EDC Cross-linking Research Articles

Fabrication and characterization of aligned nanofibrous PLGA/Collagen blends as bone tissue scaffolds

Abstract Aligned nanofibrous blends of poly ( d , l -lactide- co -glycolide) (PLGA) and collagen with various PLGA/collagen compositions (80/20, 65/35 and 50/50) were fabricated by electrospinning and characterized for bone tissue engineering. Morphological characterization showed that the addition of collagen to PLGA resulted in narrowing of the diameter distribution and a reduction in average diameter. Differential scanning calorimetric (DSC) studies showed that the triple helix structure of the native collagen was not destroyed during the fabrication process. However, the blending had a marked effect on the overall enthalpy of the blends, whereby the total enthalpy decreased as the collagen content decreased. Thermogravimetric analysis showed the addition of collagen increased the hydrophilicity of the scaffolds. The crosslinking of collagen to increase the biostability was done using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) in ethanol and an overall ∼25% degree of crosslinking was achieved. The EDC crosslinking had little effect on the nanofibrous morphology of the 80/20 blend system; however, the nanofibrous features were compromised to some extent at higher collagen concentrations. The mechanical characterization under dry and wet conditions showed that increasing collagen content resulted in a tremendous decrease in the mechanical properties. However, crosslinking resulted in the increase in elastic modulus from 47 MPa to 83 MPa for the wet PLGA/Collagen 80/20 blend system, with little effect on the tensile strength. In conclusion, the aligned nanofibrous scaffold used in this study constitutes a promising material for bone tissue engineering.

Tailoring the Properties of Cholecyst-Derived Extracellular Matrix Using Carbodiimide Cross-Linking

Modulation of properties of extracellular matrix (ECM) based scaffolds is key for their application in the clinical setting. In the present study, cross-linking was used as a tool for tailoring the properties of cholecyst-derived extracellular matrix (CEM). CEM was cross-linked with varying cross-linking concentrations of N,N-(3-dimethyl aminopropyl)-N′-ethyl carbodiimide (EDC) in the presence of N-hydroxysuccinimide (NHS). Shrink temperature measurements and ATR–FT-IR spectra were used to determine the degree of cross-linking. The effect of cross-linking on degradation was tested using the collagenase assay. Uniaxial tensile properties and the ability to support fibroblasts were also evaluated as a function of cross-linking. Shrink temperature increased from 59°C for non-cross-linked CEM to 78°C for the highest EDC cross-linking concentration, while IR peak area ratios for the free –NH2 group at 3290 cm−1 to that of the amide I band at 1635 cm−1 decreased with increasing EDC cross-linking concentration. Collagenase assay demonstrated that degradation rates for CEM can be tailored. EDC concentrations 0 to 0.0033 mmol/mg CEM were the cross-linking concentration range in which CEM showed varied susceptibility to collagenase degradation. Furthermore, cross-linking concentrations up to 0.1 mmol EDC/mg CEM did not have statistically significant effect on the uniaxial tensile strength, as well as morphology, viability and proliferation of fibroblasts on CEM. In conclusion, the degradation rates of CEM can be tailored using EDC-cross-linking, while maintaining the mechanical properties and the ability of CEM to support cells.

Enhanced biological stability of collagen with incorporation of PAMAM dendrimer

The crosslinking methods of collagen using glutaraldehyde (GTA) and 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC)/N-hydroxysuccinimide (NHS) are frequently performed in biomedical applications, but both methods still have their own disadvantages, including the GTA cytotoxicity and low degree of EDC/NHS crosslinking. In this study, we incorporated polyamidoamine (PAMAM) dendrimer with surface amine groups into the two aforementioned crosslinking methods to improve the biostability and structural integrity of collagen. Fifty micromolar of dendrimer concentration was found to have negligible in vitro cytotoxicity and was used for EDC and GTA crosslinking of collagen. The collagenase digestion assay showed that the collagen scaffolds crosslinked in the presence of PAMAM exhibited a higher denature temperature and higher resistance against collagenase digestion compared with their counterparts without dendrimer. Cell proliferation with human conjunctival fibroblasts showed that the incorporation of PAMAM in EDC crosslinking significantly increased the proliferation. All the crosslinked scaffolds also exhibited higher structural stability than the noncrosslinked scaffold. Crosslinking with EDC and PAMAM together yielded substantially higher proliferation and may be a suitable collagen scaffold for biomedical applications.

Local Release of Basic Fibroblast Growth Factor (bFGF) through Silica Nanoparticles-laden Biomimic Matrix

ABSTRACTBasic fibroblast growth factor (bFGF), a protein, plays a key role in wound healing and blood vessel regeneration. However, most negative effectsin vivo, orin vitroresult from the over dosage of bFGF. Furthermore, it needs to keep the bFGF from protein denaturant. Thus, this study aims to develop a new delivery system based on silica nanoparticles (SiO2NPs) dispersed in collagen patch for delivery of the bFGF in a local and prolonged manner. In this research, SiO2NPs are used to encapsulate bFGF through a modified water-in-oil micro-emulsion. The bFGF-loaded nanoparticles afterwards are dispersed in the collagen-based matrix through a EDC cross-linking step. Thein vitrorelease kinetics of SiO2 NPs - encapsulated bFGF with and without collagen matrix have been monitored through ELISA. In addition, the cytotoxicity of SiO2NPs is investigated by studying the viability of Human Umbilical Vein Endothelial Cells (HUVEC) under the different concentrations of SiO2NPs. It has found the average diameter (d) for SiO2NPs encapsulating bFGF is 45 ± 8 nm with a loading efficiency of 72.5±3%. The maximum concentration of bFGF locally released from SiO2NPs impregnated collagen matrix can be monitored after 30 days, while bFGF released from SiO2NPs can be detected in 20 days. The further prolonged releasing after the nanoparticle-encapsulated bFGF laden collagen matrix is possibly due to the interaction between the nanoparticles and collagen matrix. In addition, the biocompatibility of the SiO2NP has been investigated. We found that SiO2NPs at the concentration of 50 μg/ml can still keep the cell alive. The results indicate that the nanoparticle-laden collagen matrix can locally deliver growth factor in a prolonged manner. This new delivery system may benefit to blood vessel regeneration and potentiate greater angiogenesis.

Degradation of covalently cross-linked carboxymethyl chitosan and its potential application for peripheral nerve regeneration

Chitosan has been widely used in a variety of biomedical applications including peripheral nerve repair because of its excellent mechanical properties and biocompatibility. However, chitosan itself has a very slow degradation rate, and its molecules degrade in an uncontrollable manner. We hypothesized that the cross-linking of carboxymethyl chitosan (CM-chitosan), which is soluble in water, would result in a higher degradation rate in lysozyme solutions, while retaining its excellent mechanical properties and nerve cell affinity. In this study, we characterized the constructed matrix formed using a combination of carboxymethylation of chitosan chains and thereafter 1-ethyl-3(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) cross-linking. Specifically, after EDC cross-linking, the hydrophilicity and elastic modulus of the CM-chitosan films decreased. These changes are beneficial in the application of chitosan derivatives for nerve repair. The porous conduits degraded to 30% in weight during eight weeks of incubation in lysozyme solution (pH 7.4, 37 °C). In addition, the cross-linked CM-chitosan films enhanced the spread of Neuro-2a cells and provided a good proliferation substratum for Neuro-2a cells, as compared to chitosan films. Therefore, cross-linking with EDC is a promising way to modify chitosan derivatives for peripheral nerve regeneration.

Carbodiimide-mediated cross-linking of RNA to nylon membranes improves the detection of siRNA, miRNA and piRNA by northern blot

The northern blot, or RNA gel blot, is a widely used method for the discovery, validation and expression analysis of small regulatory RNA such as small interfering RNA (siRNA), microRNA (miRNA) and piwi-interacting RNA (piRNA). Although it is straightforward and quantitative, the main disadvantage of a northern blot is that it detects such RNA less sensitively than most other approaches. We found that the standard dose of UV used in northern blots was not the most efficient at immobilizing small RNA of 20–40 nt on nylon membranes. However, increasing the dose of UV reduced the detection of miRNA by hybridization in northern blotting experiments. We discovered that using the soluble carbodiimide, EDC, to cross-link RNA to nylon membranes greatly improved the detection of small RNA by hybridization. Compared to standard UV cross-linking procedures, EDC cross-linking provided a 25–50-fold increase in the sensitivity of detection of siRNA from plants and miRNA or piRNA from mammalian cells. All types of hybridization probes tested benefited from the new cross-linking procedure. Cross-linking was dependent on a terminal phosphate and so, should be applicable to other related categories of small RNA.

Characterization of the pH-dependent Interaction between the Gap Junction Protein Connexin43 Carboxyl Terminus and Cytoplasmic Loop Domains

A prevailing view regarding the regulation of connexin43 (Cx43) gap junction channels is that, upon intracellular acidification, the carboxyl-terminal domain (Cx43CT) moves toward the channel opening to interact with specific residues acting as a receptor site. Previous studies have demonstrated a direct, pH-dependent interaction between the Cx43CT and a Cx43 cytoplasmic loop (Cx43CL) peptide. This interaction was dependent on alpha-helical formation for the peptide in response to acidification; more recent studies have shown that acidification also induces Cx43CT dimerization. Whether Cx43CT dimerization is an important structural component in Cx43 regulation remains to be determined. Here we used an assortment of complimentary biophysical techniques to characterize the binding of Cx43CT or its mutants to itself and/or to a more native-like Cx43CL construct (Cx43CL(100-155), residues 100-155). Our studies expand the observation that specific Cx43CT domains are important for dimerization. We further show that properties of the Cx43CL(100-155) are different from those of the Cx43CL peptide; solvent acidification leads to Cx43CL(100-155) oligomerization and a change in the stoichiometry and binding affinity for the Cx43CT. Homo-Cx43CT and Cx43CL(100-155) oligomerization as well as the Cx43CT/Cx43CL(100-155) interaction can occur under in vivo conditions; moreover, we show that Cx43CL(100-155) strongly affects resonance peaks corresponding to Cx43CT residues Arg-376-Asp-379 and Asn-343-Lys-346. Overall, our data indicate that many of the sites involved in Cx43CT dimerization are also involved in the Cx43CT/Cx43CL interaction; we further propose that chemically induced Cx43CT and Cx43CL oligomerization is important for the interaction between these cytoplasmic domains, which leads to chemically induced gating of Cx43 channels.

Reversible Redox- and Zinc-Dependent Dimerization of the Escherichia coli Fur Protein

Fur is a bacterial regulator using iron as a cofactor to bind to specific DNA sequences. This protein exists in solution as several oligomeric states, of which the dimer is generally assumed to be the biologically relevant one. We describe the equilibria that exist between dimeric Escherichia coli Fur and higher oligomers. The dissociation constant for the dimer-tetramer equilibrium is estimated to be in the millimolar range. Oligomerization is enhanced at low ionic strength and pH. The as-isolated monomeric form of Fur is not in equilibrium with the dimer and contains two disulfide bridges (C92-C95 and C132-C137). Binding of the monomer to DNA is metal-dependent and sequence specific with an apparent affinity 5.5 times lower than that of the dimer. Size exclusion chromatography, EDC cross-linking, and CD spectroscopy show that reconstitution of the dimer from the monomer requires reduction of the disulfide bridges and coordination of Zn2+. Reduction of the disulfide bridges or Zn2+ alone does not promote dimerization. EDC and DMA cross-links reveal that the N-terminal NH2 group of one subunit is in an ionic interaction with acidic residues of the C-terminal tail and close to Lys76 and Lys97 of the other. Furthermore, the yields of cross-link drastically decrease upon binding of metal in the activation site, suggesting that the N-terminus is involved in the conformational change. Conversely, oxidizing reagents, H2O2 or diamide, disrupt the dimeric structure leading to monomer formation. These results establish that coordination of the zinc ion and the redox state of the cysteines are essential for holding E. coli Fur in a dimeric state.

Studies of Novel Hyaluronic Acid-collagen Sponge Materials Composed of Two Different Species of Type I Collagen

The authors have developed novel hyaluronic acid (HA)-collagen sponge materials (HACSMs) composed of various ratios of bird feet (BF) and pig skin (PS) collagen that are fabricated employing a combination of freezing, lyophilizing, and 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) crosslinking methods. Morphology, swelling ratio, resistance to collagenase, thermal stability, tensile strength, and free amine index are determined to evaluate the physical-chemical properties of various HACSMs. Different BF: PS ratios directly vary with the physical-chemical properties of HACSMs and control their biodegradability for multiple uses. Resistance to collagenase, thermal stability, and tensile strength of HACSMs increases as the ratio of BF collagen increases. On the contrary, the higher swelling ratio, free amine index, and pore size occur in materials composed of higher ratios of PS collagen. A linear relationship between the decreased ratio of PS collagen and the increase in tensile strength and biostability are observed. The materials of B4P1HA (BF: PS: HA=4: 1: 0.2) exhibit the highest value of tensile strength, but no significant difference exists between B4P1HA and B5P0HA (BF: PS: HA=5: 0: 0.2). These phenomena should be closely related to the BF collagen which contains a higher amount of carboxyl groups of glutamic or aspartic acid residues and forms more amine bonds under EDC cross-linking when compared to PS collagen. However, these results suggest that the B4P1HA and B5P0HA materials should be produced according to highest bio-stability and mechanical strength and, furthermore they may be suitable for artificial skin or drug delivery applications.

Gelatin microspheres cross-linked with EDC as a drug delivery system for doxycyline: Development and characterization

Chronic wounds express elevated levels of proteases, in particular matrix metalloproteinases (MMPs), which degrades de novo granulation tissue and endogenous biologically active proteins. An effective therapeutic approach for chronic wounds would be to modify this hostile environment and reduce the proteolytic imbalance. Doxycycline has been proved recently to inhibit MMPs and used topically for chronic wound ulcers, beyond their antimicrobial profile. To this end, a carrier system for controlled release of doxycycline, suitable for incorporation into various wound dressings like membranes and sponges was developed. In the present study gelatin microspheres, cross-linked with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) was proposed. The cross-linking was carried out with different concentrations of EDC (10 mM, 50 mM and 100 mM) and for different time periods (3–24 h). The cross-linked microspheres were characterized by evaluating the extent of cross-linking, the morphology, swelling behaviour and drug loading and in vitro studies of drug release, enzymatic degradation and biocompatibility. The extent of cross-linking increased as a function of both EDC concentration and the cross-linking time periods. It is found that the extent of cross-linking greatly influences the swelling and drug release behaviour of the microspheres. The cross-linked microspheres were found to be biocompatible to NIH 3T3 mouse embryonic fibroblast. The overall study indicates that the zero length cross-linker EDC can be considered as a potential alternative for cross-linking the gelatin microspheres.

EDC cross-linking improves skin substitute strength and stability

Collagen-based scaffolds are extensively utilized as an analog for the extracellular matrix in cultured skin substitutes (CSS). To improve the mechanical properties and degradation rates of collagen scaffolds, chemical cross-linking is commonly employed. In this study, freeze-dried collagen-GAG sponges were crosslinked with increasing concentrations of 1-ethyl-3-3-dimethylaminopropylcarbodiimide hydrochloride (EDC; 0, 1, 5, 10, 50mm). Cross-linking with EDC at concentrations >1mm was shown to greatly decrease degradation by collagenase up to 21 days. Ultimate tensile strength (UTS) of acellular collagen sponges scaled positively with EDC concentration up to 10mm. At 50mm EDC, the UTS decreased dramatically likely due to the brittle nature of the highly crosslinked material. Co-culture of human fibroblasts (HF) and keratinocytes (HK) on these substrates reveals an apparent cytotoxicty of the EDC at high concentrations with reduced cell viability and poor cellular organization in CSS fabricated with scaffolds crosslinked with 10 or 50mm EDC. From the data gathered in this study, intermediate concentrations of EDC, specifically 5mm, increase collagen sponge stability and strength while providing an environment in which HF and HK can attach, proliferate and organize in a manner conducive to dermal and epidermal regeneration.

Crosslinking of collagen with dendrimers

Polypropyleneimine octaamine dendrimers were studied as an alternative means of generating highly crosslinked collagen. Crosslinking was effected by using the water-soluble carbodiimide 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide hydrochloride (EDC). The multifunctional dendrimers were introduced as novel crosslinkers after the activation of the carboxylic acid groups of glutamic and aspartic acid residues in collagen. The conventional crosslinker glutaraldehyde was used as a control. EDC, itself an alternative crosslinker, which forms zero-length crosslinks by directly covalently binding collagen molecules, as well as a low molecular weight diamine and a low molecular weight triamine, were also studied. All of the resultant gels were freeze-dried to obtain sponges for characterization. Water uptake of the gels decreased from 90% to 60% after dendrimer crosslinking compared with EDC crosslinking. DSC results showed an increase of denaturation temperature of collagen after crosslinking with the various methods. The generation 2 and 3 dendrimer-crosslinked collagen samples had the highest denaturation temperature, at up to 90 degrees C compared with 50 degrees C in the uncrosslinked collagen control. The dendrimer-crosslinked collagen also showed unique thermal characteristics, with multiple denaturation temperature peaks in contrast to the single peak noted with the other crosslinked collagens. This is thought to be due to the heterogeneous nature of dendrimer crosslinking. Collagenase results revealed that the dendrimer-crosslinked collagen had a comparative resistance to proteolysis to glutaraldehyde-crosslinked collagen. Measurement of activated carboxylic acid groups before and after crosslinking indicated that 40-70% of the activated carboxylic acid was consumed during crosslinking with dendrimers. The results suggest that dendrimer crosslinking of collagen produces stable gels. The presence of a large number of excess amine groups in the dendrimers may also be useful for subsequent modification with biologically relevant groups.

Direct interaction between prion protein and tubulin

Recently published data show that the prion protein in its cellular form (PrP C) is a component of multimolecular complexes. In this report, zero-length cross-linking with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) allowed us to identify tubulin as one of the molecules interacting with PrP C in complexes observed in porcine brain extracts. We found that porcine brain tubulin added to these extracts can be cross-linked with PrP C. Moreover, we observed that the 34 kDa species identified previously as full-length diglycosylated prion protein co-purifies with tubulin. Cross-linking of PrP C species separated by Cu 2+-loaded immobilized metal affinity chromatography confirmed that only the full-length protein but not the N-terminally truncated form (C1) binds to tubulin. By means of EDC cross-linking and cosedimentation experiments, we also demonstrated a direct interaction of recombinant human PrP (rPrP) with tubulin. The stoichiometry of cosedimentation implies that rPrP molecules are able to bind both the α- and β-isoforms of tubulin composing microtubule. Furthermore, prion protein exhibits higher affinity for microtubules than for unpolymerized tubulin.

THE EVALUATION OF THERMAL PROPERTIES AND IN VITRO TEST OF CARBODIIMIDE OR GLUTARALDEHYDE CROSS-LINKED GELATIN FOR PC 12 CELLS CULTURE

The thermal and degradable properties of carbodiimide (EDC) or glutaraldehyde (GTA) cross-linked gelatin membranes have been investigated in order to evaluate the effects of different concentrations of two kinds of cross-linking reagent on the stability of membranes. In the thermogram recorded from a gelatin membrane cross-linked with EDC solution, the endothermic peak of 0.8% EDC cross-linking gelatin was centered at about 61°C that was higher than other samples treated with EDC solutions. Denaturation temperature (Td) of gelatin samples increased on increasing EDC concentration (0.2% to 0.8%), in agreement with the simultaneous increased of the extent of cross-linking. But increasing GTA concentration from 0.05% to 0.6%, the Td values of gelatin samples were decreased from 66.2°C to 56.3°C . In addition, two endothermic peaks were observed in 0.4% and 0.6% GTA cross-linking groups because of the GTA concentration was too high to complete cross-linking reaction. Therefore, partial of gelatin membrane was cross-linked completely but others were not. In the thermogravimetric analysis, the proportion of cracking endothermic peak of 0.6% GTA cross-linking gelatin (g15G0.6) was higher than the peak of 0.6% EDC cross-linking gelatin (g15C0.6). Therefore, g15G0.6 cracked to smaller molecules has to absorb more calorific capacity than g15C0.6. The increase in the strength of covalent binding on increasing the proportion of endothermic peak was evident. The results of degradable rate were in agreement with the lower concentration of cross-linked reagent the faster degraded rate of gelatin membrane. The MTT assay showed that 15% gelatin cross-linked by 0.8% EDC has the least cytotoxicity, and cell activity of this group was similar to control group (blank dish). As the concentration of GTA in gelatin membranes was down to 0.05% or 0.1% the cell viability was returned to approach the value of control group.

The LC7 light chains of Chlamydomonas flagellar dyneins interact with components required for both motor assembly and regulation.

Members of the LC7/Roadblock family of light chains (LCs) have been found in both cytoplasmic and axonemal dyneins. LC7a was originally identified within Chlamydomonas outer arm dynein and associates with this motor's cargo-binding region. We describe here a novel member of this protein family, termed LC7b that is also present in the Chlamydomonas flagellum. Levels of LC7b are reduced approximately 20% in axonemes isolated from strains lacking inner arm I1 and are approximately 80% lower in the absence of the outer arms. When both dyneins are missing, LC7b levels are diminished to <10%. In oda9 axonemal extracts that completely lack outer arms, LC7b copurifies with inner arm I1, whereas in ida1 extracts that are devoid of I1 inner arms it associates with outer arm dynein. We also have observed that some LC7a is present in both isolated axonemes and purified 18S dynein from oda1, suggesting that it is also a component of both the outer arm and inner arm I1. Intriguingly, in axonemal extracts from the LC7a null mutant, oda15, which assembles approximately 30% of its outer arms, LC7b fails to copurify with either dynein, suggesting that it interacts with LC7a. Furthermore, both the outer arm gamma heavy chain and DC2 from the outer arm docking complex completely dissociate after salt extraction from oda15 axonemes. EDC cross-linking of purified dynein revealed that LC7b interacts with LC3, an outer dynein arm thioredoxin; DC2, an outer arm docking complex component; and also with the phosphoprotein IC138 from inner arm I1. These data suggest that LC7a stabilizes both the outer arms and inner arm I1 and that both LC7a and LC7b are involved in multiple intradynein interactions within both dyneins.

Subunit proximity in the H+-translocating NADH-quinone oxidoreductase probed by zero-length cross-linking.

The proton-translocating NADH-quinone oxidoreductase (NDH-1) of Paracoccus denitrificans is composed of 14 different subunits (designated Nqo1-14), seven of which are located in the membrane domain and the other seven in the peripheral domain. It has been previously reported that membrane domain subunit Nqo7 (ND3) directly interacts with peripheral subunit Nqo6 (PSST) by using a cross-linker, m-maleimidobenzoyl-N-hydrosuccinimide ester, and heterologous expression [Di Bernardo, S., and Yagi, T. (2001) FEBS Lett. 508, 385-388]. To further explore the near-neighbor relationship of the subunits, a zero-length cross-linker, 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC), and the Paracoccus membranes were used, and the cross-linked products were examined with antibodies specific to subunits Nqo1-11. The Nqo6 subunit was cross-linked to subunit Nqo9 (TYKY). In addition, a ternary product of Nqo3 (75k), Nqo6, and Nqo7 and binary products of Nqo3 and Nqo6 and of Nqo6 and Nqo7 were observed, but a binary product of Nqo3 and Nqo7 was not detected. The Nqo4 (49k) subunit was found to be associated with the Nqo7 subunit. Furthermore, Paracoccus subunits Nqo3, Nqo6, and Nqo7 were heterologously coexpressed in Escherichia coli, and EDC cross-linking experiments were carried out using the E. coli membranes expressing these three subunits. The results were the same as those obtained with Paracoccus membranes. On the basis of the data, subunit arrangements of NDH-1 were discussed.

Novel Biomaterial from Reinforced Salmon Collagen Gel Prepared by Fibril Formation and Cross-Linking

The improvement of the thermal stability of gel prepared from salmon atelocollagen (SC) was studied. The denaturation temperature ( Td ) of the SC solution was found to be 18.6°C. Neutral buffer including 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) was mixed with acidic SC solution at 4°C, resulting in the introduction of EDC cross-linking during fibril formation. The mechanical strength and thermal stability of the resultant cross-linked SC fibrillar gels reached maximum values at an EDC concentration of 50 mM ( f -50 gel). In particular, the melting temperature of the f -50 gel was 47°C, much higher than that of the EDC cross-linked SC gel without fibril formation at the same EDC concentration. The proliferation rate of human periodontal ligament cells on the f -50 gel was higher than that of a porcine atelocollagen fibrillar gel. These results suggest that the gel employed for biomaterials can be fabricated from low Td fish collagen by EDC cross-linking during fibril formation.

The effect of chemical modifications on rheological properties of collagen from of baltic cod (Gadus morhua) skin stabilized by interaction with κ-carrageenan

The κ-carrageenan-collagen complex was precipitated by κ-carrageenan from solution of collagen sourced from Baltic cod (Gadus morhua) skin in 0.5M acetic acid. The influence of glutaraldehyde or EDC [1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide hydrochloride] concentration on rheological properties of κ-carrageen-collagen complex was examined. At 5 to 30mM glutaraldehyde content in 0.5 κ-carrageenan-collagen dispersion a rapid increase in its viscosity was observed, which remained unchanged over 120 min. Further increase in glutaraldehyde concentration to 30mM caused enhanced viscosity and shorten time of complex stability, which not exceed 45 min. The level of dispersion viscosity caused by glutaraldehyde cross-linking ranged from 70 to 420 mPa·s. For comparison, at EDC concentrations from 30 up to 180mM the viscosity of suspension changed after 200 min from 130–280 mPa·s. Glutaraldehyde not influenced on mechanical resistance of the membranes as well as elongation at the break. However, EDC cross-linking significantly increased the flexibility of the membranes and decreased the stress needed at break. It was observed that at 30 mM concentration of EDC the tensile strength of the membranes was reduced from about 21 MPa to 11 MPa, and the elongation at the break was enhanced from about 0.4 to 20%. At 60mM concentration of EDC the stress at break was reduced to 1.4 MPa, whereas the elongation at break was about 17%. Cross-linking of the membranes decreases their hydrophilic properties.

Nature of full-length HMGB1 binding to cisplatin-modified DNA.

HMGB1, a highly conserved non-histone DNA-binding protein, interacts with specific DNA structural motifs such as those encountered at cisplatin damage, four-way junctions, and supercoils. The interaction of full-length HMGB1, containing two tandem HMG box domains and a C-terminal acidic tail, with cisplatin-modified DNA was investigated by hydroxyl radical footprinting and electrophoretic gel mobility shift assays. The full-length HMGB1 protein binds to DNA containing a 1,2-intrastrand d(GpG) cross-link mainly through domain A, as revealed by footprinting, with a dissociation constant K(d) of 120 nM. Site-directed mutagenesis of intercalating residues in both HMG domains A and B in full-length HMGB1 further supports the conclusion that only one HMG box domain is bound to the site of cisplatin damage. Interaction of the C-terminal tail with the rest of the HMGB1 protein was examined by EDC cross-linking experiments. The acidic tail mainly interacts with domain B and linker regions rather than domain A in HMGB1. These results illuminate the respective roles of the tandem HMG boxes and the C-terminal acidic tail of HMGB1 in binding to DNA and to the major DNA adducts formed by the anticancer drug cisplatin.

Biochemical and structural characterization of the cross-linked complex of nitrogenase: comparison to the ADP-AlF4(-)-stabilized structure.

The transient formation of a complex between the component Fe- and MoFe-proteins of nitrogenase represents a central event in the substrate reduction mechanism of this enzyme. Previously, we have isolated an N-[3-(dimethylamino)propyl]-N'-ethylcarbodiimide (EDC) cross-linked complex of these proteins stabilized by a covalent isopeptide linkage between Glu 112 and Lys beta400 of the Fe-protein and MoFe-protein, respectively [Willing, A., et al. (1989) J. Biol. Chem. 264, 8499-8503; Willing, A., and Howard, J. B. (1990) J. Biol. Chem. 265, 6596-6599]. We report here the biochemical and structural characterization of the cross-linked complex to assess the mechanistic relevance of this species. Glycinamide inhibits the cross-linking reaction, and is found to be specifically incorporated into Glu 112 of the Fe-protein, without detectable modification of either of the neighboring residues (Glu 110 and Glu 111). This modified protein is still competent for substrate reduction, demonstrating that formation of the cross-linked complex is responsible for the enzymatic inactivation, and not the EDC reaction or the modification of the Fe-protein. Crystallographic analysis of the EDC-cross-linked complex at 3.2 A resolution confirms the site of the isopeptide linkage. The nature of the protein surfaces around the cross-linking site suggests there is a strong electrostatic component to the formation of the complex, although the interface area between the component proteins is small. The binding footprints between proteins in the cross-linked complex are adjacent, but with little overlap, to those observed in the complex of the nitrogenase proteins stabilized by ADP-AlF(4)(-). The results of these studies suggest that EDC cross-linking traps a nucleotide-independent precomplex of the nitrogenase proteins driven by complementary electrostatic interactions that subsequently rearranges in a nucleotide-dependent fashion to the electron transfer competent state observed in the ADP-AlF(4)(-) structure.