Cross-linked Products Research Articles

Chitosan-Based Anion Exchange Membranes for Direct Ethanol Fuel Cells

A series of novel cross-linked highly quaternized chitosan and quaternized poly (vinyl alcohol) membranes were successfully synthesized to be applied in alkaline direct ethanol fuel cells. Cross-linking was accomplished using two different cross-linking agents and an additional thermal process to improve both chemical and thermal properties. Equivalent blends of chitosan and poly (vinyl alcohol) membranes with various degrees of cross-linking were prepared by using different amounts of glutaraldehyde and ethylene glycol diglycidyl ether as cross-linkers. To investigate their applicability in direct ethanol fuel cells, the membranes were characterized in terms of their structural properties, chemical, thermal and alkaline stability, ion transport and ionic properties using following methods: Fourier transform infrared spectroscopy, nuclear magnetic resonance spectroscopy, scanning electron microscopy, thermogravimetric analysis, water uptake by mass change, ethanol permeability in the diffusion cell, back titration method (ion exchange capacity) and electrochemical impedance spectroscopy (anion conductivity). Despite the high degree of quaternization of the applied materials and regardless of the thin film thickness of the blend membranes, the novel cross-linked products displayed outstanding mechanical stability. The lower cross-linked membranes exhibited the best transport and ionic properties with a high anion conductivity of 0.016 S cm-1 and a high ion exchange capacity of 1.75 meq g-1, whereas membranes with a higher degree of cross-linking performed superior in terms of reduced ethanol permeability of 3.30∙10-7 cm2 s-1 at 60°C. The blend membranes - chemically and thermally cross-linked - provide excellent thermal stability with an onset degradation temperature above 280°C and superb alkaline stability in 1.0 M KOH at 60°C for 650 h. Therefore, these composite membranes exhibit high potential for application as alkaline electrolytes in fuel cells.

Cross-Linkable Epoxidized Maleinated Castor Oil: A Renewable Resin Alternative to Unsaturated Polyesters

As an alternative resin to conventional synthetic unsaturated polyesters (UPEs), epoxidized maleinated castor oil (EMACO) was synthesized in two steps. For this purpose, castor oil was reacted with maleic anhydride at 70°C to obtain maleinated castor oil (MACO). Then, epoxidation of MACO was carried out by using a mixture of formic acid and hydrogen peroxide at 0–5°C. Then, the free carboxyl groups of the synthesized EMACO were further reacted with free epoxide groups of EMACO at 90°C. At the end of the reaction, an unsaturated polyester precursor-prepolymer was obtained (P-EMACO). FTIR and1H NMR spectroscopic techniques were used to characterize the monomers synthesized. The P-EMACO was then mixed with styrene and cross-linked in the presence of AIBN at 50°C. Thermal and mechanical properties of the final cross-linked product were investigated by thermogravimetric analysis (TGA) and dynamic mechanical analysis (DMA) techniques. The degradation onset temperature of the material at which cross-linked X-EMACO loses 5% of its weight was found to be 209°C. Its dynamicTgand storage modulus at 25°C were determined as 72°C and 1.08 GPa, respectively. These results are higher than some of the different oil based polymers reported in the literature.

Preparation of Polymer Nanotube Using Self-Assembled Metal Organic Nanotube as Template

Hollow nano-material is one of the hot researches in recent years because of special optical, electrical, magnetic and catalytic properties. Polymer nanotube (PNT) is a polymer nano-material with tubular structure. Template method is an effective preparation method for tubular polymer nano-materials. In this study, we have developed a simple technique for the fabrication of polymer nanotubes by using self-assembled metal organic nanotube (MONT) as a template and multiple amine and acid as precursor molecules. An amphiphilic molecule (N-tetradecanoic glycylglycine, 1) was firstly synthesized by the coupling reaction of N-tetradecanoic acid and glycylglycine ethyl ester under the function of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC•HCl), followed by a hydration process. The amphiphilic molecule 1 was successfully obtained in high yields. MONT was then prepared by the self-assembly of 1 with copper(II) nitrate in methanol. A solution of copper(II) nitrate in water was slowly added into a solution of 1 in methanol. The mixed solution was stirred for 24 h at room temperature and MONT was obtained by filtration, washing with water and freeze-dry. And finally, template reactions were carried out as follows: a certain amount of MONT was dispersed in tetrahydrofuran (THF), and then the multiple amine was added to complex with copper ions on the surface of MONT. The mixed solution was stirred for 3 h at room temperature and a coated layer formed on the surface of MONT. The coated layer on the nanotube surface was further cross-linked by an activated ester of citric acid. Finally, the self-assembled template was removed by hot filtration and the PNTs with good dispersibility in water were obtained. The surface topography, composition and structure of PNTs were characterized by scanning electron microscope (SEM), scanning transmission electron microscopy (STEM), differential scanning calorimetry (DSC), X-ray diffraction (XRD) and diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS). The results showed that up to 80% cross-linked products form PNTs when the amount of multiple amines is 0.4 molar equivalent of MONT. The lengths of PNTs are about 500 nm~3 μm, inner diameters are 60~100 nm and outer diameters are 80~120 nm.

The Acidic Region of Factor VIII Light Chain Contains the Thrombin-Binding Site(s) Responsible for the Cleavage at Arg1689

Thrombin-catalyzed activation of factor (F)VIII by cleavages at Arg372, Arg740, and Arg1689 is essential for the propagation phase of blood coagulation cascade. Activated FVIII (FVIIIa) forms the tenase complex and markedly amplifies the activation of FX as a cofactor of FIX. We had already demonstrated that thrombin interacts with FVIII through the residues 392-394 and 484-509 in the A2 domain and the C2 domain, and each association regulates cleavage at Arg740, Arg372, and Arg1689, respectively (Nogami K, JBC 2000, 2005; BJH 2008), and recently reported that the A1 acidic clustered region 340-350 involving the sulfated tyrosine regulate the cleavage of Arg372 (Minami et al. 55th ASH). On the other hand, Fay and colleague suggested that recombinant FVIII lacking the C2 domain retains greater than 50% cofactor activity (JBC 2010), supporting the presence of other thrombin-binding region responsible for cleavage at Arg1689 of the light chain. In this study, we attempted to identify this thrombin-binding site(s). We focused on the acidic residues 1659-1669 and 1675-1685 within the light chain, which had similar sequence to the A1 residues 340-350 in terms of involving the clustered acidic residues and sulfated tyrosine as well as hirugen residues 54-65. We prepared four of synthetic peptides corresponding to the residues 1659-1669 and 1675-1685 with sulfated tyrosine, P(1659-69s) and P(1675-85s), and with non-sulfated tyrosine, P(1659-69) and P(1675-85). The inhibitory effect on the thrombin-catalyzed FVIII activation by each peptide was evaluated in a one-stage clotting assay. Each peptide showed a dose-dependent inhibition on thrombin-catalyzed activation. These inhibitory effects were greater in order of P1675-85s, P1659-69s, P1675-85, P1659-69, and the IC50 were 25, 67, 71 and 225 µM, respectively. The peptides with sulfated tyrosine had approximately 3-fold greater inhibition of the FVIII activation by thrombin than with non-sulfated tyrosine. The IC50 in the presence of mixture of P1675-85s and P1659-69s was 30.4 µM, suggesting that these peptides had no an additive effect. The impacts of P1659-69s and P1675-85s on the thrombin-catalyzed cleavage at Arg1689 were examined by SDS-PAGE/western blotting. These peptides blocked the cleavage at Arg1689 in dose-dependent fashions. In timed-course assay, the presence of P1659-69s and P1675-85s decreased the cleavage rate of Arg1689 by 61.3 % and 81.8 %, respectively compared to its absence. The direct binding of P1659-69s and P1675-85s to thrombin was examined by surface resonance plasmon (SPR)-based assay and by the zero-length cross-linking reagent EDC. In SPR-based assay using a Biacore T200TM, thrombin bound to immobilized P1659-69s and P1675-85s directly with high affinity. The Kd values adjusted to 1:1 binding model of global fitting were 203 nM and 94 nM, respectively. EDC cross-linking in fluid-phase assay revealed that formation of EDC cross-linking products between biotinylated P1659-69s or P1675-85s and thrombin were observed in dose-dependent fashions. The products between the biotinylated peptides (800 nM) and thrombin were competitively reduced by the addition of non-biotinylated peptides. Moreover, N-terminal sequence analysis of cross-linking products between both peptides-thrombin indicated that thrombin bound to the residues 1664-1669 and 1683-1684. Taken together, we demonstrated that the A3 residues 1659-1669 (QEEIDYDDTIS) and residues 1675-1685 (EDFDIYDEDEN) contained the thrombin binding-sites responsible for proteolytic cleavage at Arg1689 of the A3 domain. DisclosuresNogami:Bayer, NovoNordisk, Baxalta, Chugai, Kaketsuken, Pfizer, Biogen: Honoraria; Chugai: Membership on an entity's Board of Directors or advisory committees; Bayer, Novo Nordisk, Baxalta. Biogen: Research Funding. Shima:Chugai Pharmaceutical Co., Ltd. and F. Hoffmann-La Roche Ltd.: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

The Simultaneous Accumulation of the Extremely Cytotoxic Interstrand Cross-Links and Double-Strand Breaks Contributes to the Successful Anti-Myeloma Therapy; The Effect of DNA Repair Inhibitors

Abstract Several anticancer drugs exert their antitumor effects through the production of DNA interstrand cross-links (ICLs). During ICL repair, replication forks stall at the ICL, inducing the formation of a lethal form of DNA damage, termed DNA double-strand breaks (DSBs), which are repaired mainly by homologous recombination (HR) and non-homologous end joining (NHEJ). Herein, using the ICL-inducing drug melphalan, we investigated the underlying mechanisms in processing and repair of pharmaceutical induced DNA damage and their contribution to the successful outcome of melphalan therapy. We studied two human multiple myeloma (MM) cell lines (RPMI8226 and LR5, melphalan-sensitive and -resistant, respectively) as well as CD138+ bone marrow plasma cells (BMPCs) isolated from 15 newly diagnosed MM patients (8M/7F; median age 61 years) who underwent high-dose melphalan with autologous stem cell transplantation as first line therapy. Response assessment was based on the International Myeloma Working Group (IMWG) criteria. The patients categorized based on their outcome to responders (≥PR, n =9) and non-responders (<PR, n =6) to subsequent melphalan therapy. MM cell lines and BMPCs were ex vivo treated with melphalan alone or in combination with selective inhibitors of homologous recombination (RI-1) or non-homologous end joining (SCR7, NU7026) and the extent of the DNA damage formation/repair (monoadducts using Southern blot analysis; ICL using quantitative-PCR; DSBs using immunofluorescence quantification of γH2AX foci by confocal microscopy) as well as the induction of the apoptotic pathway (using a photometric enzyme-immunoassay) were evaluated. In all MM patients, following ex vivo treatment of BMPCs with melphalan, monoadducts were repaired over two sharply demarcated phases: a first rapid phase (0-2h), and a delayed second phase (2-48h). Interestingly, responders showed higher accumulation of monoadducts due to the significantly slower first-phase repair observed in compare to non-responders (P <0.001). Similar rates of monoadducts removal were observed in all MM patients during the second phase of repair. To note, ICL "unhooking" rates were found to be similar in all MM patients. However, accumulation of ICLs was significantly higher in responders' BMPCs compared to non-responder's (P<0.05), due to higher levels of monoadducts (precursors of ICLs) left unrepaired in responders' cells, resulting in higher formation of ICLs. Moreover, DSBs burden was significantly higher in responders compared to non-responders, due to higher accumulation of ICLs (precursors of DSBs) and lower rates of DSBs repair in responders (P <0.001). More importantly, an inverse correlation was found between the apoptotic rates and the first-phase monoadducts repair capacity of the BMPCs as well as with the DSBs repair efficiency of these cells, with the apoptotic rates being significantly higher in responders compared to non-responders (all P <0.05). In line with the BMPCs data, significantly higher accumulation of melphalan-induced monoadducts, ICLs and DSBs were found in RPMI8226 compared to LR5 cells (P <0.01). To further elucidate the mechanisms of drug-induced DSBs repair and their contribution to cell sensitivity, MM cell lines and BMPCs were treated with melphalan in combination with nontoxic doses of DSBs repair inhibitors. We found that the combined treatment of melphalan with an inhibitor significantly increased the melphalan-induced phosphorylation of H2AX and strongly enhanced the cytotoxic activity of melphalan (all P< 0.01). As expected, co-treatment with a DSB repair inhibitor had no effect on the levels of melphalan-induced monoadducts or ICLs. We conclude that responders' BMPCs are characterized by slower first-phase repair of DNA damage resulting in higher levels of monoadducts and greater accumulation of ICLs, which in combination with the deficient DSBs repair efficiency leads to much higher DSBs burden in these cells. The simultaneous accumulation of high levels of the extremely cytotoxic ICLs and DSBs in responders' cells triggers the induction of the apoptotic pathway, a priority for successful clinical outcome. Finally, the enhancement of melphalan cytotoxicity in BMPCs by DSBs repair inhibitors offers a novel therapeutic strategy toward the treatment of MM eligible for ASCT patients. Disclosures Terpos: Novartis: Honoraria; Celgene: Honoraria, Other: travel expenses; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel expenses; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Munshi:novartis: Membership on an entity's Board of Directors or advisory committees; onyx: Membership on an entity's Board of Directors or advisory committees; celgene: Membership on an entity's Board of Directors or advisory committees; millenium: Membership on an entity's Board of Directors or advisory committees. Dimopoulos:Genesis: Honoraria; Janssen: Honoraria; Onyx: Honoraria; Janssen-Cilag: Honoraria; Celgene: Honoraria; Novartis: Honoraria; Amgen: Honoraria.

Cartilage boundary lubrication synergism is mediated by hyaluronan concentration and PRG4 concentration and structure

BackgroundProteoglycan 4 (PRG4) and hyaluronan (HA) are key synovial fluid constituents that contribute synergistically to cartilage boundary lubrication; however, the effects of their concentrations as well as their structure, both of which can be altered in osteoarthritis, on this functional synergism are unknown. The objectives of this study were to evaluate cartilage boundary lubricating ability of 1) PRG4 + HA in solution at constant HA concentration in a range of PRG4 concentrations, 2) constant PRG4 concentration in a range of HA concentrations, 3) HA + reduced/alkylated (R/A) PRG4, and 4) hylan G-F 20 + PRG4.MethodsStatic and kinetic friction coefficients (μstatic,Neq, <μkinetic,Neq>) were measured using a previously characterized cartilage-cartilage boundary mode friction test for the following concentrations of purified PRG4 and HA: Test 1: HA (1.5 MDa, 3.3 mg/mL) + PRG4 from 4.5 – 1500 μg/mL; Test 2: PRG4 (450, 150, 45 μg/mL) + HA (1.5 MDa) from 0.3 – 3.3 mg/mL. Test 3: hylan G-F 20 (3. 3 mg/mL) + PRG4 (450 μg/mL). Test 4: HA (3.3 mg/mL) + R/A PRG4 (450 μg/mL). ANOVA was used to compare lubricants within (comparing 6 lubricants of interest) and between (comparing 3 lubricants of interest) test sequences, with Tukey and Fishers post-hoc testing respectively.ResultsThis study demonstrates that both PRG4 and HA concentration, as well as PRG4 disulfide-bonded structure, can alter the cartilage boundary lubricating ability of PRG4 + HA solutions. The boundary lubricating ability of high MW HA + PRG4 solutions was limited by very low concentrations of PRG4. Decreased concentrations of high MW HA also limited the cartilage boundary lubricating ability of HA + PRG4 solutions, with the effect exacerbated by low PRG4 concentrations. The reduction of friction by addition of PRG4 to a cross-linked HA viscosupplement product, but not with addition of R/A PRG4 to HA, is consistent with a non-covalent mechanism of interaction where tertiary and quaternary PRG4 structure are important.ConclusionsCollectively, these results demonstrate that deficiency of either or both PRG4 and HA, or alterations in PRG4 structure, may be detrimental to SF cartilage boundary lubricating function. This study provides further insight into the nature of cartilage boundary lubrication and advancement towards potential formulation of new intra-articular biotherapeutic treatments for osteoarthritis using PRG4 ± HA.

Dithiothreitol (DTT) Acts as a Specific, UV-inducible Cross-linker in Elucidation of Protein–RNA Interactions*

Protein-RNA cross-linking by UV irradiation at 254 nm wavelength has been established as an unbiased method to identify proteins in direct contact with RNA, and has been successfully applied to investigate the spatial arrangement of protein and RNA in large macromolecular assemblies, e.g. ribonucleoprotein-complex particles (RNPs). The mass spectrometric analysis of such peptide-RNA cross-links provides high resolution structural data to the point of mapping protein-RNA interactions to specific peptides or even amino acids. However, the approach suffers from the low yield of cross-linking products, which can be addressed by improving enrichment and analysis methods. In the present article, we introduce dithiothreitol (DTT) as a potent protein-RNA cross-linker. In order to evaluate the efficiency and specificity of DTT, we used two systems, a small synthetic peptide from smB protein incubated with U1 snRNA oligonucleotide and native ribonucleoprotein complexes from S. cerevisiae. Our results unambiguously show that DTT covalently participates in cysteine-uracil crosslinks, which is observable as a mass increment of 151.9966 Da (C(4)H(8)S(2)O(2)) upon mass spectrometric analysis. DTT presents advantages for cross-linking of cysteine containing regions of proteins. This is evidenced by comparison to experiments where (tris(2-carboxyethyl)phosphine) is used as reducing agent, and significantly less cross-links encompassing cysteine residues are found. We further propose insertion of DTT between the cysteine and uracil reactive sites as the most probable structure of the cross-linking products.

Crystal Structures of SecYEG in Lipidic Cubic Phase Elucidate a Precise Resting and a Peptide-Bound State

The bacterial SecYEG translocon functions as a conserved protein-conducting channel. Conformational transitions of SecYEG allow protein translocation across the membrane without perturbation of membrane permeability. Here, we report the crystal structures of intact SecYEG at 2.7-Å resolution and of peptide-bound SecYEG at 3.6-Å resolution. The higher-resolution structure revealed that the cytoplasmic loop of SecG covers the hourglass-shaped channel, which was confirmed to also occur in the membrane by disulfide bond formation analysis and molecular dynamics simulation. The cytoplasmic loop may be involved in protein translocation. In addition, the previously unknown peptide-bound crystal structure of SecYEG implies that interactions between the cytoplasmic side of SecY and signal peptides are related to lateral gate opening at the first step of protein translocation. These SecYEG structures therefore provide a number of structural insights into the Sec machinery for further study.

α1B-Adrenoceptor signaling molecule expression in Alzheimer’s disease brain tissue / Alzeimer hastalığı olan beyin dokusunda α1B-Adrenoseptör Sinyal Molekülü Ekspresyonu

AbstractObjective: Gαh (tissue transglutaminase; TGII), known as the α1B-adrenoceptor signal mediator, is a bifunctional enzyme possessing transglutaminase and guanosine triphosphatase activities. The microtubule-associated protein tau plays a role in formation of neurofibrillary tangles in Alzheimer’s disease (AD) brain. Tau is also known as a substrate of transglutaminase. We aimed to better understand the correlation between α1B-adrenoceptor signaling and AD.Methods: We examined not only the cross-linking ability of TGII for tau, but also the expression level of tau as well as α1B-adrenoceptor signaling molecules, TGII and phospholipase C-δ1, in the human brain.Results: When the tau protein was assayed as a transglutaminase substrate of TGII, tau proteins formed cross-linked products. However, phospholipase C-δ1 inhibited transglutaminase activity in TGII to cross-link with tau in vitro. The amount of expressed mRNA in AD brain tissue was elevated 2~10 fold for tau and 3~20 fold for TGII. Consistent with these observations, the densities of expressed proteins in AD brain tissue also increased 9 fold for tau and 15 fold for TGII. Moreover, phospholipase C-δ1, which is a negative regulator for transglutaminase activity of TGII, also increased 2~25 fold for mRNA as well as 8 fold for protein in AD brain tissue. In contrast, expressed mRNA and protein activity for α1B-adrenoceptor were almost the same between AD and normal brains.Conclusion: These results suggest that the α1B-adrenoceptor signal mediator, TGII and tau, as a substrate of TGII might have a pathophysiological role in AD, but this correlation may be independent of the α1B-adrenoceptor signaling system, including α1B-adrenoceptor and phospholipase C-δ1.

Adsorption properties of cross-linked cellulose-epichlorohydrin polymers in aqueous solution

Cellulose was cross-linked with epichlorohydrin (EP) at variable levels (CLE-0.5, CLE-2 and CLE-4), where CLE-i denotes the cellulose to EP mole ratios. The cross-linked products were characterized by TGA and FT-IR spectroscopy, pH at the point of zero charge (pHpzc), water swelling, and dye-adsorption methods employing two types of dyes [phenolphthalein (phth) and p-nitrophenol (PNP)]. The characterization methods provide evidence of cross-linking of cellulose in accordance with variations in surface area, PZC, available surface hydroxyl groups, and thermal stability when compared against pristine cellulose. The pHpzc of the sorbent materials was ∼6.5 indicating a negative surface charge occurs above pHpzc. The cross-linked polymers possess greater swelling properties relative to pristine cellulose. Detailed adsorption studies were carried out at pH 9 for cellulose and CLE-i with five types single component carboxylate anions [2-hexyldecanoic acid (S1), trans-4-pentylcyclohexanecarboxylic acid (S2), 2-dicyclohexylacetic acid (S3), adamantane carboxylic acid (S4), and cyclohexane carboxylic acid (S5)] at 295K. The uptake properties of PNP with cellulose and CLE-i were also compared at pH 5 and 9, respectively. CLE-2 had the highest uptake of PNP (Qm=1.22×10−1mmol/g, pH 9) and S1 (Qm=4.27mg/g) while cellulose and CLE-4 had the strongest binding affinity (1.43L/mmol and 5.90×10−2L/mg), respectively. Uptake of PNP by CLE-0.5 at pH 5 (Qm=5.30×10−2mmol/g) was higher than uptake at pH 9 (Qm=3.11×10−2mmol/g). Sorption of CLE-4 with S1, S2 and S3 showed that relative uptake of the surrogates had the following order: S3>S2>S1, where S2 had the strongest binding affinity to CLE-i. CLE-2 had the highest sorption capacity towards Si in an equimolar mixture with evidence of molecular selective uptake. At pH 9, low uptake was mainly related to electrostatic repulsion between the negatively charged sorbent surface and the carboxylate head groups of Si.

Thermally Induced Crosslinking of Poly(N -Propargyl Glycine)

As polypeptoids become increasingly popular, they present a more soluble and processable alternative to natural and synthetic polypeptides; the breadth of their potential functionality slowly comes into focus. This report analyzes the ability of an alkyne-functionalized polypeptoid, poly(N-propargyl glycine), to crosslink upon heating. The crosslinking process is analyzed by thermal analysis (differential scanning calorimetry and thermogravimetric analysis), Fourier-transform infrared, electron paramagnetic resonance, and solid-state NMR spectroscopy. While a precise mechanism cannot be confidently assigned, it is clear that the reaction proceeds by a radical mechanism that exclusively involves the alkyne functionality, which, upon crosslinking, yields alkene and aromatic products.

Genipin-cross-linked silk sericin/poly(N-isopropylacrylamide) IPN hydrogels: Color reaction between silk sericin and genipin, pore shape and thermo-responsibility

To investigate the color reactions between naturally cross-linking agent genipin and silk sericin (SS), a series of genipin/SS film with adjustable color were firstly fabricated and analyzed by FTIR, UV/Vis and colorimetric methods. Then under the various genipin/SS contents, double cross-linked genipin/SS/Poly(N-isopropyl- acrylamide/N,N′-Methylene-bis-acrylamide) (HMGX) hydrogels were prepared, and the SEM, DSC, gravimetric and MTT method were performed to investigate pore structure, swelling behavior, temperature-sensitivities and cell adhesion behavior of HMGX hydrogels. The results show that SS cross-linked by genipin presents various colors during reaction. With increasing genipin/SS from 0 to 20 wt% or reacted time from 0 to 250 min, the color of genipin/SS mixture solution changes from transparent yellow to green, then to skyblue and finally became dark blue. After SS was cross-linked genipin, the maximum absorption peak turns to 570–600 nm from 282 nm, the reacted gardenia blue shows thermo/pH stable and water soluble. The absorption values of genipin/SS solutions increase, especially as genipin/SS ratio is 20 wt%, sharply increase can be found. The reaction time or rate between sericin and genipin is dependent on genipin/SS feed ratios. And with increasing mass percentage of genipin/(NIPAm + SS) from 0 to 10 wt%, due to high hydrophilicity of cross-linked product between genipin and SS, the pore sizes of HMGX hydrogels increase 1–3.75 times, the maximum swelling ratios (MSR) values increase to 2243% from 1057%. The volume phase transition temperature (VPTT) values of HMGX hydrogels keep still at 32–36 °C, but the endothermic peak becomes broader. After adsorption of L929 cells for 4 h and 1 d, the hydrogel with 3 wt% of genipin/(NIPAm + SS) presents the highest adhesion percentage.

Femtosecond UV-laser pulses to unveil protein-protein interactions in living cells.

A hallmark to decipher bioprocesses is to characterize protein-protein interactions in living cells. To do this, the development of innovative methodologies, which do not alter proteins and their natural environment, is particularly needed. Here, we report a method (LUCK, Laser UV Cross-linKing) to in vivo cross-link proteins by UV-laser irradiation of living cells. Upon irradiation of HeLa cells under controlled conditions, cross-linked products of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were detected, whose yield was found to be a linear function of the total irradiation energy. We demonstrated that stable dimers of GAPDH were formed through intersubunit cross-linking, as also observed when the pure protein was irradiated by UV-laser in vitro. We proposed a defined patch of aromatic residues located at the enzyme subunit interface as the cross-linking sites involved in dimer formation. Hence, by this technique, UV-laser is able to photofix protein surfaces that come in direct contact. Due to the ultra-short time scale of UV-laser-induced cross-linking, this technique could be extended to weld even transient protein interactions in their native context.

Biophysical characterization data on Aβ soluble oligomers produced through a method enabling prolonged oligomer stability and biological buffer conditions.

The data here consists of time-dependent experimental parameters from chemical and biophysical methods used to characterize Aβ monomeric reactants as well as soluble oligomer and amyloid fibril products from a slow (3–4 week) assembly reaction under biologically-relevant solvent conditions. The data of this reaction are both of a qualitative and quantitative nature, including gel images from chemical cross-linking and Western blots, fractional solubility, thioflavin T binding, size exclusion chromatograms, transmission electron microscopy images, circular dichroism spectra, and fluorescence resonance energy transfer efficiencies of donor–acceptor pair labels in the Aβ chain. This data enables future efforts to produce the initial monomer and eventual soluble oligomer and amyloid fibril states by providing reference benchmarks of these states pertaining to physical properties (solubility), ligand-binding (thioflavin T binding), mesoscopic structure (electron microscopy, size exclusion chromatography, cross-linking products, SDS and native gels) and molecular structure (circular dichroism, FRET donor-acceptor distance).Aβ1-40 soluble oligomers are produced that are suitable for biophysical studies requiring sufficient transient stability to exist in their “native” conformation in biological phosphate-saline buffers for extended periods of time. The production involves an initial preparation of highly monomeric Aβ in a phosphate saline buffer that transitions to fibrils and oligomers through time incubation alone, without added detergents or non-aqueous chemicals. This criteria ensures that the only difference between initial monomeric Aβ reactant and subsequent Aβ oligomer products is their degree of peptide assembly. A number of chemical and biophysical methods were used to characterize the monomeric reactants and soluble oligomer and amyloid fibril products, including chemical cross-linking, Western blots, fraction solubility, thioflvain T binding, size exclusion chromatography, transmission electron micrscopy, circular dichroism spectroscopy, and fluorescence resonance energy transfer.

Constraining the Lateral Helix of Respiratory Complex I by Cross-linking Does Not Impair Enzyme Activity or Proton Translocation

Complex I (NADH:ubiquinone oxidoreductase) is a multisubunit, membrane-bound enzyme of the respiratory chain. The energy from NADH oxidation in the peripheral region of the enzyme is used to drive proton translocation across the membrane. One of the integral membrane subunits, nuoL in Escherichia coli, has an unusual lateral helix of ∼75 residues that lies parallel to the membrane surface and has been proposed to play a mechanical role as a piston during proton translocation (Efremov, R. G., Baradaran, R., and Sazanov, L. A. (2010) Nature 465, 441-445). To test this hypothesis we have introduced 11 pairs of cysteine residues into Complex I; in each pair one is in the lateral helix, and the other is in a nearby region of subunit N, M, or L. The double mutants were treated with Cu(2+) ions or with bi-functional methanethiosulfonate reagents to catalyze cross-link formation in membrane vesicles. The yields of cross-linked products were typically 50-90%, as judged by immunoblotting, but in no case did the activity of Complex I decrease by >10-20%, as indicated by deamino-NADH oxidase activity or rates of proton translocation. In contrast, several pairs of cysteine residues introduced at other interfaces of N:M and M:L subunits led to significant loss of activity, in particular, in the region of residue Glu-144 of subunit M. The results do not support the hypothesis that the lateral helix of subunit L functions like a piston, but rather, they suggest that conformational changes might be transmitted more directly through the functional residues of the proton translocation apparatus.

Advanced glycation End-products (AGEs): an emerging concern for processed food industries.

The global food industry is expected to increase more than US $ 7 trillion by 2014. This rise in processed food sector shows that more and more people are diverging towards modern processed foods. As modern diets are largely heat processed, they are more prone to contain high levels of advanced glycation end products (AGEs). AGEs are a group of complex and heterogeneous compounds which are known as brown and fluorescent cross-linking substances such as pentosidine, non-fluorescent cross-linking products such as methylglyoxal-lysine dimers (MOLD), or non-fluorescent, non-cross linking adducts such as carboxymethyllysine (CML) and pyrraline (a pyrrole aldehyde). The chemistry of the AGEs formation, absorption and bioavailability and their patho-biochemistry particularly in relation to different complications like diabetes and ageing discussed. The concept of AGEs receptor - RAGE is mentioned. AGEs contribute to a variety of microvascular and macrovascular complications through the formation of cross-links between molecules in the basement membrane of the extracellular matrix and by engaging the receptor for advanced glycation end products (RAGE). Different methods of detection and quantification along with types of agents used for the treatment of AGEs are reviewed. Generally, ELISA or LC-MS methods are used for analysis of foods and body fluids, however lack of universally established method highlighted. The inhibitory effect of bioactive components on AGEs by trapping variety of chemical moieties discussed. The emerging evidence about the adverse effects of AGEs makes it necessary to investigate the different therapies to inhibit AGEs.

Structure and property changes of soybean protein isolates resulted from the glycation and cross-linking by transglutaminase and a degraded chitosan

A glycated and cross-linked soybean protein isolate (GC-SPI) with the glucosamine content of 19.40 g/kg protein was prepared by using SPI, transglutaminase, and a degraded chitosan under fixed conditions, aiming to assess modified properties and potential application of GC-SPI. GC-SPI has less reactable –NH2 groups than SPI (0.38 versus 0.48 mol/kg protein), and is verified by electrophoretic analysis to be a glycated and cross-linked protein product. Infrared spectroscopy and circular dichroism analyses demonstrate that GC-SPI contains more –OH groups, and has a more open secondary structure. In comparison with SPI, GC-SPI has higher surface hydrophobicity but lower in vitro digestibility due to protein cross-linking, and exhibits greater water- and oil-binding capacities (9.9 versus 6.4 and 3.7 versus 2.0 kg/kg protein). It is concluded that the glycation and cross-linking confer an open structure and modified properties of SPI, and GC-SPI is a potential ingredient with better hydration and oil-binding than SPI.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) interacts with apurinic/apyrimidinic sites in DNA

Apurinic/apyrimidinic (AP) sites are some of the most frequent DNA damages and the key intermediates of base excision repair. Certain proteins can interact with the deoxyribose of the AP site to form a Schiff base, which can be stabilized by NaBH4 treatment. Several types of DNA containing the AP site were used to trap proteins in human cell extracts by this method. In the case of single-stranded AP DNA and AP DNA duplex with both 5′ and 3′ dangling ends, the major crosslinking product had an apparent molecular mass of 45kDa. Using peptide mass mapping based on mass spectrometry data, we identified the protein forming this adduct as an isoform of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) called “uracil-DNA glycosylase”. GAPDH is a glycolytic enzyme with many additional putative functions, which include interaction with nucleic acids, different DNA damages and DNA repair enzymes. We investigated interaction of GAPDH purified from HeLa cells and rabbit muscles with different AP DNAs. In spite of the ability to form a Schiff-base intermediate with the deoxyribose of the AP site, GAPDH does not display the AP lyase activity. In addition, along with the borohydride-dependent adducts with AP DNAs containing single-stranded regions, GAPDH was also shown to form the stable borohydride-independent crosslinks with these AP DNAs. GAPDH was proven to crosslink preferentially to AP DNAs cleaved via the β-elimination mechanism (spontaneously or by AP lyases) as compared to DNAs containing the intact AP site. The level of GAPDH–AP DNA adduct formation depends on oxidation of the protein SH-groups; disulfide bond reduction in GAPDH leads to the loss of its ability to form the adducts with AP DNA. A possible role of formation of the stable adducts with AP sites by GAPDH is discussed.

Analysis of glycation induced protein cross-linking inhibitory effects of some antidiabetic plants and spices.

BackgroundProtein cross-linking which occurs towards the latter part of protein glycation is implicated in the development of chronic diabetic complications. Glycation induced protein cross-linking inhibitory effects of nine antidiabetic plants and three spices were evaluated in this study using a novel, simple, electrophoresis based method.MethodsMethanol extracts of thirteen plants including nine antidiabetic plants and three spices were used. Lysozyme and fructose were incubated at 37 °C in the presence or absence of different concentrations of plant extracts up to 31 days. Standard glycation inhibitor aminoguanidine and other appropriate controls were included. A recently established sodium dodecyl polyacrylamide gel electrophoresis (SDS-PAGE) method was used to detect the products of protein cross-linking in the incubation mixtures.ResultsHigh molecular weight protein products representing the dimer, trimer and tetramer of lysozyme were detected in the presence of fructose. Among the nine antidiabetic plants, seven showed glycation induced protein cross-linking inhibitory effects namely Ficus racemosa (FR) stem bark, Gymnema sylvestre (GS) leaves, Musa paradisiaca (MP) yam, Phyllanthus debilis (PD) whole plant, Phyllanthus emblica (PE) fruit, Pterocarpus marsupium (PM) latex and Tinospora cordifolia (TC) leaves. Inhibition observed with Coccinia grandis (CG) leaves and Strychnos potatorum (SP) seeds were much low. Leaves of Gymnema lactiferum (GL), the plant without known antidiabetic effects showed the lowest inhibition. All three spices namely Coriandrum sativum (CS) seeds, Cinnamomum zeylanicum (CZ) bark and Syzygium aromaticum (SA) flower buds showed cross-link inhibitory effects with higher effects in CS and SA. PD, PE, PM, CS and SA showed almost complete inhibition on the formation of cross-linking with 25 μg/ml extracts.ConclusionsMethanol extracts of PD, PE, PM, CS and SA have shown promising inhibitory effects on glycation induced protein cross-linking.Electronic supplementary materialThe online version of this article (doi:10.1186/s12906-015-0689-1) contains supplementary material, which is available to authorized users.

Photoinduced Intermolecular Cross-Linking of Gas Phase Triacylglycerol Lipid Ions

Complex mixtures of plant derived triglycerol (TG) lipids are commonly used as feedstock components for the production of industrial polymers. However, there remains a need for the development of analytical strategies to investigate the intrinsic intermolecular cross-linking reactivity of individual TG molecules within these mixtures as a function of their structures and physicochemical properties, and for the characterization of the resultant products. Here, to address this need, we describe a novel multistage tandem mass spectrometry based method for intermolecular cross-linking and subsequent structural characterization of TG lipid ions in the gas phase. Cross-linking reactions were initiated using 266 nm ultraviolet photodissociation tandem mass spectrometry (UVPD-MS/MS) of saturated or unsaturated TG dimers introduced via electrospray ionization into a linear ion trap mass spectrometer as noncovalent complexes with protonated 3,4-, 2,4- or 3,5- diiodoaniline (diIA). UVPD resulted in the initial formation of an anilinyl biradical via the sequential loss of two iodine radicals, which underwent further reaction to yield multiple cross-linked TG products along with competing noncross-linking processes. These chemistries are proposed to occur via sequential combinations of hydrogen abstraction (H-abstraction), radical addition and radical recombination. Multistage collision induced dissociation tandem mass spectrometry (CID-MS(n)) was used to obtain evidence for the structures and mechanisms of formation for these products, as a function of both the TG lipid and diIA ion structures. The efficiency of the UVPD reaction was shown to be dependent on the number of unsaturation sites present within the TG lipids. However, when unsaturation sites were present, formation of the cross-linked and noncross-linked product ions via H-abstraction and radical addition mechanisms was found to be competitive. Finally, the identity of the anilinyl biradical (e.g., 3,4- versus 2,4-substituted) was found to significantly affect the distribution of these two types of product ions. Importantly, owing to the observed propensity for cross-linking to occur via H-abstraction-initiated processes, this novel gas-phase cross-linking reaction provides a convenient method to link two molecules covalently without the requirement of any specific functional group, and therefore could be applied to examine the gas-phase intermolecular interactions and cross-linking of a wide range of biomolecular classes.