Reactive Thiol Groups Research Articles

In vivo response to dynamic hyaluronic acid hydrogels

Tissue-specific elasticity arises in part from developmental changes in extracellular matrix over time, e.g. ∼10-fold myocardial stiffening in the chicken embryo. When this time-dependent stiffening has been mimicked in vitro with thiolated hyaluronic acid (HA-SH) hydrogels, improved cardiomyocyte maturation has been observed. However, host interactions, matrix polymerization, and the stiffening kinetics remain uncertain in vivo, and each plays a critical role in therapeutic applications using HA-SH. Hematological and histological analysis of subcutaneously injected HA-SH hydrogels showed minimal systemic immune response and host cell infiltration. Most importantly, subcutaneously injected HA-SH hydrogels exhibited time-dependent porosity and stiffness changes at a rate similar to hydrogels polymerized in vitro. When injected intramyocardially host cells begin to actively degrade HA-SH hydrogels within 1week post-injection, continuing this process while producing matrix to nearly replace the hydrogel within 1month post-injection. While non-thiolated HA did not degrade after injection into the myocardium, it also did not elicit an immune response, unlike HA-SH, where visible granulomas and macrophage infiltration were present 1month post-injection, likely due to reactive thiol groups. Altogether these data suggest that the HA-SH hydrogel responds appropriately in a less vascularized niche and stiffens as had been demonstrated in vitro, but in more vascularized tissues, in vivo applicability appears limited.

Differential anodic oxidation of single organic linkage molecules enabling orthogonal bio-immobilization

We report a novel method for preparing electrode surfaces that present thiol-containing or thiol-reactive groups using a single organic silane linkage terminated with 1,3-dithiane via differential anodic oxidation of 1,3-dithiane. We found that the surface groups resulting from the anodic oxidation reaction differed according to the concentration of H2O in the electrolyte solution during the oxidation reaction. The differential anodic oxidation was used successfully for the orthogonal immobilization of two different proteins. We demonstrated also the preparation of an immunoassay platform via orthogonal immobilization, which was then used to detect multiple target antigens based on a sandwich immunoassay.

Structure and interactions in α-crystallin probed through thiol group reactivity

a-Crystallin is the major structural protein of eye lens of vertebrates. In human lens, the ratio of aA-crystallin to aB-crystallin was found to be 3:1. aA-Crystallin contains two cysteine residues at positions 131 and 142, which are at the junction between the a-crystallin domain and the C-terminal tail. We used the accessibility of the thiol groups by Ellman’s reagent (DTNB) as a tool to gain information about the various structural perturbations of hinge region of a-crystallin and during the binding with substrates. In the native condition, the cys-142 though reacted quite fast was not fully exposed. Several reagents were used to see the accessibility of cys-131. Rate constant for cys-131 was increased gradually with increase in the concentration of reagents. The bindings of substrates are affected by the accessibility of thiol indicating that the substrates bind to the hinge region of a-crystallin. By blocking of cys-142, it was observed that the accessibility of one thiol depends on the other thiol, and they are not independent. The hinge region of a-crystallin is very important as substrate binding site and from this study we have got various structural information about that region.

Short communication: Oxidative status and incidence of mastitis relative to blood α-tocopherol concentrations in the postpartum period in dairy cows

Vitamin E supplementation, when combined with high blood α-tocopherol (>6.25μg/mL) at dry off, has been reported to unexpectedly increased the risk for clinical mastitis in dairy cows. Furthermore, higher levels of oxidative stress in the postpartum period were related to higher risk of mastitis. The objective of the present study was to determine the relationship between various serum biomarkers of oxidative status, incidence of mastitis, and blood α-tocopherol concentrations at dry off and at calving. A total of 146 dairy cows from a commercial farm were used in an observational field study. All cows were supplemented with 3,000 and 50IU/cow per day of all-rac-α-tocopherol during the dry period and lactation, respectively. Blood samples were collected at dry off and at calving. Serum was analyzed for α-tocopherol, levels of reactive oxygen metabolites (ROM), thiol groups (SH), and ferric-reducing ability. Three α-tocopherol groups at calving were created: high (>3μg/mL), medium (2–3μg/mL), and low (<2μg/mL). Three α-tocopherol groups at dry off were created: high (>6.25μg/mL), medium (4.25–6.25μg/mL), and low (<4.25μg/mL). All cases of clinical mastitis that occurred during the dry period and the entire subsequent lactation were verified by a veterinarian. No differences were observed in the incidence of mastitis between the 3 α-tocopherol groups based on the serum levels at dry off. Incidence of mastitis was 4 times lower in the high and medium groups when compared with the corresponding value for the low-α-tocopherol group based on the serum levels at calving. Lower levels of ROM and SH at dry off and at calving were found in the group of cows with the highest α-tocopherol values at dry off when compared with the corresponding values in the low-α-tocopherol group. The ROM values at dry off but not at calving were lower in the group of cows with the highest α-tocopherol values at calving when compared with the corresponding values in the low-α-tocopherol group. No differences were observed in ferric-reducing ability values between the 3 α-tocopherol groups at dry off or calving. No differences were observed in all biomarkers of oxidative status between healthy cows and those with mastitis. Thus, blood α-tocopherol is inversely related to certain biomarkers of oxidative stress in the postpartum period and incidence of mastitis. However, reduction in the incidence of mastitis is not mediated through a reduction in the levels of various biomarkers of oxidative stress.

Development of a Novel Composite Material with Carbon Nanotubes Assisted by Self-Assembled Peptides Designed in Conjunction with β-Sheet Formation

A novel composite material is developed with single-walled carbon nanotubes (SWCNTs) and artificially designed peptides, and its chemical and physicochemical characteristics are evaluated with an aim toward biomedical application. The peptides were designed to form a β-sheet structure that would be suitable for wrapping SWCNTs. The complex of SWCNTs and peptide (SWCNT-peptide) showed good dispersibility in aqueous media and was considerably stable even in the absence of an excess amount of peptide in the media. The formation of SWCNT-peptide was confirmed by its performance in water, atomic force microscopy and transmission electron microscopy observation, and molecular modeling. The possibility of introducing various functions to SWCNT-peptide was also demonstrated by several methods, such as introduction of special amino acids, chemical modification, and additional complex formation based on electrostatic interaction. These results suggest the potential of the SWCNT-peptide complex as a molecular platform on which a desirable structure and/or function can be constructed for biomedical and industrial application.

S-Protected Thiolated Chitosan for Oral Delivery of Hydrophilic Macromolecules: Evaluation of Permeation Enhancing and Efflux Pump Inhibitory Properties

The objective of this study was the investigation of permeation enhancing and P-glycoprotein (P-gp) inhibition effects of a novel thiolated chitosan, the so-named S-protected thiolated chitosan. Mediated by a carbodiimide, increasing amounts of thioglycolic acid (TGA) were covalently bound to chitosan (CS) in the first step of modification. In the second step, these thiol groups of thiolated chitosan were protected by disulfide bond formation with the thiolated aromatic residue 6-mercaptonicotinamide (6-MNA). Mucoadhesive properties of all conjugates were evaluated in vitro on porcine intestinal mucosa based on tensile strength investigations. Permeation enhancing effects were evaluated ex vivo using rat intestinal mucosa and in vitro via Caco-2 cells using the hydrophilic macromolecule FD(4) as the model drug. Caco-2 cells were further used to show P-gp inhibition effects by using Rho-123 as P-gp substrate. Apparent permeability coefficients (P(app)) were calculated and compared to values obtained from each buffer control. Three different thiolated chitosans were generated in the first step of modification, which displayed increasing amounts of covalently attached free thiol groups on the polymer backbone. In the second modification step, more than 50% of these free thiol groups were covalently linked with 6-MNA. Within 3 h of permeation studies on excised rat intestine, P(app) values of all S-protected chitosans were at least 1.3-fold higher compared to those of corresponding thiomers and more than twice as high as that of unmodified chitosan. Additional permeation studies on Caco-2 cells confirmed these results. Because of the chemical modification and higher amount of reactive thiol groups, all S-protected thiolated chitosans exhibit at least 1.4-fold pronounced P-gp inhibition effects in contrast to their corresponding thiomers. These features approve S-protected thiolated chitosan as a promising excipient for various drug delivery systems providing improved permeation enhancing and efflux inhibition effects.

Structurally Diverse Cyclisation Linkers Impose Different Backbone Conformations in Bicyclic Peptides

Combinatorial libraries of structurally diverse peptide macrocycles offer a rich source for the development of high-affinity ligands to targets of interest. In this work we have developed linkers for the generation of genetically encoded bicyclic peptides and tested whether the peptides cyclised by them have significant variations in their backbone conformations. Two new cyclisation reagents, each containing three thiol-reactive groups, efficiently and selectively cyclised linear peptides containing three cysteine moieties. When the mesitylene linker of the bicyclic peptide PK15, a potent inhibitor of plasma kallikrein (K(i)=2 nM), was replaced by the new linkers, its inhibitory activity dropped by a factor of more than 1000, suggesting that the linkers impose different conformations on the peptide. Indeed, structural analysis by solution-state NMR revealed different NOE constraints in the three bicyclic peptides, indicating that these relatively small linkers at the centres of bicyclic peptide structures significantly influence the conformations of the peptides. These results demonstrate the prominent structural role of linkers in peptide macrocycles and suggest that application of different cyclisation linkers in a combinatorial fashion could be an attractive means to generate topologically diverse macrocycle libraries.

Protein S-nitrosylation and cancer

Protein S-nitrosylation is a covalent post-translational modification through coupling of a nitric oxide (NO) moiety with the reactive thiol group of a protein cysteine residue to form an S-nitrosothiol (SNO). S-nitrosylation is a key mechanism in the transmission of NO-based cellular signals in the vital cellular processes, including transcription regulation, DNA repair, and apoptosis. Contemporary research has implicated dysregulation of S-nitrosylation in severe pathological events, including cancer onset, progression, and treatment resistance. The S-nitrosylation status may be directly linked to many cancer therapy outcomes as well as therapeutic-resistance, emphasizing the need to develop S-nitrosylation-related anti-cancer therapeutics. The role of S-nitrosylated proteins in the development and progression of cancer are varied, generating a critical need for a thorough review of the current dynamic research in this area.

The Structure of the Thioredoxin–Triosephosphate Isomerase Complex Provides Insights into the Reversible Glutathione-Mediated Regulation of Triosephosphate Isomerase

Protein-protein interactions are crucial for many biological functions. The redox interactome encompasses numerous weak transient interactions in which thioredoxin plays a central role. Proteomic studies have shown that thioredoxin binds to numerous proteins belonging to various cellular processes, including energy metabolism. Thioredoxin has cross talk with other redox mechanisms involving glutathionylation and has functional overlap with glutaredoxin in deglutathionylation reactions. In this study, we have explored the structural and biochemical interactions of thioredoxin with the glycolytic enzyme, triosephosphate isomerase. Nuclear magnetic resonance chemical shift mapping methods and molecular dynamics-based docking have been applied in deriving a structural model of the thioredoxin-triosephosphate isomerase complex. The spatial proximity of active site cysteine residues of thioredoxin to reactive thiol groups on triosephosphate isomerase provides a direct link to the observed deglutathionylation of cysteine 217 in triosephosphate isomerase, thereby reversing the inhibitory effect of S-glutathionylation of triosephosphate isomerase.

Three‐Dimensional Interdigitated Electrode Array as a Tool for Surface Reactions Registration

AbstractPossible applications of a new transducer based on a three dimensional interdigitated electrode array (3D‐IDEA) with electrode digits separated by an insulating barrier are discussed. Due to the presence of insulating barriers that separate the adjacent digits of the electrodes the main portion of the probing electrical current goes close to the surface of the barrier. Chemical modification of the barrier surface with the probe molecules permits to realise direct detection of subsequent target analytes in solution. The functional mechanism of the device is based on registration of changes in conductivity at the surface of the barrier provoked by electrical charge redistribution caused by surface chemical reactions. Three‐dimensional sensor shows considerable improvement in sensitivity compared with a standard planar IDEA design sensors. The potential of the developed device as a sensor transducer to detect various chemical and bio chemical reactions is demonstrated. Examples include enzyme immobilisation, polyelectrolytes layer‐by‐layer deposition, covalent chemical modification with molecules containing reactive thiol groups.

Homocysteine-Induced Apoptosis in Endothelial Cells Coincides With Nuclear NOX2 and Peri-nuclear NOX4 Activity

Apoptosis of endothelial cells related to homocysteine (Hcy) has been reported in several studies. In this study, we evaluated whether reactive oxygen species (ROS)-producing signaling pathways contribute to Hcy-induced apoptosis induction, with specific emphasis on NADPH oxidases. Human umbilical vein endothelial cells were incubated with 0.01–2.5 mM Hcy. We determined the effect of Hcy on caspase-3 activity, annexin V positivity, intracellular NOX1, NOX2, NOX4, and p47phox expression and localization, nuclear nitrotyrosine accumulation, and mitochondrial membrane potential (ΔΨ m). Hcy induced caspase-3 activity and apoptosis; this effect was concentration dependent and maximal after 6-h exposure to 2.5 mM Hcy. It was accompanied by a significant increase in ΔΨ m. Cysteine was inactive on these parameters excluding a reactive thiol group effect. Hcy induced an increase in cellular NOX2, p47phox, and NOX4, but not that of NOX1. 3D digital imaging microscopy followed by image deconvolution analysis showed nuclear accumulation of NOX2 and p47phox in endothelial cells exposed to Hcy, but not in control cells, which coincided with accumulation of nuclear nitrotyrosine residues. Furthermore, Hcy enhanced peri-nuclear localization of NOX4 coinciding with accumulation of peri-nuclear nitrotyrosine residues, a reflection of local ROS production. p47phox was also increased in the peri-nuclear region. The Hcy-induced increase in caspase-3 activity was prevented by DPI and apocynin, suggesting involvement of NOX activity. The data presented in this article reveal accumulation of nuclear NOX2 and peri-nuclear NOX4 accumulation as potential source of ROS production in Hcy-induced apoptosis in endothelial cells.

Redox profile in liver of Leporinus macrocephalus exposed to different dissolved oxygen levels

Aquatic organisms are continuously exposed to environmental variations, which can lead to physiological and biochemical alterations. Leporinus macrocephalus, known as piavuçu, is a migratory species that may be exposed to variations in dissolved oxygen levels. Studies evaluating oxidative changes undergone by this species in these conditions are scarce. Therefore, this investigation aimed at evaluating oxidative alterations in L. macrocephalus exposed to different oxygen levels for 96h: 6.12±0.18, 3.99±0.17, 3.22±0.17, 2.47±0.30 and 0.710±0.07mgL(-1). At the end of the experimental period, fish were euthanized and livers used to determine lipid hydroperoxides, thiobarbituric acid reactive substances, catalase, glutathione-S-transferase, superoxide dismutase and thiol groups, which are an indirect measure of reduced glutathione. Results indicated a decrease in the studied parameters in hypoxic situations, suggesting a possible metabolic depression.

Seeing the smoking gun: a sensitive and specific method to visualize loss of the tumour suppressor, fumarate hydratase, in human tissues

In this issue of the Journal of Pathology, Bardella et al report a method for identifying tumours that lack fumarate hydratase. The approach they use is immunodetection of proteins that have been modified by a non-enzymatic reaction of thiol groups in proteins with fumarate, which is termed succination. Validation included the use of mice with targeted inactivation of fumarate hydratase in the kidney, extensive studies of normal human tissues and examination of over 1000 specimens from human cancers not associated with FH mutations. Detection of protein succination is likely to provide a sensitive and specific method for pathologists to identify the small proportion of papillary renal cell carcinomas that are associated with germline mutations in the FH gene.

Multi-step surface functionalization of polyimide based evanescent wave photonic biosensors and application for DNA hybridization by Mach-Zehnder interferometer

The process of surface functionalization involving silanization, biotinylation and streptavidin bonding as platform for biospecific ligand immobilization was optimized for thin film polyimide spin-coated silicon wafers, of which the polyimide film serves as a wave guiding layer in evanescent wave photonic biosensors. This type of optical sensors make great demands on the materials involved as well as on the layer properties, such as the optical quality, the layer thickness and the surface roughness. In this work we realized the binding of a 3-mercaptopropyl trimethoxysilane on an oxygen plasma activated polyimide surface followed by subsequent derivatization of the reactive thiol groups with maleimide-PEG 2-biotin and immobilization of streptavidin. The progress of the functionalization was monitored by using different fluorescence labels for optimization of the chemical derivatization steps. Further, X-ray photoelectron spectroscopy and atomic force microscopy were utilized for the characterization of the modified surface. These established analytical methods allowed to derive information like chemical composition of the surface, surface coverage with immobilized streptavidin, as well as parameters of the surface roughness. The proposed functionalization protocol furnished a surface density of 144 fmol mm −2 streptavidin with good reproducibility (13.9% RSD, n = 10) and without inflicted damage to the surface. This surface modification was applied to polyimide based Mach-Zehnder interferometer sensors to realize a real-time measurement of streptavidin binding validating the functionality of the MZI biosensor. Subsequently, this streptavidin surface was employed to immobilize biotinylated single-stranded DNA and utilized for monitoring of selective DNA hybridization. These proved the usability of polyimide based evanescent photonic devices for biosensing application.

Redox agents and N-ethylmaleimide affect the extractability of gluten proteins during fresh pasta processing

The gluten protein network is of great importance for pasta cooking quality. Redox agents were used as a tool to impact the protein network formation during laboratory scale fresh pasta making (mixing and sheet rolling) and cooking. SE- and RP-HPLC data showed that disulphide bonds are formed in the pre-existing gluten protein network during cooking of fresh pasta and that, in the process, glutenin polymerisation occurs faster than gliadin–glutenin copolymerisation. The thiol blocking agent N-ethylmaleimide (245ppm, expressed on semolina, dry basis) and, to a lesser extent, the oxidising agent potassium iodate (70ppm), hindered glutenin polymerisation and gliadin–glutenin copolymerisation during cooking. However, the introduction of reactive thiol groups, by addition of the reducing agent glutathione (100ppm), resulted in faster gliadin–glutenin copolymerisation during cooking.

Immobilization of biomolecules onto surfaces according to ultraviolet light diffraction patterns

We developed a method for immobilization of biomolecules onto thiol functionalized surfaces according to UV diffraction patterns. UV light-assisted molecular immobilization proceeds through the formation of free, reactive thiol groups that can bind covalently to thiol reactive surfaces. We demonstrate that, by shaping the pattern of the UV light used to induce molecular immobilization, one can control the pattern of immobilized molecules onto the surface. Using a single-aperture spatial mask, combined with the Fourier transforming property of a focusing lens, we show that submicrometer (0.7 μm) resolved patterns of immobilized prostate-specific antigen biomolecules can be created. If a dual-aperture spatial mask is used, the results differ from the expected Fourier transform pattern of the mask. It appears as a superposition of two diffraction patterns produced by the two apertures, with a fine structured interference pattern superimposed.

Controlling Surface Functionality through Generation of Thiol Groups in a Self-Assembled Monolayer

A lithographic method to generate reactive thiol groups on functionalized synthetic diamond for biosensor and molecular electronic applications is developed. We demonstrate that ultrananocrystalline diamond (UNCD) thin films covalently functionalized with surface-generated thiol groups allow controlled thiol-disulfide exchange surface hybridization processes. The generation of the thiol functional head groups was obtained by irradiating phenylsulfonic acid (PSA) monolayers on UNCD surfaces. The conversion of the functional headgroup of the self-assembled monolayer was verified by using X-ray photoelectron spectroscopy (XPS), near-edge X-ray absorption fine structure (NEXAFS), and fluorescence microscopy. Our findings indicate the selective generation of reactive thiol surface groups. Furthermore, we demonstrate the grafting of yeast cytochrome c to the thiol-modified diamond surface and the electron transfer between protein and electrode.

Arraying prostate specific antigen PSA and Fab anti‐PSA using light‐assisted molecular immobilization technology

We here report for the first time the creation of prostate specific antigen (PSA) and Fab anti-PSA biosensor arrays using UV light-assisted molecular immobilization (LAMI), aiming at the detection and quantification of PSA, a cancer marker. The technology involves formation of free, reactive thiol groups upon UV excitation of protein aromatic residues located in spatial proximity of disulphide bridges, a conserved structural feature in both PSA and Fab molecules. The created thiol groups bind onto thiol reactive surfaces leading to oriented covalent protein immobilization. Protein activity was confirmed carrying out immunoassays: immobilized PSA was recognized by Fab anti-PSA in solution and immobilized Fab anti-PSA cross-reacted with PSA in solution. LAMI technology proved successful in immobilizing biomedically relevant molecules while preserving their activity, highlighting that insight into how light interacts with biomolecules may lead to new biophotonic technologies. Our work focused on the application of our new engineering principles to the design, analysis, construction, and manipulation of biological systems, and on the discovery and application of new engineering principles inspired by the properties of biological systems.

Use of modified whey protein in yoghurt formulations

Substituting native or denatured whey protein ingredients for milk protein in yoghurt formulations resulted in excessive elasticity and grainy texture. These characteristics were incompatible with the smooth and short texture expected from yoghurt. Whey protein ingredients were modified to produce yoghurts with acceptable texture properties. Alteration of the ratio of α-lactalbumin to β-lactoglobulin, heat denaturation and hydrolysis treatments were applied to whey protein to improve their behaviour in yoghurt formulations. Ingredients with increased proportion of α-lactalbumin or made from partially hydrolyzed protein produced yoghurts that closely matched the characteristics of control yoghurt. The effect of whey protein ingredients on yoghurt rheological properties and dispersibility was related to the concentrations of reactive thiol groups that determined the extent of cross linking during acidification. During storage, yoghurt firmness and viscosity increased and syneresis decreased. Yoghurt microstructure was altered by whey protein ingredients, which significantly reduced void spaces and increased gel matrix compactness.

New insights on the formation of colloidal whey protein particles

This paper describes the formation and properties of whey protein particle suspensions having different particle sizes and different abilities to form S–S bridges. Simple shear flow was used to control the protein particles size. The ability to form S–S bridges was steered by blocking the reactive thiol groups of the whey proteins with N-ethylmaleimide. Microscopy and light scattering showed that simple shear flow applied during the formation of whey protein particles give irregularly shaped particles. Especially small particles aggregated into particle clusters. Microscopy and rheological measurements (strain and shear rate sweeps) showed that the protein particle clustering was favoured by the ability of the protein to form S–S bridges and to a lesser extend by a smaller particle size. From the study, it can be concluded that the formation of S–S bridges has no effect on the formation process of protein particles, but S–S bridges are important for the ability of the whey protein particles to form particle clusters.