Effects of psoralen on hepatic bile acid transporters in rats.

Fructus Psoraleae (FP), widely used in traditional medicine, is increasingly reported to cause serious hepatotoxicity in recent years. However, the main toxic constituents responsible for hepatotoxicity and the underlying mechanisms are poorly understood. In the present study, psoralen, a main and quality-control constituent of FP, was intragastrically administered to Sprague-Dawley rats at a dose of 60 mg/kg for 1, 3 and 7 days. Blood and selected tissue samples were collected and analyzed for biochemistry and histopathology to evaluate hepatotoxicity. The results showed that psoralen could induce hepatotoxicity by enhanced liver-to-body weight ratio and alterations of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST) and total cholesterol after administration for 3 days. In addition, histopathological examinations also indicated the hepatotoxicity induced by psoralen. Furthermore, the mRNA and protein levels of hepatic bile acid transporters were significantly changed, in which MRP4, ABCG5 and ABCG8 were repressed, while the protein level of NTCP tended to increase in the rat liver. Taken together, psoralen caused liver injury possibly through affecting bile acid transporters, leading to the disorder of bile acid transport and accumulation in hepatocytes.

The influence of pH and enzyme cross-linking on protein delivery properties of WPI-beet pectin complexes.

The incorporation of bioactive proteins and peptides into food is associated with the loss of bioactivity due to deactivation in complex food matrices and in digestion systems. In this study, two different types of protein carriers, i.e. biopolymer complexation and complex coacervation were fabricated using whey protein isolation (WPI, 6wt%) and beet pectin (BP, 1.25 and 1.00wt%) at pH5.5 and 3.5, respectively. The release of the encapsulated FITC-BSA, a model bioactive protein, in both carriers in the absence and presence of laccase was investigated at both pH7.0 and 4.0. Release of FITC-BSA from both lyophilized WPI-beet pectin biopolymer complexation and complex coacervation were biphasic with an initial burst release followed by a slower release phase. The addition of laccase in biopolymer complexation increased the loading efficiency from 44.95% to 52.15% and slowed down the burst release of FITC-BSA but did change the biphasic release pattern. Laccase-cross linked WPI (6wt%)-BP (1wt%) complex coacervation had highest FITC-BSA loading efficiency (96.90%). The release of the embedded FITC-BSA in this carrier at both pH4 and 7 was in a gradual manner and the profile can be fit to zero order kinetics over the 72h study period suggesting enzymatically reinforced complex coacervation between the protein and the negatively charged beet pectin can restrain the burst release of FITC-BSA. These results indicate that laccase cross-linked WPI-beet pectin complex coacervation can be a good carrier system for delivering hydrophilic bioactive proteins or peptides successfully with enhanced loading parameters and sustained release profiles.

Mammals divert endogenous genotoxic formaldehyde into one-carbon metabolism.

The folate-driven one-carbon (1C) cycle is a fundamental metabolic hub in cells that enables the synthesis of nucleotides and amino acids and epigenetic modifications. This cycle might also release formaldehyde, a potent protein and DNA crosslinking agent that organisms produce in substantial quantities. Here we show that supplementation with tetrahydrofolate, the essential cofactor of this cycle, and other oxidation-prone folate derivatives kills human, mouse and chicken cells that cannot detoxify formaldehyde or that lack DNA crosslink repair. Notably, formaldehyde is generated from oxidative decomposition of the folate backbone. Furthermore, we find that formaldehyde detoxification in human cells generates formate, and thereby promotes nucleotide synthesis. This supply of 1C units is sufficient to sustain the growth of cells that are unable to use serine, which is the predominant source of 1C units. These findings identify an unexpected source of formaldehyde and, more generally, indicate that the detoxification of this ubiquitous endogenous genotoxin creates a benign 1C unit that can sustain essential metabolism.

Inhibitor mechanisms in the S1 binding site of the dopamine transporter defined by multi-site molecular tethering of photoactive cocaine analogs.

Dopamine transporter (DAT) blockers like cocaine and many other abused and therapeutic drugs bind and stabilize an inactive form of the transporter inhibiting reuptake of extracellular dopamine (DA). The resulting increases in DA lead to the ability of these drugs to induce psychomotor alterations and addiction, but paradoxical findings in animal models indicate that not all DAT antagonists induce cocaine-like behavioral outcomes. How this occurs is not known, but one possibility is that uptake inhibitors may bind at multiple locations or in different poses to stabilize distinct conformational transporter states associated with differential neurochemical endpoints. Understanding the molecular mechanisms governing the pharmacological inhibition of DAT is therefore key for understanding the requisite interactions for behavioral modulation and addiction. Previously, we leveraged complementary computational docking, mutagenesis, peptide mapping, and substituted cysteine accessibility strategies to identify the specific adduction site and binding pose for the crosslinkable, photoactive cocaine analog, RTI 82, which contains a photoactive azide attached at the 2ß position of the tropane pharmacophore. Here, we utilize similar methodology with a different cocaine analog N-[4-(4-azido-3-I-iodophenyl)-butyl]-2-carbomethoxy-3-(4-chlorophenyl)tropane, MFZ 2-24, where the photoactive azide is attached to the tropane nitrogen. In contrast to RTI 82, which crosslinked into residue Phe319 of transmembrane domain (TM) 6, our findings show that MFZ 2-24 adducts to Leu80 in TM1 with modeling and biochemical data indicating that MFZ 2-24, like RTI 82, occupies the central S1 binding pocket with the (+)-charged tropane ring nitrogen coordinating with the (-)-charged carboxyl side chain of Asp79. The superimposition of the tropane ring in the three-dimensional binding poses of these two distinct ligands provides strong experimental evidence for cocaine binding to DAT in the S1 site and the importance of the tropane moiety in competitive mechanisms of DA uptake inhibition. These findings set a structure-function baseline for comparison of typical and atypical DAT inhibitors and how their interactions with DAT could lead to the loss of cocaine-like behaviors.

Poly (methyl vinyl ether-co-maleic acid) - Pectin based hydrogel-forming systems: Gel, film, and microneedles.

Cross-linking of natural and synthetic polymers is widely explored to achieve the desired material properties (mechanical strength, drug loading capacity, swelling and erosion rates). However, the potential of polymers produced by crosslinking poly (methyl vinyl ether-co-maleic acid) (PMVE/MA) and pectin (PE) in pharmaceutics is mainly unexplored so far. We have investigated the effect of various esterification conditions and pectin content on the physicochemical properties. Materials have been characterized by fourier transform infrared, differential scanning calorimetry and scanning electron microscopy. In addition, swelling and bioadhesive features of PMVE/MA-PE hydrogel systems were investigated. A band shift for the carbonyl group from 1706 to 1776cm-1, and glass transition (Tg) increased from 55.4±0.9°C to 119.5±0.3°C confirmed the formation of esterification reaction within the cross-linked films. Cross-linked PMVE/MA:PE films with a ratio of 5 demonstrated a superior mass increase when compared to 2.5, 3.125, 3.75, 6.25, and 7.5 ratios of the same hydrogel film. Formulations containing PMVE/MA and pectin with a ratio of 3.75 showed superior bioadhesive features. For the first time, we engineered three-dimensional printing based swell-able microneedle arrays made out of cross-linked PMVE/MA-PE. Microneedle arrays height and aspect ratio were ranged from 702.5±11.9µm to 726±23.3µm and 3.12±0.20 to 3.29±0.21, respectively. Cross-linked PMVE/MA-PE Microneedle arrays (10-2, 24h) indicated the least height loss, 22.33±4.15%, during axial compression test; whilst, transverse failure of cross-linked PMVE/MA-PE Microneedle arrays was varied from 0.15±0.05 to 0.25±0.04N/needle. In conclusion, we obtained a novel cross-linked polymer system with promising features of drug delivery and bio-analytical applications.

Calcium gluconate as cross-linker improves survival and shelf life of encapsulated and dried Metarhizium brunneum and Saccharomyces cerevisiae for the application as biological control agents.

Calcium chloride (CC) is the most common cross-linker for the encapsulation of biocontrol microorganisms in alginate beads. The aim of this study was to evaluate if calcium gluconate (CG) can replace CC as cross-linker and at the same time improve viability after drying and rehydration, hygroscopic properties, shelf life and nutrient supply. Hence, the biocontrol fungi Metarhizium brunneum and Saccharomyces cerevisiae were encapsulated in Ca-alginate beads supplemented with starch. Beads were dried and maximum survival was found in beads cross-linked with CG. Beads prepared with CG showed lower hygroscopic properties, but a higher shelf life for encapsulated fungi. Moreover, we demonstrated that gluconate has a nutritive effect on encapsulated fungi, leading to increased mycelium growth of M. brunneum and to enhanced CO2 release from beads containing Saccharomyces cerevisiae. The application of CG as cross-linker will pave the way towards increasing drying survival and shelf life of various, especially drying-sensitive microbes.

Effects of suppressed bone remodeling by minodronic acid and alendronate on bone mass, microdamage accumulation, collagen crosslinks and bone mechanical properties in the lumbar vertebra of ovariectomized cynomolgus monkeys.

Collagen crosslinking is an important determinant of the quality of bone material. We have previously shown that suppressed bone turnover by high doses of minodronic acid and alendronate increases compressive strength of vertebra, but also increases microdamage accumulation, in monkey bone. The aim of this study is to examine the effects of these bisphosphonates on collagen crosslinks and intrinsic material properties, in addition to microdamage accumulation, in vertebral cancellous bone in ovariectomized cynomolgus monkeys. Sixty female monkeys aged 9-17years were divided into five groups: sham and ovariectomized groups were treated daily for 17months with lactose vehicle, and the other three groups were given minodronic acid daily at 0.015 or 0.15mg/kg or alendronate daily at 0.5mg/kg orally. After sacrifice, lumbar vertebrae were subjected to histomorphometry, microdamage measurement, analysis of collagen crosslinking and compressive mechanical tests. Minodronic acid caused dose-dependent suppression of increased bone remodeling due to ovariectomy, and low-dose minodronic acid suppressed remodeling same level as alendronate. However, low-dose minodronic acid did not change microdamage accumulation, collagen maturity and the pentosidine level, whereas high-dose minodronic acid and alendronate increased these parameters. Compressive ultimate load was increased following high-dose minodronic acid and alendronate, but no treatment altered the reduction in intrinsic material properties caused by ovariectomy. These findings suggest that deterioration of bone material and formation of pentosidine and microdamage induced by minodronic acid is less than that expected based on the extent of remodeling suppression, in comparison with alendronate, but this was not reflected in any significant change of mechanical properties.

Correlation of Phosphorus Cross-Linking to Hydration Rates in Sodium Starch Glycolate Tablet Disintegrants Using MRI.

Understanding the behavior of tablet disintegrants is valuable in the development of pharmaceutical solid dosage formulations. In this study, high-resolution magnetic resonance imaging has been used to understand the hydration behavior of a series of commercial sodium starch glycolate (SSG) samples, providing robust estimates of tablet disintegration rate that could be correlated with physicochemical properties of the SSGs, such as the extent of phosphorus (P) cross-linking as obtained from infra-red spectroscopy. Furthermore, elemental analysis together with powder X-ray diffraction has been used to quantify the presence of carboxymethyl groups and salt impurities, which also contribute to the disintegration behavior. The utility of Fast Low Angle SHot magnetic resonance imaging has been demonstrated as an approach to rapidly acquire approximations of the volume of a disintegrating tablet and, together with a robust voxel analysis routine, extract tablet disintegration rates. In this manner, a complete characterization of a series of SSG grades from different sources has been performed, showing the variability in their physicochemical properties and demonstrating a correlation between their disintegration rates and intrinsic characteristics. The insights obtained will be a valuable aid in the choice of disintegrant source as well as in managing SSG variability to ensure robustness of drug products containing SSG.

Subunit-directed click coupling via doubly cross-linked hemoglobin efficiently produces readily purified functional bis-tetrameric oxygen carriers.

While cross-linked hemoglobin (Hb) tetramers can deliver oxygen as a supplement to red cells, they also cause unacceptable increases in blood pressure, presumably from their penetration of the linings of blood vessels (endothelia) where the internal hemes bind endogenous nitric oxide (NO). This penetration would lower the local concentration of NO that normally induces vasodilation. Enlarging the effective size of the oxygen-carrying protein by coupling two Hbs can prevent their extravasation. Efficient and selective protein-protein coupling to produce those species has been a significant challenge. Introduction of an azide within a protein provides a directionally-oriented reaction site for utilization of the Cu(i)-catalyzed azide-alkyne cycloaddition (CuAAC) in the protein-protein-coupling process based on solubility-directed sequential addition to a bis-alkyne. However, it is known that Hb with an azide-containing cross-link between α-subunits is unreactive in CuAAC. To direct reaction away from the α-subunits of Hb, a specific fumaryl cross-link is installed exclusively between the most reactive sites on those subunits, thereby blocking the α-99 lysyl groups and preventing any further reaction. This modification allows installation of an azide-containing cross-link exclusively between lysine-82 ε-amino groups of the ß-subunits of Hb. The multiply interconnected sites establish a geometry that permits initial interfacial interaction of the cross-linked Hb-azide with Cu(i) and a bis-alkyne. After coupling, the protein-linked azide product undergoes CuAAC at the remaining alkyne with a second cross-linked Hb-azide, producing a fully functional cross-linked Hb bis-tetramer whose oxygenation and structural properties include cooperativity and oxygen affinity that should be suitable for testing as an alternative to red cells in transfusions.

The major birch pollen allergen Bet v 1 induces different responses in dendritic cells of birch pollen allergic and healthy individuals.

Dendritic cells play a fundamental role in shaping the immune response to allergens. The events that lead to allergic sensitization or tolerance induction during the interaction of the major birch pollen allergen Bet v 1 and dendritic cells are not very well studied. Here, we analyzed the uptake of Bet v 1 and the cross-reactive celery allergen Api g 1 by immature monocyte-derived dendritic cells (iMoDCs) of allergic and normal donors. In addition, we characterized the allergen-triggered intracellular signaling and transcriptional events. Uptake kinetics, competitive binding, and internalization pathways of labeled allergens by iMoDCs were visualized by live-cell imaging. Surface-bound IgE was detected by immunofluorescence microscopy and flow cytometry. Allergen- and IgE-induced gene expression of early growth response genes and Th1 and Th2 related cytokines and chemokines were analyzed by real-time PCR. Phosporylation of signaling kinases was analyzed by Western blot. Internalization of Bet v 1 by iMoDCs of both donor groups, likely by receptor-mediated caveolar endocytosis, followed similar kinetics. Bet v 1 outcompeted Api g 1 in cell surface binding and uptake. MoDCs of allergic and healthy donors displayed surface-bound IgE and showed a pronounced upregulation of Th2 cytokine- and NFκB-dependent genes upon non-specific Fcε receptor cross-linking. In contrast to these IgE-mediated responses, Bet v 1-stimulation increased transcript levels of the Th2 cytokines IL-4 and IL-13 but not of NFκB-related genes in MoDCs of BP allergic donors. Cells of healthy donors were either unresponsive or showed elevated mRNA levels of Th1-promoting chemokines. Moreover, Bet v 1 was able to induce Erk1/2 and p38 MAPK activation in BP allergics but only a slight p38 activation in normal donors. In conclusion, our data indicate that Bet v 1 favors the activation of a Th2 program only in DCs of BP allergic individuals.

In vivo evaluation of folate decorated cross-linked micelles for the delivery of platinum anticancer drugs.

The biodistribution of micelles with and without folic acid targeting ligands were studied using a block copolymer consisting of acrylic acid (AA) and polyethylene glycol methyl ether acrylate (PEGMEA) blocks. The polymers were prepared using RAFT polymerization in the presence of a folic acid functionalized RAFT agent. Oxoplatin was conjugated onto the acrylic acid block to form amphiphilic polymers which, when diluted in water, formed stable micelles. In order to probe the in vivo stability, a selection of micelles were cross-linked using 1,8-diamino octane. The sizes of the micelles used in this study range between 75 and 200 nm, with both spherical and worm-like conformation. The effects of cross-linking, folate conjugation and different conformation on the biodistribution were studied in female nude mice (BALB/c) following intravenous injection into the tail vein. Using optical imaging to monitor the fluorophore-labeled polymer, the in vivo biodistribution of the micelles was monitored over a 48 h time-course after which the organs were removed and evaluated ex vivo. These experiments showed that both cross-linking and conjugation with folic acid led to increased fluorescence intensities in the organs, especially in the liver and kidneys, while micelles that are not conjugated with folate and not cross-linked are cleared rapidly from the body. Higher accumulation in the spleen, liver, and kidneys was also observed for micelles with worm-like shapes compared to the spherical micelles. While the various factors of cross-linking, micelle shape, and conjugation with folic acid all contribute separately to prolong the circulation time of the micelle, optimization of these parameters for drug delivery devices could potentially overcome adverse effects such as liver and kidney toxicity.

Structure-property relationships and biocompatibility of carbohydrate crosslinked polyurethanes.

Biocompatible and biodegradable polyurethanes (PUs) based on castor oil and polypropylene glycols (PPGs) were prepared using various carbohydrate crosslinkers: monosaccharide (glucose), disaccharide (sucrose) and polysaccharides (starch and cellulose). The mechanical and thermal properties were investigated and interpreted on the basis of SEM study. The advantage of incorporating various carbohydrates is to have tunable mechanical properties and biodegradability due to variety in their structure. The glass transition temperature and sorption behavior were dominated by the type of polyol than by the type of crosslinker. All the PUs were observed to be biodegradable as well as non-cytotoxic as revealed by MTT assay in normal lung cell line L132. The study supports the suitability of carbohydrates as important components of biocompatible PUs for development of biomedical devices.

Aliphatic ß-nitroalcohols for therapeutic corneoscleral cross-linking: corneal permeability considerations.

PURPOSE: Our recent tissue cross-linking studies have raised the possibility of using aliphatic ß-nitroalcohols (BNAs) for pharmacologic, therapeutic corneal cross-linking. The present study was performed to determine the permeability of BNAs and to explore the use of permeability-enhancing agents. METHODS: Ex vivo rabbit corneas were mounted in a typical Franz diffusion chamber. BNA permeability was determined by assaying the recipient chamber over time using a modification of the Griess nitrite colorimetric assay. The apparent permeability coefficient (Ptot) was determined for 2 mono-nitroalcohols [2-nitroethanol (2ne) and 2-nitro-1-propanol (2nprop)], a nitrodiol [2-methyl-2-nitro-1,3-propanediol (MNPD)], and a nitrotriol [2-hydroxymethyl-2-nitro-1,3-propanediol (HNPD)]. Permeability-enhancing effects using benzalkonium chloride (BAC) (0.01% and 0.02%), ethylenediaminetetraacetic acid (0.05%), and a combination of 0.01% BAC + 0.5% tetracaine were also studied. RESULTS: The Ptot (±SE) values (in centimeters per second) were as follows: 4.33 × 10 (±9.82 × 10) for 2ne [molecular weight (MW) = 91 Da], 9.34 × 10 (±2.16 × 10) for 2nprop (MW = 105 Da), 4.37 × 10 (±1.86 × 10) for MNPD (MW = 135 Da), and 8.95 × 10 (±1.93 × 10) for HNPD (MW = 151 Da). Using the nitrodiol, permeability increased approximately 2-fold using 0.01% BAC, 5-fold using 0.02% BAC, and 5-fold using the combination of 0.01% BAC + 0.5% tetracaine. No effect was observed using 0.05% ethylenediaminetetraacetic acid. CONCLUSIONS: The results indicate that the corneal epithelium is permeable to BNAs, with the apparent permeability corresponding to MW. The findings are consistent with previous literature indicating that the small size of these compounds (<10Å) favors their passage through the corneal epithelium via the paracellular route. This information will help to guide dosing regimens for in vivo topical cross-linking studies.

Reduced bone breakage and increased bone strength in free range laying hens fed omega-3 polyunsaturated fatty acid supplemented diets.

INTRODUCTION: The omega-3 and omega-6 polyunsaturated fatty acids (PUFAs) are the immediate precursors to a number of important mediators of immunity, inflammation and bone function, with products of omega-6 generally thought to promote inflammation and favour bone resorption. Western diets generally provide a 10 to 20-fold deficit in omega-3 PUFAs compared with omega-6, and this is thought to have contributed to the marked rise in incidence of disorders of modern human societies, such as heart disease, colitis and perhaps osteoporosis. Many of our food production animals, fed on grains rich in omega-6, are also exposed to a dietary deficit in omega-3, with perhaps similar health consequences. Bone fragility due to osteoporotic changes in laying hens is a major economic and welfare problem, with our recent estimates of breakage rates indicating up to 95% of free range hens suffer breaks during lay. METHODS: Free range hens housed in full scale commercial systems were provided diets supplemented with omega-3 alpha linolenic acid, and the skeletal benefits were investigated by comparison to standard diets rich in omega-6. RESULTS: There was a significant 40-60% reduction in keel bone breakage rate, and a corresponding reduction in breakage severity in the omega-3 supplemented hens. There was significantly greater bone density and bone mineral content, alongside increases in total bone and trabecular volumes. The mechanical properties of the omega-3 supplemented hens were improved, with strength, energy to break and stiffness demonstrating significant increases. Alkaline phosphatase (an osteoblast marker) and tartrate-resistant acid phosphatase (an osteoclast marker) both showed significant increases with the omega-3 diets, indicating enhanced bone turnover. This was corroborated by the significantly lower levels of the mature collagen crosslinks, hydroxylysyl pyridinoline, lysyl pyridinoline and histidinohydroxy-lysinonorleucine, with a corresponding significant shift in the mature:immature crosslink ratio. CONCLUSIONS: The improved skeletal health in laying hens corresponds to as many as 68million fewer hens suffering keel fractures in the EU each year. The biomechanical and biochemical evidence suggests that increased bone turnover has enhanced the bone mechanical properties, and that this may suggest potential benefits for human osteoporosis.

Transglutaminase catalyzed cross-linking of sodium caseinate improves oxidative stability of flaxseed oil emulsion.

Sodium caseinate was modified by transglutaminase catalyzed cross-linking reaction prior to the emulsification process in order to study the effect of cross-linking on the oxidative stability of protein stabilized emulsions. The extent of the cross-linking catalyzed by different dosages of transglutaminase was investigated by following the ammonia production during the reaction and using SDS-PAGE gel. O/W emulsions prepared with the cross-linked and non-cross-linked sodium caseinates were stored for 30 days under the same conditions. Peroxide value measurement, oxygen consumption measurement, and headspace gas chromatography analysis were used to study the oxidative stability of the emulsions. The emulsion made of the cross-linked sodium caseinate showed an improved oxidative stability with reduced formation of fatty acid hydroperoxides and volatiles and a longer period of low rate oxygen consumption. The improving effect of transglutaminase catalyzed cross-linking could be most likely attributed to the enhanced physical stability of the interfacial protein layer against competitive adsorption by oil oxidation products.

Transepithelial corneal collagen crosslinking for keratoconus: qualitative investigation by in vivo HRT II confocal analysis.

PURPOSE: This was a qualitative investigation of corneal microstructural modifications in keratoconic patients undergoing experimental transepithelial crosslinking (TE CXL). METHODS: Ten patients with keratoconus intolerant to gas-permeable rigid contact lenses were enrolled. Corneal thickness was in the range 350-390 µm at the thinnest point measured by Visante AC optical coherence tomography system (Zeiss, Jena, Germany). All patients underwent TE CXL with 0.1% riboflavin-15% dextran solution supplemented with TRIS plus sodium EDTA (Ricrolin TE, Sooft Italia) according to Siena protocol. In vivo Heidelberg retinal tomograph II laser scanning confocal analysis (Rostock Cornea Module, Heidelberg, Germany) was performed with the following follow-up: preoperative and postoperative assessments at 1, 3, and 6 months. The following morphologic parameters were evaluated: epithelium, subepithelial, and anterior stroma nerve plexi, keratocytes apoptosis, stromal changes, and the endothelium. RESULTS: After TE CXL, epithelial cells showed apoptosis, with mosaic alterations gradually disappearing. Keratocytes apoptosis was variable, superficial, and uneven, with a maximum depth of penetration at about 140 µm, measured from the surface of epithelium. Treatment respected subepithelial and stromal nerves that did not disappear. No variation in cell count or endothelial mosaic was observed. CONCLUSIONS: In vivo confocal analysis of corneal modifications induced by TE CXL showed a limited apoptotic affect of this treatment, about one-third of classic epi-off crosslinking procedure. The TE CXL respected sub-basal and anterior stroma nerve fibers, resulting safe for corneal endothelium. According to limited penetration, its mid- to long-term efficacy needs to be determined in different clinical settings related to patient age and keratoconus progression.

Cross-linking of interfacial layers affects the salt and temperature stability of multilayered emulsions consisting of fish gelatin and sugar beet pectin.

This study assessed the stabilizing effect of enzymatic cross-linking on double-coated emulsions (beet pectin-fish gelatin). The beet pectin layer was cross-linked via ferulic acid groups using laccase (an enzyme that is known to catalyze the oxidation of phenolic groups). Fish gelatin-coated oil droplets (primary emulsion) were mixed at pH 3.5 to promote electrostatic deposition of the beet pectin molecules onto the surfaces of the oil droplets (secondary emulsion). Laccase was then added to promote cross-linking of the adsorbed beet pectin layer. Cross-linked pectin-coated oil droplets had similar or significantly better stability (p < 0.05) than oil droplets of primary or secondary emulsions to NaCl addition (0-500 mM), CaCl(2) addition (0-250 mM), and thermal processing (30-90 °C for 30 min). Freeze-thaw stability and creaming behavior of enzyme-treated, secondary emulsions after two cycles (-8 °C for 22 h; 25 °C for 2 h) were significantly improved (p < 0.05). These results may have important implications for food manufacturers that are in need of emulsions with improved physical stability, for example, emulsions used in frozen foods for sauces or dips.

Extracellular TG2: emerging functions and regulation.

Tissue transglutaminase (TG2) is a ubiquitously expressed member of the transglutaminase family of Ca(2+)-dependent crosslinking enzymes. Unlike other family members, TG2 is a multifunctional protein, which has several other well documented enzymatic and non-enzymatic functions. A significant body of evidence accumulated over the last decade reveals multiple and complex activities of this protein on the cell surface and in the extracellular matrix (ECM), including its role in the regulation of cell-ECM interactions and outside-in signaling by several types of transmembrane receptors. Moreover, recent findings indicate a dynamic regulation of the levels and functions of extracellular TG2 by several complementary mechanisms. This review summarizes and assesses recent research into the emerging functions and regulation of extracellular TG2.

Improving laccase catalyzed cross-linking of whey protein isolate and their application as emulsifiers.

Whey protein isolate (WPI) was chemically modified by vanillic acid in order to enhance its cross-linkability by laccase enzyme. Incorporation of methoxyphenol groups created reactive sites for laccase on the surface of the protein and improved the efficiency of cross-linking. The vanillic acid modified WPI (Van-WPI) was characterized using MALDI-TOF mass spectrometry, and the laccase-catalyzed cross-linking of Van-WPI was studied. Furthermore, the vanillic acid modification was compared with the conventional approach to improve laccase-catalyzed cross-linking by adding free phenolic compounds. A small extent of the vanillic acid modification significantly improved the cross-linkability of the protein and made it possible to avoid color formation in a system that is free of small phenolic compounds. Moreover, the potential application of Van-WPI as emulsifier and the effect of cross-linking on the stability of Van-WPI emulsion were investigated. The post-emulsification cross-linking by laccase was proven to enhance the storage stability of Van-WPI emulsion.