Breakage Of Disulfide Bonds Research Articles

Effect of UV-Radiation on the Packaging-Related Properties of Whey Protein Isolate Based Films and Coatings

The high oxygen barrier properties of whey protein based films and coatings means these materials are of great interest to the food and packaging industry. However, these materials have poor mechanical properties such as the tensile strength, Young's modulus and elongation at break. Up until now, the influence of ultraviolet (UV) radiation on whey protein films has not been reported in the literature. This study thus investigates the influence of UV-radiation on the properties of whey protein based films. UV-irradiated films showed increased tensile strength and a yellowing that was dependent on the radiation time. After irradiation, the films showed no significant change in the barrier properties, Young's modulus or elongation at break. In addition, a protein solubility study was undertaken to characterize and quantify changes in structure-property relationships. The significant decrease in protein solubility in buffer systems which break disulfide and non-covalent bonds indicates that additional molecular interactions arise with increasing radiation dose. This study provides new data for researchers and material developers to tailor the characteristics of whey protein based films according to their intended application and processing. Copyright © 2015 John Wiley & Sons, Ltd.

Residue Specific Radiation Damage of Protein Structures using High-Resolution Cryo-Electron Microscopy

High-resolution structures of proteins and protein complexes are currently determined using either X-ray crystallography, NMR spectroscopy, or now also cryo-electron microscopy (cryo-EM). The highest resolutions achieved by cryo-EM have been typically restricted to large, well-ordered entities such as helical or icosahedral assemblies or 2D crystals. However, we show that emerging methods in single-particle cryo-EM now allow structure determination at near-atomic resolution, even for much smaller protein complexes with low symmetry. We solved the structure of the ∼465-kDa Escherichia coli β-galactosidase at ∼3.2-Å resolution using single-particle cryo-EM. At this resolution, the majority of the side-chains, the N-termini, and the geometry of the active sites, including a catalytic Mg2+-ion, can be clearly discerned in the density map. Inspection of the map reveals that while densities for residues with positively charged and neutral side-chains are well resolved, systematically weaker densities are observed for residues with negatively charged side-chains. The negatively charged glutamate and aspartate show on average 30% less density than the similarly sized neutral glutamine and asparagine. This observation is independent of the exposure of these residues to solvent. Analysis of other high-resolution cryo-EM structures reveals similar weaker densities for these types of residues. Radiation damage in X-ray crystallography has been linked to decarboxylation of glutamate and aspartate residues, breakage of disulfide bonds, loss of hydroxyl-groups from tyrosine and methylthio-group of methionine. We now show that negatively charged residues exhibit more pronounced effects of radiation damage in structures solved by cryo-EM. We determined that the degree of damage is dose dependent by comparison of density maps obtained using electron doses ranging from 10-30 electrons/Å2. In summary, we establish the feasibility of determining structures at near-atomic resolution and provide a measure of the effects of radiation damage in high-resolution cryo-electron microscopy.

Double-network gels and the toughness of terrestrial slug glue.

The terrestrial slug Arion subfuscus produces a defensive secretion that is sticky and tough, despite being a dilute gel. It is unusual in having high stiffness for a gel, yet retaining the high extensibility typical of mucus. In tensile tests, it sustains an average peak stress of 101 kPa, and fails at an average strain of 9.5. This gives the gel toughness; it requires much greater strain energy to fracture than most gels. This toughness may arise from a double-network type mechanism. In this mechanism, two separate, interpenetrating networks of polymers with different properties combine to give toughness that can be several orders of magnitude greater than either network individually. Native gel electrophoresis suggests that A. subfuscus glue consists of two networks: a network of negatively charged proteins ranging in Mr from 40×10(3) to 220×10(3) that can be dissociated by hydroxylamine and a network of heparan sulfate-like proteoglycans. The two networks are not tightly linked, though proteins of Mr 40×10(3) and 165×10(3) may associate with the carbohydrates. Targeted disruption of either network separately, using enzymatic hydrolysis, disulfide bond breakage or imine bond disruption completely disrupted the glue, resulting in no measurable toughness. Thus, the two networks separately provide little toughness, but together they work synergistically to create a tough material, as predicted in the double-network mechanism.

Biomodification of Rubberized Asphalt and Its High Temperature Properties

Asphalt rubber has been commonly used in the United States for modifying asphalt binder. There have been several studies on improving the interaction and adhesion between rubber and asphalt matrix. The modification of asphalt binder with crumb rubber (CR) is mainly controlled by the exchange equilibrium between components of asphalt binder and CR. This phenomenon increases the size of rubber particles (swelling) up to three times their original size and leads to a significant increase in the viscosity of the resulting asphalt. Swelling and increased viscosity not only promote separation of the modified matrix into two distinguishable phases but also increase the difficulty of pumping and application of rubberized asphalt. This study investigated the effectiveness of treating rubber with an amide-enriched biomodifier to facilitate breakage of disulfide bonds in rubber polymer while promoting interactions between free radicals and amide groups of biomodifier. Promoting interactions between rubber polymer and asphalt aromatic compounds by adding a biomodifier could alleviate phase separation while enhancing overall matrix elasticity. This modification enhanced the rheological properties of rubberized asphalt, including high-temperature properties as well as its palpability. The rotational viscometer and dynamic shear rheometer were used to evaluate the effect of biomodifier on the rheological properties of asphalt rubber at high and intermediate temperatures. Fourier transform infrared spectroscopy was used to examine the presence of rubber polymer compounds inside the asphalt matrix. The results showed that introduction of biomodifier facilitated the application of higher CR percentages while improving the overall rheological properties of the asphalt rubber.

Self-assembly of plasma protein through disulfide bond breaking and its use as a nanocarrier for lipophilic drugs

A disulfide bond breaking strategy used to fabricate HSA nanoparticles with dithiothreitol as the reducing agent is presented. The hydrophobic interaction is a dominating factor in the assembly process. These nanoparticles are suitable lipophilic drug delivery systems, which have significant tumor-targeting capabilities.

Observing Real-Time Molecular Event Dynamics of Apoptosis in Living Cancer Cells using Nuclear-Targeted Plasmonically Enhanced Raman Nanoprobes

Apoptosis is a biological process that plays important roles in embryogenesis, aging, and various diseases. During the process of apoptosis, cells undergo a series of morphological and molecular events such as blebbing, cell shrinkage, proteolysis, and nuclear DNA fragmentation. Investigating these events on a molecular level is crucial for gaining a more complete understanding of the intricate mechanism of apoptosis; however, the simultaneous direct observation of morphological and molecular events in real-time on a single living cell scale still remains a challenge. Herein, we directly monitored morphological and molecular events during cellular apoptosis in real-time after the treatment of an apoptosis-inducing agent, by utilizing our previously described plasmonically enhanced Rayleigh/Raman spectroscopic technique. Spectroscopic analysis of the DNA/protein composition around the cell nucleus revealed the occurrence and dynamics of three apoptotic molecular events: protein denaturation, proteolysis, and DNA fragmentation. The molecular event dynamics were used to create a temporal profile of apoptotic events in single cells. It is found that the sequence of events occurring in the apoptotic process induced by hydrogen peroxide addition is protein denaturation through disulfide bond breakage as well as DNA fragmentation, followed in time by protein unraveling with hydrophobic amino acid exposure, and finally protein degradation. These results demonstrate the potential of using this time-dependent plasmonically enhanced vibrational imaging technique to study the detailed mechanism of other apoptosis molecular pathways induced by different agents (e.g., anticancer drugs). A note is given in the conclusion discussing the expected large difference between the SERS spectrum of biological molecules in solution and that observed in live cells which are enhanced by the plasmonic field of the aggregated nanoparticles.

Effectiveness of MESNA on the success of cholesteatoma surgery

ImportanceIt is important that chronic otitis media with cholesteatoma be treated successfully in patients to protect them from having repeated surgeries with related surgical co-morbidities and hearing loss. ObjectiveTo evaluate the effectiveness of MESNA usage on the residual cholesteatoma rates of the patients who underwent surgery due to chronic otitis media with cholesteatoma. DesignRetrospective single-institution study of a prospectively collected database. SettingTertiary University Hospital. ParticipantsNine hundred and thirty-four patients underwent surgery due to chronic otitis media between September 2000 and March 2012 by the same surgeon. One hundred and forty-one cases out of 934 patients were selected who had cholesteatoma for the study. These randomly selected 141 cases were divided into two groups as follows: I. Forty-six cases were applied MESNA (Sodium 2-mercaptoethanesulfonate) intraoperatively, and II. Ninety-five cases were not applied MESNA intraoperatively. The cases that were followed-up at least one year were included in this study. InterventionMESNA (Ureomitexan, MESNA, Baxter oncology, Germany) was diluted with saline (20% MESNA and 80% saline) that was applied, and then a waiting period of approximately 5min followed to start to dissect cholesteatoma matrix. Main outcomes and measuresResidual cholesteatoma rates between intraoperative MESNA, a disulfide bond breaking chemical agent, applied and MESNA non-applied cases in the postoperative follow-up period were compared for the success of the surgery. ResultsMESNA was used in 46 patients out of 141 cases intraoperatively. Twenty-four of these patients underwent CWD (canal wall down), and twenty-two patients underwent CWU (canal wall up) mastoidectomy. For the other 95 subjects, 56 patients with CWD and 39 with CWU mastoidectomy, MESNA was not applied. Residual cholesteatoma rates were found to be significantly higher in MESNA non-applied group than MESNA applied group (p<0.05). Residual cholesteatoma rates between CWD and CWU mastoidectomy procedures were not statistically significant (p>0.05). Conclusions and relevanceMESNA application that breaks disulfide bonds in the structure of the matrix in cholesteatoma surgery may assist the elimination of the disease, and increase surgical success by facilitating the elevation of the epithelium. Thereby, it causes a decrease in the possibility of remaining residual epithelium after surgery, which decreases the need for second-look surgery. Trial registrationThe retrospective research protocol was approved by the Inonu University Clinical Research Ethics Committee. Registration number:………

Co-delivery of 5-fluorocytosine and cytosine deaminase into glioma cells mediated by an intracellular environment-responsive nanovesicle

The diblock copolymer (PEI-PAsp(DIP/MEA)) of branched polyethyleneimine (PEI) and biodegradable poly(2-diisopropylamino/2-mercaptoethylamine) ethyl aspartate (PAsp(DIP/MEA)) was synthesized and assembled into nanovesicles for the co-delivery of cytosine deaminase (CD) gene and 5-fluorocytosine (pCMVCD/5-FC) into glioma cells. The nanovesicle comprises a hydrophilic outerlayer of branched PEI for pCMVCD complexation, a hydrophilic inner cavity for prodrug 5-FC loading, and a pH-sensitive membrane crosslinked by disulfide to prevent drug leakage into the bloodstream-simulating environment with neutral pH and without reducing agent. Yet, once the PPDM (PEI-PAsp(DIP/MEA)) nanovesicle was internalized into cancer cells and entrapped inside lysosomes featuring a low pH (∼5) and enriched reducing agent (∼10 mM GSH), it dissociated as a result of tertiary amine protonation and disulfide bond breakage to release the loaded drug. The in vitro release studies showed that less than 10% of 5-FC was released from the nanovesicle in 24 h at pH 7.4, whereas about 98% of 5-FC was released in the presence of 10 mM GSH at pH 5.0. The prodrug 5-FC was converted into its toxic active metabolite 5-fluorouracil (5-FU) by cytosine deaminase inside C6 glioma cells, owing to the effective CD gene transfection and expression. As a result, the 5-FC/pCMVCD-loaded nanovesicle induced effective apoptosis and generated a significant cytotoxic effect in the cancer cells. Our results indicated that this multifunctional nanovesicle is a promising carrier for the co-delivery of gene and drug or prodrug in cancer therapy.

Extraction of Keratins from Human Hair at Low Pressure and their Characterization

Keratins are natural proteins that can be found in hair, nails, feathers and horn. Moreover, they could be used as flame retardants for cotton fabrics. Human hairs wastes from salon were discarded in a large amount. The extraction of keratins from human hairs should be claimed as an effective waste management.It is difficult to break disulfide bonds that hold keratins together. The specific solvents used to break these chemical bonds are needed. In this study, keratins were extracted from human hairs using 2.6 M thiourea, 5 M urea and 5% of 2-mercaptoethanol. The effects of temperature, time and pressureon keratin extraction were investigated. The yields of protein extracted were determined bydry weight method. In addition, the chemical composition of keratin was also examined using FTIR. The morphological changes of the hair surface after extraction were examined using anoptical microscope.

Abstract 5513: Discovery of an orally active small-molecule irreversible inhibitor of protein disulfide isomerase (PDI) for ovarian cancer treatment.

Abstract Protein disulfide isomerase (PDI), an endoplasmic reticulum (ER) chaperone protein, catalyzes disulfide bond breakage, formation and rearrangement. The effect of PDI inhibition on ovarian cancer progression is not yet clear, and there is a need for potent, selective and safe small-molecule inhibitors of PDI. Here, we report a new class of novel propynoic acid carbamoyl methyl amides (PACMAs) that are active against a panel of human ovarian cancer cell lines. Using fluorescent derivatives, 2D gel electrophoresis, and mass spectrometry, we established that PACMA 31, one of the most active analogues, acts as an irreversible small-molecule inhibitor of PDI, forming a covalent bond with PDI's active site cysteines. We also demonstrated that PDI activity is essential for the survival and proliferation of human ovarian cancer cells. In vivo, PACMA 31 showed tumor targeting ability and significantly suppressed ovarian tumor growth without causing toxicity to normal tissues. These irreversible small-molecule PDI inhibitors represent a new approach for the development of targeted anticancer agents for ovarian cancer therapy, and can also serve as useful probes for investigating the biology of PDI-implicated pathways. Citation Format: Shili Xu, Alexey N. Butkevich, Roppei Yamada, Yu Zhou, Bikash Debnath, Roger Duncan, Ebrahim Zandi, Nicos A. Petasis, Nouri Neamati. Discovery of an orally active small-molecule irreversible inhibitor of protein disulfide isomerase (PDI) for ovarian cancer treatment. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5513. doi:10.1158/1538-7445.AM2013-5513

Tissue Factor De-encryption, Thrombus Formation, and Thiol-disulfide Exchange

Tissue factor (TF) by forming a complex with factor VIIa (FVIIa) initiates blood coagulation. It was traditionally believed that the separation of FVIIa in circulation from subendothelial TF was the main control that was preventing spontaneous initiation of thrombosis and that circulating cells and endothelium did not express TF protein at rest in healthy individuals. However, TF has been detected in healthy human plasma and animal models of thrombosis, which indicate that TF in circulation can contribute to thrombin generation and fibrin formation after an activation event. Circulating TF-and indeed, most of the TF on the cell surface-is "encrypted" or coagulation inactive. The de-encryption step involves exposure of phosphatidylserine (PS), but PS exposure alone is insufficient for full TF activity. Allosteric disulfide bonds control protein function by mediating conformal change through the formation and breaking of disulfide bonds. TF contains a typical surface exposed allosteric bond in the membrane proximal fibronectin type III domain. Thiol-disulfide exchange involving this disulfide is implicated in TF activation with the formation of the disulfide bond corresponding with the active conformation of TF and free thiol or thiol-modified forms corresponding with encryption. Although the exact mechanism by which TF de-encryption occurs remains a subject of debate, thiol blockade and inhibition of oxidoreductases show an important role for thiol-disulfide reactions in platelet-independent pathways of coagulation in vitro and in vivo. In particular, redox active extracellular protein disulfide isomerase is involved in the earliest stages of thrombus initiation and has proven to be a potential target for antithrombotic drug development.

Molecular Mechanics of Disulfide Bonded Alpha-Helical Protein Filaments

Keratin, an alpha-helical protein, is an abundant material that forms the basis of hair and hoof, and is a composite of alpha-helical coiled coils with dense disulfide bonding between helical protein domains. Here, we report a molecular analysis of the mechanics of disulfide bonded alpha-helical protein filaments, focusing on a systematic assessment of structure–property relationships and deformation and failure mechanisms, carried out using a full atomistic explicit water model based on the CHARMM force field, extended here to capture the breaking of disulfide bonds in varied chemical microenvironments. By considering a three-strand alpha-helical model of an assembly of disulfide bonds under an external loading, we demonstrate that weak disulfide cross-link results in a highly cooperative behavior. Strong disulfide bonding resist greater external load, but the cooperative behavior is reduced. We compare the mechanical behavior of the disulfide bonded systems to a molecule with weaker H-bonds between alpha-helix domains. Under mechanical loading, H-bonds between the protein filaments are easily sacrificed and the alpha-helical structure is maintained, but the system has a lower strength. Our atomistic models provide fundamental insight into the effect of disulfide cross-link on mechanical properties of alpha-helix-based protein filament and reveals that the dependence of disulfide bond strength on the chemical microenvironment enables a tunable fiber strength by a factor of ≈2.5.

Discovery of an orally active small-molecule irreversible inhibitor of protein disulfide isomerase for ovarian cancer treatment

Protein disulfide isomerase (PDI), an endoplasmic reticulum chaperone protein, catalyzes disulfide bond breakage, formation, and rearrangement. The effect of PDI inhibition on ovarian cancer progression is not yet clear, and there is a need for potent, selective, and safe small-molecule inhibitors of PDI. Here, we report a class of propynoic acid carbamoyl methyl amides (PACMAs) that are active against a panel of human ovarian cancer cell lines. Using fluorescent derivatives, 2D gel electrophoresis, and MS, we established that PACMA 31, one of the most active analogs, acts as an irreversible small-molecule inhibitor of PDI, forming a covalent bond with the active site cysteines of PDI. We also showed that PDI activity is essential for the survival and proliferation of human ovarian cancer cells. In vivo, PACMA 31 showed tumor targeting ability and significantly suppressed ovarian tumor growth without causing toxicity to normal tissues. These irreversible small-molecule PDI inhibitors represent an important approach for the development of targeted anticancer agents for ovarian cancer therapy, and they can also serve as useful probes for investigating the biology of PDI-implicated pathways.

Determination of dalcetrapib by liquid chromatography–tandem mass spectrometry

The cholesteryl ester transfer protein modulator dalcetrapib is currently under development for the prevention of dyslipidemia and cardiovascular disease. Dalcetrapib, a thioester, is rapidly hydrolyzed in vivo to the corresponding thiophenol which in turn is further oxidized to the dimer and mixed disulfides (where the thiophenol binds to peptides, proteins and other endogenous thiols). These forms co-exist in an oxidation-reduction equilibrium via the thiol and cannot be stabilized without influencing the equilibrium, hence specific determination of individual components, i.e., in order to distinguish between the free thiol, the disulfide dimer and mixed disulfide adducts, was not pursued for routine analysis. The individual forms were quantified collectively as dalcetrapib-thiol (dal-thiol) after reduction under basic conditions with dithiothreitol to break disulfide bonds and derivatization with N-ethylmaleimide to stabilize the free thiol. The S-methyl and S-glucuronide metabolites were determined simultaneously with dal-thiol with no effect from the derivatization procedure. Column-switching liquid chromatography-tandem mass spectrometry provided a simple, fast and robust method for analysis of human and animal plasma and human urine samples. Addition of the surfactant Tween 80 to urine prevented adsorptive compound loss. The lower limits of quantitation (LLOQ) were 5 ng/mL for dal-thiol, and 5 ng/mL for the S-methyl and 50 ng/mL for the S-glucuronide metabolites. Using stable isotope-labeled internal standards, inter- and intra-assay precisions were each <15% (<20% at LLOQ) and accuracy was between 85 and 115%. Recovery was close to 100%, and no significant matrix effect was observed.

Electrochemistry-Assisted Top-Down Characterization of Disulfide-Containing Proteins

Covalent disulfide bond linkage in a protein represents an important challenge for mass spectrometry (MS)-based top-down protein structure analysis as it reduces the backbone cleavage efficiency for MS/MS dissociation. This study presents a strategy for solving this critical issue via integrating electrochemistry (EC) online with a top-down MS approach. In this approach, proteins undergo electrolytic reduction in an electrochemical cell to break disulfide bonds and then undergo online ionization into gaseous ions for analysis by electron-capture dissociation (ECD) and collision-induced dissociation (CID). The electrochemical reduction of proteins allows one to remove disulfide bond constraints and also leads to increased charge numbers of the resulting protein ions. As a result, sequence coverage was significantly enhanced, as exemplified by β-lactoglobulin A (24 vs 75 backbone cleavages before and after electrolytic reduction, respectively) and lysozyme (5 vs 66 backbone cleavages before and after electrolytic reduction, respectively). This methodology is fast and does not need chemical reductants, which would have an important impact in high-throughput proteomics research.

Adherence analysis between human erythroleukemia cells and human lung fibroblasts

Initially, human erythroluekemia (HEL) cells were mixed with human lung fibroblasts (WI‐38 cells) to create a simulated in vivo environment. Normally, HEL cells grow in suspension while WI‐38 cells grow attached. When the two cell types grow together, HEL cells adhere to the WI‐38 cells (80%). This mixed cellular environment was analyzed for viability, growth, and adherence via trypan blue exclusion and cell enumeration. The viability of the HEL cells that grew attached and unattached to the WI‐38 cells was significantly elevated and depressed, respectively. Growth remains constant for HEL cells with and without WI‐38 cells. To determine adherence, HEL cells were grown in chamber slides coated with and without fibronectin (FN). Approximately 57% of HEL cells adhered to the fibronectin‐coated plate suggesting that FN is the extracellular matrix protein that is responsible for HEL adhesion. Breakage of disulfide bonds with dithiothreitol did not disrupt the attachment between the two cell types, indicating the presence of an additional linkage. Most of the WI‐38 cells that detached were nonviable suggesting that HEL cells reroute WI‐38 cell nutrients to increase their own viability. Support from College of Health, Environment and Science and Slippery Rock University.

Preparation of Genomic DNA from Mammalian Sperm

Semen consists of spermatozoa and the seminal fluid, also called seminal plasma. This fluid is important for the survival of the spermatozoa, but may decrease the purity and thus quality of the DNA due to its fructose and protein content. In this protocol, spermatozoa are washed with ethanol to remove the fluid. The spermatozoa themselves are protected by a membrane rich in disulfide bonds, which impede cell lysis and thus hamper DNA isolation. To break disulfide bonds a strong antioxidant, such as dithiothreitol (DDT), is necessary. Similar to other protocols for DNA isolation from other mammalian tissues, proteinase K and SDS are included in the lysis reaction. After lysis of the sperm cells, the DNA is precipitated using ethanol and then redissolved in TE-buffer or ddH(2)O.

Protein conformation driven biomimetic synthesis of semiconductor nanoparticles

The present investigation demonstrates the role of protein conformation in synthesizing nanoparticles (NPs) through biomimetic route. Highly water-soluble and biocompatible CdS and CdSe nanoparticles in bovine serum albumin (BSA) matrix have been synthesized using a simple and controllable method at room temperature. Fourier transform infrared (FTIR) data are used to envisage the binding of the semiconducting particles with amide and OH groups of the protein molecule. Optical absorption and emission spectra confirm that particles formed lie within the size quantization regime. Circular dichroism spectroscopy reveals that BSA adopts different conformations at different pH which in turn controls the particle size. Further, addition of sodium borohydride (NaBH4) in BSA solution results in breakage of disulfide bonds generating increased number of thiolate groups which provide better stabilization and increased passivation of electronic defects on particle surface. In the process, better quality semiconductor NPs with higher quantum yield are produced. Thus, by modulating the protein conformation, the size and quality of the nanoparticles can be controlled.

A Molecular Dynamics Study on the Effect of Disulfide Bonds in Cys-Loop Ligand-Gated GABAA Receptors

GABAA-receptors (GABARs) are chloride ion channels in the ‘cys-loop receptor’ superfamily that are major inhibitory neuroreceptors. Upon agonist binding, GABARs open and increase intraneuronal chloride ion concentration, hyperpolarize the cell and inhibit neural transmission. Reducing agents will break the disulfide bonds in the cys-loop of the receptor. Indeed, it has been speculated that sulfhydryl compounds modify proteins via thiol/disulfide redox reactions and serve as neuromodulators (1). Reducing reagents were found to decrease the GABA EC50 in the highly homologous GABAC-receptor, while oxidizing reagents increased the EC50 (2). Conversely, conflicting findings suggests the mechanism of action of redox modulation does not alter GABAR agonist-binding affinity (3). However, studies are complicated by the fact that mutagenesis of the cysteines commonly produces failure of subunit assembly (4). Thus, to investigate the effect of disulfide bond breakage on the receptor we generated various GABAR homology models with and without disulfide bonds. These systems were subjected to ligand docking and extensive molecular dynamics simulations to determine if the GABA affinity was modulated. This work performed under the auspices of the U.S. Department of Energy by Lawrence Livermore National Laboratory under Contract DE-AC52-07NA27344.1. Allan, A. M. and L. D. Baier. 1992. Effect of thiol group modification on ion flux and ligand binding properties of the GABAA-benzodiazepine receptor chloride channel complex. Synapse 10:310-316.2. Calero, C. I. and D. J. Calvo. 2008. Redox modulation of homomeric rho1 GABA receptors. J Neurochem 105:2367-2374.3. Pan, Z. H., et al. 2000. Redox modulation of recombinant human GABA(A) receptors. Neuroscience 98:333-338.4. Amin, J., et al. 1994. The agonist binding site of the gamma-aminobutyric acid type A channel is not formed by the extracellular cysteine loop. Mol Pharmacol 45:317-323.LLNL-ABS-502871

Effects of erdosteine and its metabolites on bacterial adhesiveness.

Erdosteine (CAS 84611-23-4) is administered as a mucolytic drug in patients with pulmonary disorders who suffer from a thickening of bronchial mucus with altered physico-chemical characteristics. Erdosteine itself does not have a free thiol group but its metabolization produces active metabolites with a -SH group that is capable of breaking disulfide bonds of mucins and improving the mucociliary clearance of the airways, and thus reproducing the effects of the class of muco-active drugs having a thiol group. It has also been reported that muco-active drugs with this group reduce bacterial adhesiveness to human mucosal cells. The aim of this study was to investigate whether erdosteine and its SH-metabolites are capable of interfering with bacterial adhesiveness. Metabolite I significantly reduces both S. aureus and E. coli adhesiveness to human mucosal epithelial cells at concentrations of 2.5, 5 and 10 micrograms/ml. The same concentrations of erdosteine, metabolite II, metabolite III and N-acetylcysteine (as a control drug) were devoid of such activity, whereas the results of hemagglutination and hydrophobicity assays showed that the behaviour of metabolite I overlapped that of bacterial adhesiveness, thus indicating that interference takes place at a fimbrial level. This is confirmed by the fact that the incubation of human buccal cells with drugs does not reduce the adhesiveness of untreated bacteria. The presence of this additional activity in a muco-active drug is useful because bacteria not only adhere to epithelial cells but also to tracheobronchial secretions.