Xylan from Pineapple Stem Waste: a Potential Biopolymer for Colonic Targeting of Anti-inflammatory Agent Mesalamine.

We have successfully conjugated mesalamine (5-aminosalicylic acid, 5-ASA) with xylan, a biopolymer isolated from pineapple stem waste, to form xylan-5-ASA conjugate. The biopolymer was used to provide colon-targeting properties for 5-ASA, a golden standard anti-inflammatory agent commonly used for ulcerative colitis treatment. A series of data from FTIR spectroscopy, UV-Vis spectrophotometry, and HPLC confirmed the xylan-5-ASA conjugate formation. To ensure successful colon targeting properties, in vitro and in vivo drug release studies after oral administration of xylan-5-ASA conjugate to Wistar rats were performed. Xylan-5-ASA conjugate was able to retain 5-ASA release in the upper gastrointestinal tract fluid simulation but rapidly released 5-ASA in the rat colon fluid simulation. In vivo release profile shows a very low peak plasma concentration, reached at 6 h after xylan-5-ASA conjugate administration. The delayed release and the lower bioavailability of 5-ASA from xylan-5-ASA conjugate administration compared to free 5-ASA administration confirmed the successful local colon delivery of 5-ASA using xylan-5-ASA conjugate. The administration of xylan-5-ASA conjugate also exhibited greater efficacy in recovering 2,4,6-trinitrobenzene sulfonic acid-induced colon ulcer compared to free 5-ASA administration. Taken together, xylan isolated from pineapple stem waste is promising to obtain colon targeting property for 5-ASA.

Analysis of Trinitrophenylated Adenosine and Inosine by Capillary Electrophoresis and γ-Cyclodextrin-Enhanced Fluorescence Detection.

Monitoring molecules such as adenosine (Ado) and inosine (Ino) in the central nervous system has enabled the field of neuroscience to correlate molecular concentrations dynamics to neurological function, behavior, and disease. In vivo sampling techniques are commonly used to monitor these dynamics; however, many techniques are limited by the sensitivity and sample volume requirements of currently available detection methods. Here, we present a novel capillary electrophoresis-laser-induced fluorescence detection (CE-LIF) method that analyzes Ado and Ino by derivatization with 2,4,6-trinitrobenzenesulfonic acid to form fluorescent trinitrophenylated complexes of Ado (TNP-Ado) and Ino (TNP-Ino). These complexes exhibit ∼25-fold fluorescence enhancement upon the formation of inclusion complexes with γ-cyclodextrin (γ-CD). Association constants were determined as 4600 M(-1) for Ado and 1000 M(-1) for Ino by CE-LIF. The structure of the TNP-Ado:γ-CD complex was determined by 2D nuclear magnetic resonance (NMR) spectroscopy. Optimal trinitrophenylation reaction conditions and CE-LIF parameters were determined and resulted in the limit of detection of 1.6 µM for Ado and 4 µM for Ino. Ado and Ino were simultaneously quantified in homogenized rat forebrain samples to illustrate application of the technique. Simulated biological samples, desalted by ultrafiltration in the presence γ-CD, were concentrated on-capillary by large-volume sample stacking (LVSS) to achieve detection limits of 32 and 38 nM for TNP-Ado and TNP-Ino, respectively.

5-Aminosalicylic Acid Azo-Linked to Procainamide Acts as an Anticolitic Mutual Prodrug via Additive Inhibition of Nuclear Factor kappaB.

To improve the anticolitic efficacy of 5-aminosalicylic acid (5-ASA), a colon-specific mutual prodrug of 5-ASA was designed. 5-ASA was coupled to procainamide (PA), a local anesthetic, via an azo bond to prepare 5-(4-{[2-(diethylamino)ethyl]carbamoyl}phenylazo)salicylic acid (5-ASA-azo-PA). 5-ASA-azo-PA was cleaved to 5-ASA and PA up to about 76% at 10 h in the cecal contents while remaining stable in the small intestinal contents. Oral gavage of 5-ASA-azo-PA and sulfasalazine, a colon-specific prodrug currently used in clinic, to rats showed similar efficiency in delivery of 5-ASA to the large intestine, and PA was not detectable in the blood after 5-ASA-azo-PA administration. Oral gavage of 5-ASA-azo-PA alleviated 2,4,6-trinitrobenzenesulfonic acid-induced rat colitis. Moreover, combined intracolonic treatment with 5-ASA and PA elicited an additive ameliorative effect. Furthermore, combined treatment with 5-ASA and PA additively inhibited nuclear factor-kappaB (NFκB) activity in human colon carcinoma cells and inflamed colonic tissues. Finally, 5-ASA-azo-PA administered orally was able to reduce inflammatory mediators, NFκB target gene products, in the inflamed colon. 5-ASA-azo-PA may be a colon-specific mutual prodrug acting against colitis, and the mutual anticolitic effects occurred at least partly through the cooperative inhibition of NFκB activity.

The neurotensin-HIF-1α-VEGFα axis orchestrates hypoxia, colonic inflammation, and intestinal angiogenesis.

The expression of neurotensin (NT) and its receptor (NTR1) is up-regulated in experimental colitis and inflammatory bowel disease; NT/NTR1 interactions regulate gut inflammation. During active inflammation, metabolic shifts toward hypoxia lead to the activation of hypoxia-inducible factor (HIF)-1, which enhances vascular endothelial growth factor (VEGF) expression, promoting angiogenesis. We hypothesized that NT/NTR1 signaling regulates intestinal manifestations of hypoxia and angiogenesis by promoting HIF-1 transcriptional activity and VEGFα expression in experimental colitis. We studied NTR1 signaling in colitis-associated angiogenesis using 2,4,6-trinitrobenzenesulfonic acid-treated wild-type and NTR1-knockout mice. The effects of NT on HIF-1α and VEGFα were assessed on human colonic epithelial cells overexpressing NTR1 (NCM460-NTR1) and human intestinal microvascular-endothelial cells. NTR1-knockout mice had reduced microvascular density and mucosal integrity score compared with wild-type mice after 2,4,6-trinitrobenzenesulfonic acid treatment. VEGFα mRNA levels were increased in NCM460-NTR1 cells treated with 10(-7) mol/L NT, at 1 and 6 hours post-treatment. NT exposure in NCM460-NTR1 cells caused stabilization, nuclear translocation, and transcriptional activity of HIF-1α in a diacylglycerol kinase-dependent manner. NT did not stimulate tube formation in isolated human intestinal macrovascular endothelial cells but did so in human intestinal macrovascular endothelial cells cocultured with NCM460-NTR1 cells. Our results demonstrate the importance of an NTR1-HIF-1α-VEGFα axis in intestinal angiogenic responses and in the pathophysiology of colitis and inflammatory bowel disease.

Comparison of noncontrast MRI magnetization transfer and T2 -Weighted signal intensity ratios for detection of bowel wall fibrosis in a Crohn's disease animal model.

PURPOSE: To compare the abilities of magnetization transfer magnetic resonance imaging (MT-MRI) and T2 -weighted signal intensity (T2 WSI) ratios to detect intestinal fibrosis in a Crohn's disease animal model. MATERIALS AND METHODS: Ten rats ("Group 1") received one trinitrobenzenesulfonic acid enema to induce acute colonic inflammation, while 10 additional animals ("Group 2") received multiple enemas to induce colonic inflammation and fibrosis. Gradient recalled-echo MT-MRI (5 and 10 kHz off-resonance) and T2 -weighted spin-echo imaging were performed 2 days after the last enema. MT ratios (MTR) and T2 WSI ratios were calculated in the area of greatest colonic thickening. Bowel wall MTR, bowel wall MTR normalized to paraspinous muscle MTR ("normalized MTR"), and T2 WSI ratios were compared between animal groups using Student's t-test. RESULTS: At 10 kHz off-resonance, mean bowel wall MTR for Group 1 was 24.8 ± 3.1% vs. 30.3 ± 3.2% for Group 2 (P = 0.001). Mean normalized MTR was 0.45 ± 0.05 for Group 1 and 0.58 ± 0.08 for Group 2 (P = 0.0003). At 5 kHz off-resonance, mean bowel wall MTR for Group 1 was 34.7 ± 5.2% vs. 40.3 ± 3.6% for Group 2 (P = 0.015). Mean normalized MTR was 0.53 ± 0.08 for Group 1 and 0.64 ± 0.07 for Group 2 (P = 0.003). Mean T2 WSI ratio was 5.32 ± 0.98 for Group 1 and 3.01 ± 0.66 for group 2 (P < 0.0001). Mean T2 WSI ratio/MTR (10 kHz off-resonance) was 12.06 ± 2.70 for Group 1 and 5.22 ± 1.29 for Group 2 (P < 0.0001), with an ROC c-statistic of 0.98. CONCLUSION: MTR and T2 WSI ratios detect bowel wall fibrosis in a Crohn's disease animal model.

Use of the guanidination reaction for determining reactive lysine, bioavailable lysine and gut endogenous lysine.

Determining the bioavailability of lysine in foods and feedstuffs is important since lysine is often the first limiting indispensable amino acid in diets for intensively farmed livestock (pigs and poultry) and also in many cereal-based diets consumed by humans. When foods or feedstuffs are heat processed, lysine can undergo Maillard reactions to produce nutritionally unavailable products. The guanidination reaction, the reaction of O-methylisourea with the side chain amino group of lysine that produces homoarginine, has been used to determine the unmodified lysine (reactive lysine) in processed foods and feedstuffs and also true ileal digestible reactive lysine (bioavailable lysine). The advantages of the guanidination method in comparison with other reactive lysine methods such as the fluorodinitrobenzene, trinitrobenzenesulphonic acid and dye-binding methods are that it is very specific for reactive lysine and also that the method is relatively straightforward to conduct. The specificity of the guanidination reaction for the lysine side chain amino group is particularly important, since ileal digesta will contain N-terminal groups in the form of free amino acids and peptides. The main disadvantage is that complete conversion of lysine to homoarginine is required, yet it is not straightforward to test for complete guanidination in processed foods and feedstuffs. Another disadvantage is that the guanidination reaction conditions may vary for different food types and sometimes within the same food type. Consequently, food-specific guanidination reaction conditions may be required and more work is needed to optimise the reaction conditions across different foods and feedstuffs.

Cysteine-functional polymers via thiol-ene conjugation.

A thiofunctional thiazolidine is introduced as a new low-molar-mass building block for the introduction of cysteine residues via a thiol-ene reaction. Allyl-functional polyglycidol (PG) is used as a model polymer to demonstrate polymer-analogue functionalization through reaction with the unsaturated side-chains. A modified trinitrobenzenesulfonic acid (TNBSA) assay is used for the redox-insensitive quantification and a precise final cysteine content can be predetermined at the polymerization stage. Native chemical ligation at cysteine-functional PG is performed as a model reaction for a chemoselective peptide modification of this polymer. The three-step synthesis of the thiofunctional thiazolidine reactant, together with the standard thiol-ene coupling and the robust quantification assay, broadens the toolbox for thiol-ene chemistry and offers a generic and straightforward approach to cysteine-functional materials.

In Vivo Evaluation of 5-ASA Colon-Specific Tablets Using Experimental-Induced Colitis Rat Animal Model.

Colonic drug delivery is intended not only for local treatment in inflammatory bowel disease (IBD) but also for systemic delivery of therapeutics. Intestinal myeloperoxidase (MPO) determination could be used to estimate the average level of inflammation in colon as well as to determine the efficacy of drugs to be used in the treatment of inflammatory bowel diseases or study the specificity of dosage forms to be used for colonic targeting of anti-inflammatory drugs. Colonic prodrug sulfasalazine (SASP) gets metabolized to give 5-aminosalicylic acid (5-ASA), which is the active portion of SASP. However, when given orally, 5-ASA is absorbed in upper part of gastrointestinal tract (GIT) and not made available in colon. In the present study, colon-targeted delivery of 5-ASA was achieved by formulating tablets with two natural polymers namely guar gum and pectin using compression coating method. Colonic specificity of 5-ASA tablets (prepared using guar gum and pectin as polymers) was evaluated in vitro using simulated fluids mimicking in vivo environment as well as in vivo method using chemically (2,4,6-trinitrobenzenesulfonic acid and acetic acid)-induced colitis rat model. Both colon-specific formulations of 5-ASA (guar gum and pectin) were observed to be more effective in reducing inflammation in chemically induced colitis rat models when compared to colon-specific prodrug sulfasalazine as well as conventional 5-ASA administered orally.

Colitis generates remote antinociception in rats: the role of the L-arginine/NO/cGMP/PKG/KATP pathway and involvement of cannabinoid and opioid systems.

OBJECTIVE AND DESIGN: The aim of this study was to investigate the possible involvement of the NO/cGMP/PKG/KATP+ pathway, cannabinoids and opioids in remote antinociception associated with 2,4,6-trinitrobenzene sulph onic acid (TNBS)-induced colitis. METHODS: TNBS-induced colitis was induced by intracolonic administration of 20 mg of TNBS in 50% ethanol. After induction, carrageenan (500 µg/paw) or prostaglandin (PG) E2 (100 ng/paw) was injected in the rat's plantar surface and hypersensitivity was evaluated by the electronic von Frey test. Rats were pre-treated with L-Noarg one hour before carrageenan injection. L-Arginine was given 10 min before L-Noarg injections. ODQ, KT 5823, glibenclamide (Glib), naloxone and AM 251 or AM 630 were administered 30 min prior to carrageenan or PGE2 treatments. RESULTS: Colitis induction by TNBS reduced PGE2 or carrageenan-induced hypersensitivity. Antinociception produced by TNBS-induced colitis was reversed significantly (P<0.05) by L-Noarg, ODQ, KT 5823, glibenclamide, naloxone, AM251 and AM630 treatments. CONCLUSIONS: TNBS-induced colitis causes antinociception in the rat paw. This disorder appears to be mediated by activation of the NO/cGMP/PKG/KATP pathway, endocannabinoids and endogenous opioids. This information may contribute to a better understanding of peripheral neurological dysfunctions occurring in Crohn's disease.

Highly-sensitive detection of free advanced glycation end-products by liquid chromatography-electrospray ionization-tandem mass spectrometry with 2,4,6-trinitrobenzene sulfonate derivatization.

The common derivatization method of primary amino groups by 2,4,6-trinitrobenzene sulfonate (TNBS) was applied to the detection of free adducts of advanced glycation end-products (AGEs), in combination with liquid chromatography-tandem mass spectrometry-multiple reaction monitoring (LC-MS/MS-MRM). The proposed TNBS-MS method provided a surprisingly significant improvement of the detection of AGE-free adducts (e.g., by a factor of >1000 for methylglyoxal-derived hydroimidazolone) with a detection limit of 1.0 nM (10 fmol/injection volume), which was due to the high ionization efficiency of the derived trinitrophenol moiety and its hydrophobicity. With the aid of stable-isotope-labeled internal standard (MG-H1-d3), the convenient TNBS method that allowed the derivation of AGE-free adducts bearing amino groups under mild reaction conditions (30 mM, pH 8.5, 30 min, 30 °C) also permitted successive detection and quantification of five typical AGE-free adducts at nanomolar levels in rat plasma (50 µL) with high reproducibility (2-9% of RSD) and recovery (93-113%), using LC-MS/MS-MRM.

External surface area determination of lipid vesicles using trinitrobenzene sulfonate and ultraviolet/visible spectrophotometry.

The lamellarity of liposomes is an important parameter to be controlled in liposomal delivery-release applications. A practical estimate of the degree of liposome lamellarity can be obtained by measuring the relative external surface area of the liposomes using a chemical assay. All such assays are based on a signal change caused by exposed marker lipids on reaction with a specific externally added reagent. However, a quantitative determination is often distorted by background reactions and contributions of internal lipid labeling. In the so-called TNBS assay, the marker lipid is phosphatidylethanolamine (PE) and the externally added reagent is TNBS (2,4,6-trinotrobenzene sulfonate). Mechanistic aspects of the TNBS assay were considered for improving the assay. Internal lipid labeling via PE flip-flop and/or TNBS permeation was minimal not only in cholesterol-containing liposomes but also in cholesterol-free liposomes if in the latter case membrane fluidity was decreased by slightly increasing the PE content. Compared with earlier versions of the TNBS assay, the amount of marker lipid and the time for analysis could be reduced considerably. The elaborated protocol was also applied to liposomes prepared from lipidic egg yolk isolates, offering a simple and inexpensive method for the development and in-process control of new liposome formation technologies.

Improved detection of di-peptides by liquid chromatography-tandem mass spectrometry with 2,4,6-trinitrobenzene sulfonate conversion.

Highly sensitive detection of small peptides at the pM level was achieved by liquid chromatography-multiple reaction monitoring-tandem mass spectrometry (LC-MRM-MS/MS) in combination with the 2,4,6-trinitrobenzene sulfonate (TNBS) conversion technique. Six di-peptides having Tyr at the C-terminal (i.e., Gly-Tyr, Val-Tyr, Met-Tyr, Glu-Tyr, Lys-Tyr and His-Tyr) were subjected to the TNBS-MRM analysis in this study. The TNBS conversion conditions of pH 8.0, 30 °C and 60-min incubation enabled the di-peptides to be successfully converted to a trinitrophenyl (TNP) form with the mass increment of +212 Da. The proposed TNBS-MRM method enabled di-peptide detection that was highly improved by a factor of 3-55 in signal-to-noise ratio due to increased hydrophobicity by the induced TNP moiety. The method also permitted highly sensitive detection of di-peptides with a detection limit of >54 pM (>1.35 fmol/injection), achieving high reproducibility (<5% coefficient of variation) and rapidity (<30 min) by LC-MRM-MS/MS.

Comparison of methods for analysis of proteolysis by plasmin in milk.

Sensitive methods that are currently used to monitor proteolysis by plasmin in milk are limited due to their high cost and lack of standardisation for quality assurance in the various dairy laboratories. In this study, four methods, trinitrobenzene sulphonic acid (TNBS), reverse phase high pressure liquid chromatography (RP-HPLC), gel electrophoresis and fluorescamine, were selected to assess their suitability for the detection of proteolysis in milk by plasmin. Commercial UHT milk was incubated with plasmin at 37°C for one week. Clarification was achieved by isoelectric precipitation (pH 4.6 soluble extracts) or 6% (final concentration) trichloroacetic acid (TCA). The pH 4.6 and 6% TCA soluble extracts of milk showed high correlations (R2 > 0.93) by the TNBS, fluorescamine and RP-HPLC methods, confirming increased proteolysis during storage. For gel electrophoresis, extensive proteolysis was confirmed by the disappearance of α- and ß-casein bands on the seventh day, which was more evident in the highest plasmin concentration. This was accompanied by the appearance of α- and ß-casein proteolysis products with higher intensities than on previous days, implying that more products had been formed as a result of casein breakdown. The fluorescamine method had a lower detection limit compared with the other methods, whereas gel electrophoresis was the best qualitative method for monitoring ß-casein proteolysis products. Although HPLC was the most sensitive, the TNBS method is recommended for use in routine laboratory analysis on the basis of its accuracy, reliability and simplicity.

A liquid chromatographic method for determination of the modification degree of proteins: PEGylated arginase as an example.

A liquid chromatographic method was developed to determine the modification degree of PEGylated proteins. This method effectively separated free polyethylene glycol (PEG) from other species in conjugation mixtures on a C4 reversed-phase column using water-acetonitrile gradient elution. Then the concentrations of free PEG were determined according to the integrated area under the curve of its evaporative light scattering detector (ELSD) signal, which was normalized by the PEG standard with similar molecular weights. The actual numbers of PEG attached to proteins, not those of lysines modified, were calculated. This method was performed with PEGylated arginase mixtures as an example and showed clear advantages over 2,4,6-trinitrobenzenesulfonic acid (TNBS) assays.

Methodology for determining degree of hydrolysis of proteins in Hydrolysates: a review.

Degree of hydrolysis (DH) is defined as the proportion of cleaved peptide bonds in a protein hydrolysate. Several methods exist for determining DH; the most commonly used of these include the pH-stat, trinitrobenzenesulfonic acid (TNBS), o-phthaldialdehyde (OPA), trichloroacetic acid soluble nitrogen (SN-TCA), and formol titration methods. The pH-stat method is based on the number of protons released during hydrolysis; the TNBS, OPA, and formol titration methods are based on the measurement of amino groups generated from hydrolysis. The SN-TCA method measures the amount of TCA-soluble nitrogen, rather than DH. The pH-stat is the simplest and most commonly used method, but does not determine peptide bonds directly. In addition, the accuracy of the method depends on the type of hydrolytic enzymes used, the size of the hydrolyzed peptides, and the reaction temperature. Generally, the TNBS and OPA methods compare well and do directly determine DH. However, the assumption that the response factor for all derivatized N-terminal amino acids is similar may lead to inaccuracies. In conclusion, there is no consensus as to the best method for determining the DH of protein hydrolysates; consequently, there is a need for a standardized approach if interstudy comparisons are to be made.

Development and Characterization of Polymeric Peptides for Antibody Tagging of Bacterial Targets.

BACKGROUND: Microbe-Binding Peptides (MBPs) are currently being investigated to address the problem of antimicrobial resistance. Strategies enhancing their antimicrobial activity have been developed, including peptide dimerization. Here, we present an alternative approach based on peptide polymerization, yielding hapten-labelled polymeric MBPs that mediate tagging of bacteria with anti-hapten antibodies, for enhanced immune recognition by host phagocytes. METHODS: C-terminally amidated analogs of the bacterial-binding peptide IIGGR were synthesized, with or without addition of cysteine residues at both N- and C-termini. Peptides were subjected to oxidizing conditions in a dimethyl-sulfoxide/water solvent system, and polymerization was demonstrated using SDS-PAGE. Peptides were then N-terminally labelled with a trinitrophenyl (TNP) group using trinitrobenzene sulfonate (TNBS). Binding to representative bacteria was demonstrated by ELISA using anti-TNP antibodies and was quantified as half-maximal effective concentration (EC50). Minimum Inhibitory Concentration (MIC) and concentration yielding 50% hemolysis (H50) were estimated. Neutrophil phagocytic index was determined for TNP-labelled polymeric bacterial- binding peptide (Pbac) with anti-TNP antibodies and/or serum complement. RESULTS: Polydisperse Pbac was synthesized. EC50 was lower for Pbac than for the corresponding monomeric form (Mbac), for both Staphylococcus aureus ATCC 29213 and Escherichia coli ATCC 25922. MIC and H50 were >250µg/mL for both Pbac and Mbac. A complement-independent increase in neutrophil phagocytic index was observed for E. coli treated with TNP-labelled Pbac in conjunction with anti-TNP antibodies. CONCLUSION: Our data suggest that hapten-labelled polymeric bacterial-binding peptides may easily be produced from even crude synthetic oligopeptide precursors, and that such bacterial-binding peptides in conjunction with cognate anti-hapten antibodies can enhance immune recognition of bacteria by host phagocytes.

The construction of thiol-functionalized DNAsomes with small molecules response and protein release.

In this work, a poly(N-isopropylacrylamide) polymer (PNIPAAm) was prepared via the photoinduced reversible addition-fragmentation chain transfer (RAFT) polymerization using Ru(bpy)3Cl2·6H2O as photoinitiator. The design and spontaneous assembly of thiol-functionalized DNA-Thiol/PNIPAAm polymeric capsule (DNAsomes) by water-in-oil Pickering emulsion method and effective response with small molecules (Sybr green and phenanthrene) were described. The intermediate product, DNA-Thiol/PNIPAAm conjugates and DNAsomes were characterized by using 1H NMR, dynamic light scattering (DLS), SEM, TEM and UV-vis methods. The obtained results indicated that DNA-Thiol/PNIPAAm constructs assembled in a Pickering emulsion could produce DNA-based spherical DNAsomes with typically 3.3-267.7 µm in diameter. The DNAsomes showed a vesicle formation approximately 2 µm in diameter, resulting in phenanthrene molecule intercalating with DNAsomes. The phenomenon indicated that the DNA-Thiol/PNIPAAm conjugates may have potential applications in recognition polycyclic aromatic hydrocarbon molecules. The membrane of the DNAsomes could effective response toward small molecules such as Sybr green or phenanthrene, and DNAsomes has release capability of protein (BSA) under reductive agent glutathione (GSH). Our results highlight the potential of integrating aspects of supramolecular and polymer chemistry into the design and construction of DNA-polymeric capsule, guest molecule encapsulation, control delivery of drugs, recognition organic polycyclic aromatic hydrocarbon molecules and gene-directed capsule synthesis.

Smart gated magnetic silica mesoporous particles for targeted colon drug delivery: New approaches for inflammatory bowel diseases treatment.

Magnetic mesoporous silica microparticles were loaded with safranin O (S1) and with hydrocortisone (S2) and the outer surface functionalized with a bulky azo derivative bearing urea moieties. Aqueous suspensions of both solids at pH 7.4 showed negligible payload release whereas a marked delivery was observed in the presence of sodium dithionite due to the rupture of the azo bonds. Besides, a moderate cargo release was observed at acidic pH due to the hydrolysis of the urea bonds that linked the azo derivative onto the external surface of the inorganic scaffolds. In vitro digestion models showed that S1 and S2 microparticles could be used for the controlled release of payload in the reducing colon environment (in which azoreductase enzymes are present). On the other hand, in vivo pharmacokinetic studies in rats showed that safranine O release from S1 microparticles was concentrated in colon. The performance of S2 microparticles for the treatment of colitis in rats (induced by oral administration of a 2,4,6-trinitrobenzenesulfonic acid solution) was tested. The controlled release of hydrocortisone from S2 in the colon of injured rats induced marked reduction in colon/body weight ratio and in clinical activity score. Also, histological studies showed a marked decrease in inflammation followed by intensive regeneration and almost normal mucosal structure of the individuals treated with S2. Besides, the use of a magnetic belt increased the therapeutic performances of S2 due to an enhanced retention time of the particles in the colon.

Biochemical characterization of a novel beta-1--3, 1--4 glucan 4-glucanohydrolase from Thermomonospora sp. having a single active site for lichenan and xylan.

A bifunctional high molecular weight (Mr, 64,500 Da) beta-1-3, 1-4 glucan 4-glucanohydrolase was purified to homogeneity from Thermomonospora sp., exhibiting activity towards lichenan and xylan. A kinetic method was used to analyze the active site that hydrolyzes lichenan and xylan. The experimental data was in agreement with the theoretical values calculated for a single active site. Probing the conformation and microenvironment at active site of the enzyme by fluorescent chemo-affinity label, OPTA resulted in the formation of an isoindole derivative with complete inactivation of the enzyme to hydrolyse both lichenan and xylan confirmed the results of kinetic method. OPTA forms an isoindole derivative by cross-linking the proximal thiol and amino groups. The modification of cysteine and lysine residues by DTNB and TNBS respectively abolished the ability of the enzyme to form an isoindole derivative with OPTA, indicating the participation of cysteine and lysine in the formation of isoindole complex.

Characterisation of heat-induced protein aggregation in whey protein isolate and the influence of aggregation on the availability of amino groups as measured by the ortho-phthaldialdehyde (OPA) and trinitrobenzenesulfonic acid (TNBS) methods.

Whey protein isolate (WPI) solutions, with different levels of aggregated protein, were prepared by heating (5% protein, pH 7, 90°C for 30min) WPI solutions with either 20mM added NaCl (WPI+NaCl), 5mM N-ethylmaleimide (WPI+NEM) or 20mM added NaCl and 5mM NEM (WPI+NaCl+NEM). Gel electrophoresis demonstrated that the heated WPI and WPI+NaCl solutions had higher levels of aggregated protein, due to more covalent interactions between proteins, than the heated WPI+NEM and WPI+NaCl+NEM solutions. There were marked differences in the levels of amino groups between all heated WPI solutions when measured by the OPA and TNBS methods, with lower levels being measured by the TNBS method than by the OPA method. These results demonstrate that the measurement of available amino groups by the OPA method is less impacted than by the TNBS method after heat-induced structural changes, arising from disulfide or sulfhydryl-disulfide bond-mediated aggregation of whey protein molecules.