Natural Cross-linker Research Articles

Effect of microbial transglutaminase on the natural actomyosin cross-linking in chicken and beef

The objective of this research was to investigate the difference between chicken and beef in the interaction of actomyosin (myosin B) with microbial transglutaminase (MTG). The gel strength of myosin B was improved in both species and was significantly greater in beef than in chicken ( P < 0.01). The degree of protein viscosity and the ε(γ-glutamyl)lysine (G–L) content were significantly higher in beef than in chicken ( P < 0.01). Myosin heavy chain (MHC) bands visualized by SDS–PAGE revealed that the same proteins in various meat species vary in their size and structure. Scanning electron microscope images (SEMI) revealed that myosin B in both species was polymerized, and formed multi-projection structures of G–L; surprisingly, more of these structures were found in beef than in chicken. It is possible that the proteins in chicken are folded into a strand shape that tightly encases a considerable number of glutamine and lysine residues, whereas MTG substrate cannot couple glutamine and lysine. This suggests that the reactivity of MTG is dependent on the residual amino acids present on the surface of myosin B in meat. Some protein components (peptides with long reiterated methylene groups attached) joined by disulfide bonds (cysteine) in chicken samples were inhibitory and reduced MTG activity. SEMI also suggested that all MTG-dependent mega-structures of protein molecules generated in chicken and beef may vary greatly in size, configuration and complexity after treatment with MTG. We concluded that the optimal cross-links in myosin B induced by MTG are heterogeneous in chicken and beef.

The proteoglycan (heparan sulfate proteoglycan) binding domain of APRIL serves as a platform for ligand multimerization andcross‐linking

A proliferation-inducing ligand (APRIL) (also known as TALL-2 and TRDL-1) is a member of the tumor necrosis factor (TNF) superfamily that has tumorigenic properties but is also important for the induction of humoral immune responses. APRIL binds two TNF receptors: transmembrane activator and calcium modulator and cyclophilin ligand interactor (TACI) and B-cell maturation antigen (BCMA) as well as heparan sulfate proteoglycans (HSPGs). The aim of this study was to clarify the role of the HSPG interaction in canonical APRIL signaling, because it has been proposed to act as a docking site and also to play a role in direct signaling. In this study, we generated point mutants of soluble APRIL that lack either the capacity to bind HSPGs or TACI and BCMA and then tested the function of these mutants in mouse B-cell assays. In contrast to previous reports, we found that APRIL alone is sufficient to costimulate B-cell proliferation and drive IgA production and does not require artificial antibody cross-linking. We found no evidence that APRIL requires signaling through HSPGs but, notably, were able to show that binding of APRIL to HSPGs is crucial for mediating natural APRIL cross-linking to allow for optimal activation of murine B cells.

天然ゴム 天然ゴムの構造

Natural rubber, obtained from Hevea brasiliensis, consists of ω’-terminal, two trans-1,4-isoprene units, long sequence of cis-1,4-isoprene units and α’-terminal (ω’-(trans)2-(cis)n-α’), in this order, in which the ω’ and α’ terminals represent unknown terminal groups attracted with proteins and phospholipids. The ω’ and α’ terminals are analyzed in relation to structures of polyprenols such as (CH3)2C=CHCH2-(trans)2-(cis)n-OH and (CH3)2C=CHCH2-(trans)3-(cis)n-OH. The ω’ and α’ terminals are found to play an important role in a formation of three dimensional networks of natural rubber, i.e. branching and crosslinking; the higher the molecular weight, the larger is the number of branching points. The formation of three dimensional networks is concerned with a biosynthesis of natural rubber on a surface of a rubber particle in latex stage.

Characterization of Protein Cross-Links via Mass Spectrometry and an Open-Modification Search Strategy

Protein-protein interactions are key to function and regulation of many biological pathways. To facilitate characterization of protein-protein interactions using mass spectrometry, a new data acquisition/analysis pipeline was designed. The goal for this pipeline was to provide a generic strategy for identifying cross-linked peptides from single LC/MS/MS data sets, without using specialized cross-linkers or custom-written software. To achieve this, each peptide in the pair of cross-linked peptides was considered to be "post-translationally" modified with an unknown mass at an unknown amino acid. This allowed use of an open-modification search engine, Popitam, to interpret the tandem mass spectra of cross-linked peptides. False positives were reduced and database selectivity increased by acquiring precursors and fragments at high mass accuracy. Additionally, a high-charge-state-driven data acquisition scheme was utilized to enrich data sets for cross-linked peptides. This open-modification search based pipeline was shown to be useful for characterizing both chemical as well as native cross-links in proteins. The pipeline was validated by characterizing the known interactions in the chemically cross-linked CYP2E1-b5 complex. Utility of this method in identifying native cross-links was demonstrated by mapping disulfide bridges in RcsF, an outer membrane lipoprotein involved in Rcs phosphorelay.

Gelatin microspheres of rifampicin cross-linked with sucrose using thermal gelation method for the treatment of tuberculosis

Oral controlled release microspheres of rifampicin (RIF) were prepared in order to circumvent the required regular high dose of the conventional dosage forms for the treatment of tuberculosis. Rifampicin containing microspheres were designed by using a biodegradable and biocompatible polymer, gelatin B, using a thermal gelation method. The microspheres were cross-linked with natural cross-linker, sucrose, to avoid the toxicities due to the synthetic di- and poly-aldehydes. This formulation was found to be controlled release for drug in the gastro-intestinal tract. Drug encapsulation efficiency was found to be in the range of 52–83%. These microspheres were characterized for; particle size analysis by optical microscopy and scanning electron microscopy; in vitro release study by USP paddle apparatus and drug polymer interaction study using DSC and FT-IR. The results suggested that microspheres prepared by the above method were smaller in size, i.e. less than 60 µm and sucrose could be used as an interesting means to cross-link gelatin B microspheres, allowing the use of this formulation for controlled release of rifampicin. Microspheres could be observed in the intestinal lumen at 4 h and were detectable in the intestine 24 h post-oral administration, although the percentage of radioactivity had significantly decreased (t1/2 of 99mTc = 4–5 h). Dissolution and scintigraphy studies have shown promising results, proving the utility of the formulation for the whole intestine.

Neurite growth in 3D collagen gels with gradients of mechanical properties

We have designed and developed a microfluidic system to study the response of cells to controlled gradients of mechanical stiffness in 3D collagen gels. An 'H'-shaped, source-sink network was filled with a type I collagen solution, which self-assembled into a fibrillar gel. A 1D gradient of genipin--a natural crosslinker that also causes collagen to fluoresce upon crosslinking--was generated in the cross-channel through the 3D collagen gel to create a gradient of crosslinks and stiffness. The gradient of stiffness was observed via fluorescence. A separate, underlying channel in the microfluidic construct allowed the introduction of cells into the gradient. Neurites from chick dorsal root ganglia explants grew significantly longer down the gradient of stiffness than up the gradient and than in control gels not treated with genipin. No changes in cell adhesion, collagen fiber size, or density were observed following crosslinking with genipin, indicating that the primary effect of genipin was on the mechanical properties of the gel. These results demonstrate that (1) the microfluidic system can be used to study durotactic behavior of cells and (2) neurite growth can be directed and enhanced by a gradient of mechanical properties, with the goal of incorporating mechanical gradients into nerve and spinal cord regenerative therapies.

Effect of phenolic compounds on the properties of porcine plasma protein-based film

Properties of porcine plasma protein-based film incorporated with tannic acid, caffeic acid and ferulic acid at different concentrations (1–3% (w/w) of protein content) were studied. Film-forming solution (FFS) containing 3% protein (w/v) and 70% glycerol (w/w of protein content) was preheated at 70 °C for 30 min and adjusted pH to 10 followed by the addition of phenols and film casting. Tensile strength (TS) of resulting film increased by 123.3, 194.3 and 19.5% and elongation at break (EAB) increased by 71.1, 86.3 and 10.2%, respectively, compared with the control film, when tannic acid, caffeic acid and ferulic acid at a level of 3% was added. The use of all phenolic compounds slightly increased water vapor permeability (WVP) of resulting films ( p < 0.05). The increases in a ∗- and b ∗-values of films were observed as the higher concentrations of tannic acid and caffeic acid were used. This was associated with the lowered transparency of resulting films. FFS containing 3% caffeic acid with prior oxygenation, especially with pH 10, yielded the film with increased TS but lowered EAB ( p < 0.05). Oxygenation of FFS was associated with the lower L ∗-value and higher a ∗-value of resulting films. Therefore, phenolic compounds could be used as natural cross-linkers which affected the properties of porcine plasma protein-based film differently.

Towards a biosensor immunoassay of protein-bound isopeptides in human plasma

This study demonstrates that synthetic isopeptides formed on BSA can be quantitatively analyzed by a surface plasmon resonance-based biosensor method. A monoclonal IgM antibody 81D4, that reacts with the synthetic isopeptide and also with the natural isopeptide cross-link in D-dimer (but not with its non-cross-linked fibrin monomer), was covalently immobilized to a carboxymethylated dextran surface, a CM5 surface. Its immunocapturing efficiency was found to be good. The affinity of the interaction between the monoclonal 81D4 and the synthetic isopeptide was estimated to approximately 4 × 10 −7 M. Good reactivity was also observed when human plasma spiked with this isopeptide was used as test solution. Cross-linked D-dimer in the plasma of patients is a marker of disseminated intravascular coagulation (DIC) which occurs late in sepsis. This biosensor method has the potential to be developed into a rapid sensitive assay for measuring the level of natural isopeptide cross-links in proteins in the plasma of patients with a suspected diagnosis of sepsis.

Effects of Natural Cross-Linkers on the Stability of Dentin Collagen and the Inhibition of Root Caries in vitro

Purpose: To evaluate the effects of dentin collagen modifications induced by various cross-linkers on the stability of collagen matrix and the inhibition of root caries. Materials and Methods: The following cross-linkers were tested: 5% glutaraldehyde (GA), 0.5% proanthocyanidin (PA), 0.625% genipin (GE). In the first experiment, cross-linker-treated demineralized human root dentin was digested with bacterial collagenase, centrifuged, and the supernatants were subjected to amino acid analysis to determine collagen content. The residues were analyzed by SDS-PAGE and hydroxyproline analysis. In the second experiment, bovine root surfaces were conditioned with phosphoric acid, treated with the cross-linkers, incubated with Streptococcus mutans and Lactobacillus acidophilus for 1 week and the root caries inhibition was evaluated with confocal microscopy. Lastly, the ability of the bacteria to colonize the root surface was evaluated. In this experiment slabs of bovine root were treated with the cross-linkers and incubated in a suspension of S. mutans and L. acidophilus. The slabs were washed, resuspended in water, glucose was added, and the pH measured. Results: While all collagen was digested with collagenase in the control groups, only a small proportion was solubilized in the GA-, PA-, and GE-treated groups. The root caries was significantly inhibited by treatment with PA or GA. Drops in pH in the cross-linker-treated groups were essentially the same as in the untreated group. Conclusion: Naturally occurring cross-linkers, especially PA, could be used to modify root dentin collagen to efficiently stabilize collagen and to increase its resistance against caries.

Development of a stable hydrogel network based on agar–kappa-carrageenan blend cross-linked with genipin

Blend of food hydrocolloids, viz. agar and kappa-carrageeenan (agar/kC), was treated with the natural cross-linker genipin in aqueous medium to impart functional stability. The genipin-fixed blend exhibited remarkable stability in the wide range of pH 1–12, as well as in Ringer's solution. Ratios of the hydrocolloids and genipin in the blend as well as in the cross-linked product were optimized (agar/kC 25:75 and agar/kC/genipin 25:75:0.8, w/w) on the basis of swelling properties. Compatibilities of unmodified blend (agar/kC) and the cross-linked blend product in aqueous solution were studied by measuring apparent and intrinsic viscosities and UV absorptions. Properties of agar/kC blend and the genipin cross-linked blend in solid state were evaluated by bulk and true density, pore volume, porosity, thermogravimetric analysis (TGA), swelling ability, X-ray diffraction, degradation rate in vitro, optical microscopy, scanning electron microscopy (SEM) and rheological measurements. The cross-linked blend exhibited higher viscosity, thermal stability and swelling ability as well as low weight loss ratio compared to those of the un-modified blend. The presence of genipin in the cross-linked polymer network was confirmed by MS/MS analysis. This genipin cross-linked blend presents an immense potential for food applications and in other pH-specific applications as well.

Recombinant human gelatin nanoparticles as a protein drug carrier

Considerable attention has been directed to the application of natural gelatin-based nanoparticles. However, there are still critical problems associated with a safety issue of animal-origin gelatins, heterogeneous molecular weight, subsequent nanoparticle size distributions and use of toxic cross-linker. The purpose of this study was to develop a simple, safe and reproducible preparation method of recombinant human gelatin (rHG) nanoparticles via a modified desolvation method using a natural cross-linker for protein drug delivery. The model protein, FITC-BSA, was released from rHG nanoparticles in a biphasic and sustained-release pattern without initial burst. Optimized nanoparticles based on rHG and a natural cross-linker were prepared for the first time and showed great potential for protein drug delivery in terms of sustained release, less initial burst, and safety. In addition, rHG nanoparticles were efficiently internalized in the cell and mainly localized in the cytoplasm without significant cytotoxicity. Overall, these results suggest that rHG nanoparticles are appropriate for a protein drug delivery system with minimal toxicity.

Effects of Natural Cross-Linkers on the Stability of Dentin Collagen and the Inhibition of Root Caries in vitro

Effects of Natural Cross-Linkers on the Stability of Dentin Collagen and the Inhibition of Root Caries in vitro

In Vivo Production of scFv-Displaying Biopolymer Beads Using a Self-Assembly-Promoting Fusion Partner

Recombinant production and, in particular, immobilization of antibody fragments onto carrier materials are of high interest with regard to diagnostic and therapeutic applications. In this study, the recombinant production of scFv-displaying biopolymer beads intracellularly in Escherichia coli was investigated. An anti-beta-galactosidase scFv (single chain variable fragment of an antibody) was C-terminally tagged with the polymer-synthesizing enzyme PhaC from Cupriavidus necator by generating the respective hybrid gene. The functionality of the anti-beta-galactosidase scFv-PhaC fusion protein was assessed by producing the respective soluble fusion protein in an Escherichia coli AMEF mutant strain. AMEF (antibody-mediated enzyme formation) strains contain an inactive mutant beta-galactosidase, which can be activated by binding of an anti-beta-galactosidase antibody. In vivo activation of AMEF beta-galactosidase indicated that the scFv is functional with the C-terminal fusion partner PhaC. It was further demonstrated that polymer biosynthesis and bead formation were mediated by the scFv-PhaC fusion protein in the cytoplasm of recombinant E. coli when the polymer precursor was metabolically provided. This suggested that the C-terminal fusion partner PhaC acts as a functional insolubility partner, providing a natural cross-link to the bead and leading to in vivo immobilization of the scFv. Overproduction of the fusion protein at the polymer bead surface was confirmed by SDS-PAGE and MALDI-TOF/MS analysis of purified beads. Antigen binding functionality and specificity of the beads was assessed by analyzing the binding of beta-galactosidase to scFv-displaying beads and subsequently eluting the bound protein at pH 2.7. A strong enrichment of beta-galactosidase suggested the functional display of scFv at the bead surface as well as the applicability of these beads for antigen purification. Binding of beta-galactosidase to the scFv-displaying beads was quantitatively analyzed by enzyme-linked assays measuring beta-galactosidase activity. These indicated that the anti-beta-galactosidase scFv-displaying beads bound a maximum of 38 ng of beta-galactosidase per 1 microg of bead protein, showing an apparent equilibrium dissociation constant ( KD) of 12 x 10 (-7) M. This study clearly demonstrated that anti-beta-galactosidase scFv-displaying polymer beads can be produced in engineered E. coli in a one-step process by using PhaC as a self-assembly-promoting fusion partner.

A fibrinogen-based precision microporous scaffold for tissue engineering

Fibrin has been long used as an effective scaffolding material to grow a variety of cells and tissue constructs. It has been utilized mainly as a hydrogel in varying concentrations to provide an environment in which suspended cells work to rearrange the fibers and lay down their own extracellular matrix. For these fibrin hydrogels to be useful in many tissue-engineering applications, the gels must be cultured for long periods of time in order to increase their mechanical strength to the levels of native tissues. High concentrations of fibrinogen increase the mechanical strength of fibrin hydrogels, but at the same time reduce the ability of cells within the scaffold to spread and survive. We present a method to create a microporous, nanofibriliar fibrin scaffold that has controllable pore size, porosity, and microstructure for applications in tissue engineering. Fibrin has numerous advantages as a scaffolding material as it is normally used by the body as temporary scaffolding for tissue regeneration and healing, and can be autologously sourced. We present here a scaffolding process which enhances the mechanical properties of the fibrin hydrogel by forming it surrounding poly(methyl-methacrylate) beads, then removing the beads with acetone to form an interconnected microporous network. The acetone serves the dual purpose of precipitating and fixing the fibrinogen-based scaffolds as well as adding strength to the network during polymer bead removal. Effects of fibrinogen concentration and time in acetone were examined as well as polymerization with thrombin. A natural crosslinker, genipin, was also used to add strength to the scaffolds, producing a Young's modulus of up to 184±5 kPa after 36 h of reaction. Using these methods we were able to produce microporous fibrin scaffolds that support cell growth and have mechanical properties similar to many native tissues.

Protocol for the fabrication of enzymatically crosslinked gelatin microchannels for microfluidic cell culture

We have developed a technique for fabricating microfluidic devices from gelatin using a natural crosslinking process. By producing reusable poly(dimethyl siloxane) molds using standard photolithography, gelatin can be molded into microchannel geometries. The gelatin is crosslinked with the naturally occurring enzyme transglutaminase via a straightforward process that can produce devices suitable for cell culture. The protocol takes approximately 1 day from the start of gelatin preparation to cell seeding. Using these devices, the effects of both the extracellular matrix and soluble factors on cellular behavior and differentiation can be studied in microenvironments that more closely mimic the in vivo environment.

Effects of formalin fixation and collagen cross‐linking on T2 and magnetization transfer in bovine nasal cartilage

Endogenous collagen cross-links influence cartilage biomechanical properties and resistance to degradation. Formalin fixation modifies collagen residues and forms new cross-links in a dose-dependent manner. We tested the hypothesis that magnetization transfer (MT) effects and T(2) depend on collagen cross-linking in cartilage. These parameters were measured in bovine nasal cartilage (BNC) prior to fixation, after 9 weeks of immersion in formalin solutions ranging in concentration from 0% to 10%, and after NaBH(3)CN reduction and washing. T(2) decreased by 59.4% +/- 1.1% upon fixation in 10% formalin, and was 32.2% +/- 5.2% shorter than initial values after washing. The apparent MT rate increased 25.9% +/- 3.7% and 52.8% +/- 7.1% over baseline under these conditions. Biochemical assays showed no significant differences in water, proteoglycan, natural cross-link, or collagen content between the 0% and 10% formalin-treated samples, while amino acid analysis demonstrated losses in (hydroxy)lysine and tyrosine, and new peaks consistent with methylene cross-links in fixed samples only. We conclude that formalin fixation of cartilage results in significant decreases in T(2) and increases in MT parameters that persist after removal of unreacted formaldehyde. The collagen cross-links thus created are associated with large changes in MT and T(2), indicating that interpretation of T(2) and MT values in terms of cartilage macromolecular content must be made with caution.

Photoactivated Coumaryl-diazopyruvate Fluorescent Label for Amine Functional Groups of Tissues Containing Type-I Collagen¶

The design, synthesis and application of a new fluorescent-labeling reagent for collagen has been developed as a prerequisite for the design of a photoactivated collagen-crosslinking compound for surgical wound closure. The amine groups in collagen are the targets of a rational design for a new fluorophore because natural collagen crosslinks are formed between primary (1°) amine groups of lysine and hydroxylysine. The availability of 1° amines for crosslinking in native collagenous tissues was evaluated by reacting tendon and corneal samples with o-phthalaldehyde and dansyl chloride, fluorophores commonly used for the detection of 1° and 2° amines. The resulting fluorescent collagen fibrils indicated the presence of amines in native tissue. Subsequently, a photoactivated fluorescent label for 1° and 2° amines, coumaryl gamma-amino-butyric acid diazopyruvate (CGDP), was designed and synthesized. CGDP was first used to photolabel poly-l-lysine, forming a fluorescent, covalent bond to the 1° amine. CGDP was then photoreacted with corneal and tendon tissue samples to produce CGDP fluorescent–labeled samples that were statistically significantly more fluorescent than were the controls. These experiments support the postulate that 1° or 2° (or both) amines in native collagenous tissues are available to serve as targets for photoactivated collagen crosslinkers for wound closure.

The effect of cross-linking of chitosan microspheres with genipin on protein release

Genipin, a natural and non-toxic cross-linking reagent, was evaluated for its effects on the drug/protein release and swelling of chitosan microspheres. Chitosan microspheres, using albumin as a model protein, were prepared and cross-linked with 0.5 mM genipin for 4 to 16 h or for 4 h using 0.5 to 2.0 mM genipin. The degree of cross-linking, swelling and the release of albumin from the microspheres was determined by the ninhydrin assay, measuring change in mass between dry and wet spheres, and in 31-day elution tests, respectively. The degree of cross-linking increased up to maximum of 33% to 34% with up to 8 hour cross-linking time or with up to 1.0 mM genipin concentration. Additional cross-linking time or concentration did not significantly increase degree of cross-linking. Swelling ratios decreased significantly from 119.2% in the uncross-linked condition to 108.8% at 16 h cross-linking time. However, increasing the genipin concentration resulted in much smaller decreases in swelling. The release of albumin was reduced with as little as 4 h cross-linking time to 30.9% of uncross-linked microspheres for up to 24 days and by as much as 52.3–60.0% for up to 31 days with 8–16 h cross-linking time. Using genipin concentrations of 1.0 to 2.0 mM for 4 h, greatly reduced albumin release to only 12.4% to 27.1% on day 24. These data demonstrate that protein and drug delivery rates from chitosan microspheres may be controlled and extended by controlling the degree of cross-linking with genipin.

Specific Covalent Immobilization of Proteins through Dityrosine Cross-Links

Dityrosine cross-links are widely observed in nature in structural proteins such as elastin and silk. Natural oxidative cross-linking between tyrosine residues is catalyzed by a diverse group of metalloenzymes. Dityrosine formation is also catalyzed in vitro by metal-peptide complexes such as Gly-Gly-His-Ni(II). On the basis of these observations, a system was developed to specifically and covalently surface immobilize proteins through dityrosine cross-links. Methacrylate monomers of the catalytic peptide Gly-Gly-His-Tyr-OH (GGHY) and the Ni(II)-chelating group nitrilotriacetic acid (NTA) were copolymerized with acrylamide into microbeads. Green fluorescent protein (GFP), as a model protein, was genetically tagged with a tyrosine-modified His6 peptide on its carboxy terminus. GFP-YGH6, specifically associated with the NTA-Ni(II) groups, was covalently coupled to the bead surface through dityrosine bond formation catalyzed by the colocalized GGHY-Ni(II) complex. After extensive washing with EDTA to disrupt metal coordination bonds, we observed that up to 75% of the initially bound GFP-YGH6 remained covalently bound to the bead while retaining its structure and activity. Dityrosine cross-linking was confirmed by quenching the reaction with free tyrosine. The method may find particular utility in the construction and optimization of protein microarrays.

In vitro and in vivo tissue repair with laser‐activated chitosan adhesive

Sutures are currently the gold standard for wound closure but they are still unable to seal tissue and may induce scarring or inflammation. Biocompatible glues, based on polysaccharides such as chitosan, are a possible alternative to conventional wound closure. In this study, the adhesion of laser-activated chitosan films is investigated in vitro and in vivo. In particular we examine the effect of varying the laser power, as well as adding a natural cross-linker (genipin) to the adhesive composition. Flexible and insoluble strips of chitosan films (surface area approximately 34 mm(2), thickness approximately 20 microm) were bonded to sheep intestine using several laser powers (0, 80, 120, and 160 mW) at 808-nm wavelength. The strength of repaired tissue was tested by a calibrated tensiometer to select the best power. A natural cross-linker (genipin) was also added to the film and the tissue repair strength compared with the strength of plain films. The adhesive was also bonded in vivo to the sciatic nerve of rats and the thermal damage induced by the laser assessed 4 days post-operatively. Chitosan adhesives successfully repaired intestine tissue, attaining a maximum repair strength of 14.7+/-4.3 kPa (n = 30) at the laser power of 120 mW. The chitosan-genipin films achieved lower repair strength (9.1+/-2.9 kPa). The laser caused partial demyelination of axons at the site of operation, but the myelinated axons retained a normal morphology proximally and distally. The chitosan adhesive effectively bonded to tissue causing only localized thermal damage in vivo, when the appropriate laser parameters were selected.