport. We apply the proposed theory to study the transendothelial fluid pressure in the in vivo cornea, which is a crucial factor maintaining the hydration and transparency of the tissue. The results show the importance of the proposed pressure mechanism in mediating stromal fluid pressure and provide a new interpretation of the pressure modulation mechanism in the in vivo cornea.     

Rapid Genotyping of Alpha 1 Antitrypsin Deletion Mutation (PI*Mmalton) Using Bi-directional PCR Allele-specific Amplification

Alpha 1 antitrypsin deficiency (AATD) is a well recognized genetic risk factor for pulmonary disease and less common liver disease. The two most common deficiency alleles worldwide PI*S and PI*Z can be easily detected using several molecular methods. However, there are at least 30 other AATD variants, which are only detectable by alpha 1 antitrypsin (AAT) gene sequencing and, therefore, seem to be more under-recognized than the PI*S and PI*Z alleles. PI*Mmalton is the most frequent AATD variant in different regions of the Southern Mediterranean basin countries, where its prevalence seems to prevail over PI*S and PI*Z. In this work, we report the development of a simple PCR-based analysis designed for the detection of the PI*Mmalton deficiency alleles using two specific primers. A one-tube reaction enables the distinction between the different genotypes. This reliable, easy, fast, and low-cost technique might be useful for laboratories involved in the study of AATD-related diseases, especially those of the Southern Mediterranean basin area with modest budget or where sophisticated equipment is not available. This will allow larger targeted screening for PI*Mmalton in order to better understand this mutation epidemiology and its origin.     

Estrogen Receptor Alpha as a Key Target of Red Wine Polyphenols Action on the Endothelium

Background

A greater reduction in cardiovascular risk and vascular protection associated with diet rich in polyphenols are generally accepted; however, the molecular targets for polyphenols effects remain unknown. Meanwhile evidences in the literature have enlightened, not only structural similarities between estrogens and polyphenols known as phytoestrogens, but also in their vascular effects. We hypothesized that alpha isoform of estrogen receptor (ERα) could be involved in the transduction of the vascular benefits of polyphenols.

Methodology/Principal Findings

Here, we used ERα deficient mice to show that endothelium-dependent vasorelaxation induced either by red wine polyphenol extract, Provinols™, or delphinidin, an anthocyanin that possesses similar pharmacological profile, is mediated by ERα. Indeed, Provinols™, delphinidin and ERα agonists, 17-beta-estradiol and PPT, are able to induce endothelial vasodilatation in aorta from ERα Wild-Type but not from Knock-Out mice, by activation of nitric oxide (NO) pathway in endothelial cells. Besides, silencing the effects of ERα completely prevented the effects of Provinols™ and delphinidin to activate NO pathway (Src, ERK 1/2, eNOS, caveolin-1) leading to NO production. Furthermore, direct interaction between delphinidin and ERα activator site is demonstrated using both binding assay and docking. Most interestingly, the ability of short term oral administration of Provinols™ to decrease response to serotonin and to enhance sensitivity of the endothelium-dependent relaxation to acetylcholine, associated with concomitant increased NO production and decreased superoxide anions, was completely blunted in ERα deficient mice.

Conclusions/Significance

This study provides evidence that red wine polyphenols, especially delphinidin, exert their endothelial benefits via ERα activation. It is a major breakthrough bringing new insights of the potential therapeutic of polyphenols against cardiovascular pathologies.     

A Mathematical Model of Electrolyte and Fluid Transport across Corneal Endothelium

To predict the behavior of a transporting epithelium by intuitive means can be complex and frustrating. As the number of parameters to be considered increases beyond a few, the task can be termed impossible. The alternative is to model epithelial behavior by mathematical means. For that to be feasible, it has been presumed that a large amount of experimental information is required, so as to be able to use known values for the majority of kinetic parameters. However, in the present case, we are modeling corneal endothelial behavior beginning with experimental values for only five of eleven parameters. The remaining parameter values are calculated assuming cellular steady state and using algebraic software. With that as base, as in preceding treatments but with a distribution of channels/transporters suited to the endothelium, temporal cell and tissue behavior are computed by a program written in Basic that monitors changes in chemical and electrical driving forces across cell membranes and the paracellular pathway. We find that the program reproduces quite well the behaviors experimentally observed for the translayer electrical potential difference and rate of fluid transport, (a) in the steady state, (b) after perturbations by changes in ambient conditions HCO₃^–, Na⁺, and Cl^– concentrations), and (c) after challenge by inhibitors (ouabain, DIDS, Na^+- and Cl^–-channel inhibitors). In addition, we have used the program to compare predictions of translayer fluid transport by two competing theories, electro-osmosis and local osmosis. Only predictions using electro-osmosis fit all the experimental data.     

Discovery of an Inhibitor of Z-Alpha1 Antitrypsin Polymerization

Polymerization of the Z variant alpha-1-antitrypsin (Z-α1AT) results in the most common and severe form of α1AT deficiency (α1ATD), a debilitating genetic disorder whose clinical manifestations range from asymptomatic to fatal liver and/or lung disease. As the altered conformation of Z-α1AT and its attendant aggregation are responsible for pathogenesis, the polymerization process per se has become a major target for the development of therapeutics. Based on the ability of Z-α1AT to aggregate by recruiting the reactive center loop (RCL) of another Z-α1AT into its s4A cavity, we developed a high-throughput screening assay that uses a modified 6-mer peptide mimicking the RCL to screen for inhibitors of Z-α1AT polymer growth. A subset of compounds from the Library of Pharmacologically Active Compounds (LOPAC) with molecular weights ranging from 300 to 700 Da, was used to evaluate the assay’s capabilities. The inhibitor S-(4-nitrobenzyl)-6-thioguanosine was identified as a lead compound and its ability to prevent Z-α1AT polymerization confirmed by secondary assays. To further investigate the binding location of S-(4-nitrobenzyl)-6-thioguanosine, an in silico strategy was pursued and the intermediate α1AT M* state modeled to allow molecular docking simulations and explore various potential binding sites. Docking results predict that S-(4-nitrobenzyl)-6-thioguanosine can bind at the s4A cavity and at the edge of β-sheet A. The former binding site would directly block RCL insertion whereas the latter site would prevent β-sheet A from expanding between s3A/s5A, and thus indirectly impede RCL insertion. Altogether, our investigations have revealed a novel compound that inhibits the formation of Z-α1AT polymers, as well as in vitro and in silico strategies for identifying and characterizing additional blocking molecules of Z-α1AT polymerization.     

Coordinated Network Changes across the Catalytic Cycle of Alpha Tryptophan Synthase

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PCR-Based Screening for the Most Prevalent Alpha 1 Antitrypsin Deficiency Mutations (PI S, Z, and Mmalton) in COPD Patients from Eastern Tunisia

It is generally agreed that the protease inhibitor (PI) alleles PI*S (Val264Glu) and PI*Z (Lys342Glu) are the most common alpha 1 antitrypsin deficiency variants worldwide, but the PI*Mmalton allele (ΔPhe52) prevails over these variants in some Mediterranean regions. In eastern Tunisia (Mahdia), we screened 100 subjects with chronic obstructive pulmonary disease for these variants. The PI*S and PI*Z alleles were genotyped by the previously described SexAI/Hpγ99I RFLP–PCR. We provide here a new method for PI*Mmalton genotyping using mismatched RFLP–PCR. These methods are suitable for routine clinical application and can easily be reproduced by several laboratories, since they do not require extensive optimization, unlike the previously described bidirectional allele-specific amplification PCR for PI*Mmalton genotyping. Our results were in agreement with previous reports from central Tunisia (Kairouan), suggesting that the PI*Mmalton mutation is the most frequent alpha 1 antitrypsin deficiency-related mutation in Tunisia.     

Alpha Adrenergic Induction of Transport of Lysosomal Enzyme across the Blood-Brain Barrier

The impermeability of the adult blood-brain barrier (BBB) to lysosomal enzymes impedes the ability to treat the central nervous system manifestations of lysosomal storage diseases.Here, we found that simultaneous stimulation of the alpha1 and alpha2 adrenoreceptor restores in adult mice the high rate of transport for the lysosomal enzyme P-GUS that is seen in neonates but lost with development. Beta adrenergics, other monoamines, and acetylcholine did not restore this transport. A high dose (500 microg/mouse) of clonidine, a strong alpha2 and weak alpha1 agonist, was able to act as monotherapy in the stimulation of P-GUS transport. Neither use of alpha1 plus alpha2 agonists nor the high dose clonidine disrupted the BBB to albumin. In situ brain perfusion and immunohistochemistry studies indicated that adrengerics act on transporters already at the luminal surface of brain endothelial cells. These results show that adrenergic stimulation, including monotherapy with clonidine, could be key for CNS enzyme replacement therapy.     

Active transport of ions across sunflower roots

Summary The electrical potential difference across exuding roots of Helianthus annuus in two strengths of complete culture solution was measured. The determination of the concentration of the major nutrient ions in the outside solution and the xylem sap enabled the Nernst potential for each ion to be calculated. A comparison of the measured and calculated potentials indicated that the anions NO₃, SO₄, H₂PO₄ and HPO₄ were actively transported into the sap against the electrochemical potential gradient. The cations Ca and Mg, on the other hand, appeared to move passively into the sap. The behaviour of potassium depended on its concentration in the medium. With a relatively low external concentration (0.75 mM) it appeared to be actively tansported into the sap, whilst at higher outside concentrations (7.5 mM) it was apparently moving passively into the xylem down the electrochemical potential gradient. The possibility of potassium being pumped out of the sap with relatively high external concentrations is discussed.     

Trafficking dynamics of glycosylated pannexin 1 proteins

Pannexins are mammalian orthologs of innexins and have a predicted topological folding pattern similar to that of connexins, except they are glycosylated. Rat pannexin 1 is glycosylated at N254 and this residue is important for plasma membrane targeting. Here we demonstrate that cell surface expression levels of the rat pannexin 1 N254Q mutant are rescued by coexpression with the wild-type protein. In paired Xenopus oocytes, the functional effect of this rescue is inconsequential; however, cell surface deglycosylation by PNGase F significantly enhanced functional gap junction formation. In mammalian cells, wild-type oligomers traffic at a slower rate than Myc-or tetracysteine domain-tagged versions, a behavior opposite to that of tagged connexins. The temporal differences of Panx1 trafficking correlate with spatial differences of intracellular localizations induced by Golgi blockage by Brefeldin-A or glycosylation prevention by tunicamycin. Therefore, Panx1 has kinetics and dynamics that make it unique to serve distinct functions separate from connexin-based channels.     

Secretomic Analysis Identifies Alpha-1 Antitrypsin (A1AT) as a Required Protein in Cancer Cell Migration,Invasion, and Pericellular Fibronectin Assembly for Facilitating Lung Colonization of Lung Adenocarcinoma Cells

Metastasis is a major obstacle that must be overcome for the successful treatment of lung cancer. Proteins secreted by cancer cells may facilitate the progression of metastasis, particularly within the phases of migration and invasion. To discover metastasis-promoting secretory proteins within cancer cells, we used the label-free quantitative proteomics approach and compared the secretomes from the lung adenocarcinoma cell lines CL1-0 and CL1-5, which exhibit low and high metastatic properties, respectively. By employing quantitative analyses, we identified 660 proteins, 68 of which were considered to be expressed at different levels between the two cell lines. High levels of A1AT were secreted by CL1-5, and the roles of A1AT in the influence of lung adenocarcinoma metastasis were investigated. Molecular and pathological confirmation demonstrated that altered expression of A1AT correlates with the metastatic potential of lung adenocarcinoma. The migration and invasion properties of CL1-5 cells were significantly diminished by reducing the expression and secretion of their A1AT proteins. Conversely, the migration and invasion properties of CL1-0 cells were significantly increased through the overexpression and secretion of A1AT proteins. Furthermore, the assembly levels of the metastasis-promoting pericellular fibronectin (FN1), which facilitates colonization of lung capillary endothelia by adhering to the cell surface receptor dipeptidyl peptidase IV (DPP IV), were higher on the surfaces of suspended CL1-5 cells than on those of the CL1-0 cells. This discovery reflects previous findings in breast cancer. In line with this finding, FN1 assembly and the lung colonization of suspended CL1-5 cells were inhibited when endogenous A1AT protein was knocked down using siRNA. The major thrust of this study is to demonstrate the effects of coupling the label-free proteomics strategy with the secretomes of cancer cells that differentially exhibit invasive and metastatic properties. This provides a new opportunity for the effective identification of metastasis-associated proteins that are secreted by cancer cells and promote experimental metastasis.Lung cancer is the leading cause of cancer death, and ∼90% of all lung cancer deaths are attributed to metastases (1). Approximately 95% of lung cancer patients are not diagnosed until they develop symptoms, and 85% of the newly diagnosed lung cancer patients are already in the advanced stages of the disease (2, 3). Once the tumor cells have metastasized and spread throughout the lungs, the cancer is considerably more difficult to treat. Invasiveness and metastasis are major threats to successful treatment. Cancer metastasis is an intricate, multi-step process in which the tumor cells must gain both migratory and invasive properties (4). In metastasis research, there are two common in vivo models, spontaneous and experimental metastasis (5–7). In brief, spontaneous metastasis refers to primary tumor cells that are able to dissociate from the primary tumor and metastasize to the secondary organ via the circulatory system. In contrast, experimental metastasis refers to the injection of tumor cells directly into the systemic circulation. Many researchers have attempted to determine the molecular basis of these transitions in hopes of developing target-specific drugs or biomarkers for the prevention and diagnosis of metastasis. Although there have been many discoveries regarding a particular protein''s influence on metastasis, the contribution of many protein targets to the metastatic process remains poorly defined.The term “secretome” was originally coined to refer to the secretory proteins from the entire genome of Bacillus subtilis (8). The word secretome has developed a broader meaning and now refers to the proteins released by a cell, tissue, or organism through various mechanisms, which include classical secretion, nonclassical secretion, membrane protein shedding, and secretion via exosomes (9–11). Each step involved in tumor metastasis, including migration and invasion, requires specific molecular interactions by both the tumor cells and the surrounding extracellular matrix (12). Some interactions are mediated by secretory factors that function as catalytic agents or by specific recognitions. For example, cathepsins, a family of lysosomal cysteine and aspartic proteases, plays a role in breaking down the connective barriers in the extracellular matrix and basement membranes, effectively enhancing the metastasis of tumor cells (13). These unique functions correlate with invasive activity and are otherwise known as the promigratory and pro-invasive effects on cells (14, 15). With respect to cancer progression, chronic changes or abnormal secretions of certain proteins may indicate a pathologic condition and, therefore, provide suitable targets for therapeutic and biomarker discoveries (16).Proteomic tools have been proposed as a new platform for studying complex biological functions, which entail large numbers and networks of proteins (17). Moving beyond the imposing burden of providing lists of proteins identified in certain samples, the field of quantitative proteomics yields information that specifically recognizes the differences between samples and has emerged as a very important area of research in the study of cancer. These proteomics approaches have been extensively applied to cell secretome analyses for the elucidation of disease mechanisms, diagnoses, and new drug developments (16, 18–23). To comprehensively understand the roles of the secretion-related regulations in metastatic progression, the label-free quantitative proteomics approach was used to identify metastatic-associated proteins secreted by lung adenocarcinoma cells. Comparative secretome analysis was conducted in lung adenocarcinoma cells with differing levels of migration and invasiveness (CL1-0 versus CL1-5) (24). The CL1 cell lines have been used in previous metastasis research as novel protein targets associated with lung cancer metastasis discovery (25–28). The characterized protein, A1AT, was validated for its association and functions involved with lung adenocarcinoma metastasis by subjecting the cells to experimental metastasis assays in vitro and in vivo.     

Active Transport of Ions across the Root of Ricinus communis

The electrical potential difference between the exuding sapof detopped castor oil plants and the external I mM KC1 bathingsolution has been measured, together with the concentrationsof potassium and chloride. A typical value for the electricalpotential of the sap with respect to the external solution was—56 mV, while the sap concentrations of potassium andchloride were 8 mM and 3.5 mM respectively. The total cationconcentration, made up of K, Na, Ca, and Mg, was about Io m.equiv.1.;the anion deficit is made up by SO₄, NO₃, and PO₄. A comparisonof the measured potential difference with the Nernst potentialsfor potassium and chloride suggests that the movement of potassiuminto the sap is a passive process while the movement of chlorideis an active process against the electrochemical potential gradient.It is suggested that the potassium and chloride ions pass throughthe diffusion barrier at which active transport takes placebefore they exchange with the other ions which appear in thesap. On the basis of this assumption it is shown that potassiumis close to passive equilibrium while chloride is accumulatedagainst an electrochemical potential difference of 110 mV or2, 500 cal.mole^–1.     

Microparticle-Induced Activation of the Vascular Endothelium Requires Caveolin-1/Caveolae

Microparticles (MPs) are small membrane fragments shed from normal as well as activated, apoptotic or injured cells. Emerging evidence implicates MPs as a causal and/or contributing factor in altering normal vascular cell phenotype through initiation of proinflammatory signal transduction events and paracrine delivery of proteins, mRNA and miRNA. However, little is known regarding the mechanism by which MPs influence these events. Caveolae are important membrane microdomains that function as centers of signal transduction and endocytosis. Here, we tested the concept that the MP-induced pro-inflammatory phenotype shift in endothelial cells (ECs) depends on caveolae. Consistent with previous reports, MP challenge activated ECs as evidenced by upregulation of intracellular adhesion molecule-1 (ICAM-1) expression. ICAM-1 upregulation was mediated by activation of NF-κB, Poly [ADP-ribose] polymerase 1 (PARP-1) and the epidermal growth factor receptor (EGFR). This response was absent in ECs lacking caveolin-1/caveolae. To test whether caveolae-mediated endocytosis, a dynamin-2 dependent process, is a feature of the proinflammatory response, EC’s were pretreated with the dynamin-2 inhibitor dynasore. Similar to observations in cells lacking caveolin-1, inhibition of endocytosis significantly attenuated MPs effects including, EGFR phosphorylation, activation of NF-κB and upregulation of ICAM-1 expression. Thus, our results indicate that caveolae play a role in mediating the pro-inflammatory signaling pathways which lead to EC activation in response to MPs.