Reduced immunogenicity of beta-lactoglobulin by conjugating with chitosan

Bovine beta-lactoglobulin (beta-LG) was conjugated with chitosan (CHS) by means of a water-soluble carbodiimide to reduce the immunogenicity of beta-LG. Each beta-LG-CHS conjugate was purified by ion-exchange chromatography and hydrophobic chromatography. The conjugation between beta-LG and CHS was confirmed by SDS-PAGE, the isoelectric point of the conjugate being higher than that of beta-LG. Two types of the beta-LG-CHS conjugate were obtained with molar ratios of beta-LG to CHS of 1:1 (F1) and 1:2 (F2). Structural analyses by fluorescence measurement, ELISA with monoclonal antibodies and retinol-binding activity indicated that the conjugates had almost maintained the native structure of beta-LG. The antigenicity of the beta-LG-CHS conjugates was similar to that of beta-LG in C3H/He mice. Reduction of the immunogenicity of beta-LG was achieved by conjugation with CHS. In particular, F2 showed very low immunogenicity. B cell epitopes of beta-LG and the conjugates recognized in C3H/He mice were determined with 15-mer multi-pin peptide; the linear epitope profiles of the conjugates were found to be similar to those of beta-LG, while the antibody response to each epitope was dramatically reduced. Conjugation of beta-LG with chitosan was effective for reducing the immunogenicity of beta-LG.     

Improved physical and biochemical features of a collagen membrane by conjugating with soluble egg shell membrane protein

Soluble egg shell membrane protein (SEP) prepared from the egg shell membrane by performic acid oxidation and pepsin digestion was applied to prepare a membrane conjugated with pepsin-solubilized collagen (PSC) by cross-linking with water-soluble carbodiimide. SEP enabled a conjugated membrane to be constructed with a well-developed network structure having thicker collagen fibrils and higher stability than the PSC membrane alone, as indicated by reduced solubility and collagenase-digestion, increased denaturation temperature, and superior flexibility. The SEP-PSC membrane (SEP:PSC of 1:1, w/w) had lower sweatiness than the PSC membrane. Fibroblasts and keratinocytes adhered to the SEP-PSC-coated wells (SEP:PSC of 1:10, w/w) as a fundamental model of an artificial skin similar to that to the PSC-coated wells due to similar expression level of several cell-adhesion proteins. An SEP-PSC coating accelerated the cell growth of fibroblasts, whereas the cell growth of keratinocytes did not change, or decreased slightly due to more rapid differentiation indicated by a more substantial expression of differentiation marker proteins than a PSC-coating without SEP.     

Improved functional and nutritional properties of tomato fruit during cold storage

The use of synthetic antioxidants has been associated with serious concerns for human and environmental health. During ripening stages, tomato fruit is exposed to different abiotic stresses which not only influence its nutritional, mechanical, and functional properties at harvest, but also affect the quality and shelf life of the fruit during storage. This study investigated the pattern of changes in dietary antioxidants during various ripening stages of tomato fruit (cv. Red Rose) and their impact on storage behavior of the fruit during cold storage. Tomato fruits were harvested at mature green, breaker, turning, pink, light-red and red stages of maturity. Then, they were analysed for flesh firmness, soluble solids content, titratable acidity, total sugars, pH, dry matter content, lipophilic (lycopene, β-carotene, and total carotenoids), and hydrophilic (ascorbic acid, phenolic and flavonoids) antioxidants. Additional fruits were harvested at each maturity stage and divided into three equal lots, then were subjected to low-temperature (10 ± 1 °C) storage with 80 ± 5% RH, for 7, 14, and 21 days. Flesh firmness, and the levels of dietary antioxidants were analysed following the subsequent storage periods. The results revealed that the peak of hydrophilic antioxidants such as ascorbic acid, phenolic compounds, and flavonoids was between the ‘pink’ and the ‘light-red’ stages of fruit maturity. Whereas tomatoes harvested at the ‘red’ stage of maturity had the highest levels of lycopene and β-carotene. Both the stage of fruit maturity at harvest and duration of cold storage influenced flesh firmness, organoleptic and functional properties of ‘Red Rose’ tomato fruit. In conclusion, the results of the current investigation have practical implications in formulating foods with improved functional properties at processing industries.     

Rheological properties of guar galactomannan solutions during hydrolysis with galactomannanase and alpha-galactosidase enzyme mixtures

Guar galactomannan, a naturally occurring polysaccharide, is susceptible to hydrolysis by three enzymes: beta-mannosidase, beta-mannanase, and alpha-galactosidase. The beta-mannosidase cleaves a single mannose unit from the nonreducing end of the guar molecule, the beta-mannanase cleaves interior glycosidic bonds between adjacent mannose units, and the alpha-galactosidase cleaves the galactose side branches off the guar. In this study, hydrolysis of guar solutions using hyperthermopilic versions of these enzymes together in different proportions and combinations are examined. The enzymatic reactions are carried out in situ in a rheometer, and the progress of the reaction is monitored through measuring the variation in zero shear viscosity. We find the presence of alpha-galactosidase to affect the action of both beta-mannanase and beta-mannosidase with respect to solution rheology. However, this effect is more pronounced when the alpha-galactosidase and beta-mannanase or beta-mannosidase enzymes were added sequentially rather than simultaneously. This likely is the result of debranching of the guar, which facilitates attack on beta-1,4-linkages by both the beta-mannanase and the beta-mannosidase enzymes and increases hydrolytic rates by the individual enzymes. A rheology-based kinetic model is developed to estimate the reaction rate constants and interpret synergistic effects of multiple enzyme contributions. The model fits the experimental data well and reveals that both the native and the debranched guar have the same activation energy for beta-mannanase action, although debranching considerably increases the frequency of enzyme-guar interactions.     

Degradation of galactomannan by aClostridium butyricum strain

During staphylococcal pneumonia massive destruction of lung tissue is often observed. Staphylococcal serine proteinase (SSP) inactivates -1-proteinase inhibitor (1PI) a major factor which protects lungs from phagocyte proteases. We investigated the effect of SSP on elastin degradation by porcine pancreatic elastase (PE) and crude extract of human neutrophil elastase (NE) in solution and gel containing 1PI. SSP having no elastase activity enhanced PE and NE-induced elastinolysis in solution when added to 1PI before mixing with elastase and then with elastin. SSP added simultaneously with 1PI to PE had no influence on elastin degradation. However, SSP added simultaneously, 30 min before or 30 min after PE significantly increased elastin digestion in elastin-agarose plate with 1PI. Maximal increase in elastinolysis about 3-fold was for SSP added 30 min prior to PE. Since elastin is the major component of the alveolar walls it is possible that lung damage in the course of staphylococcal infection may partly depend on action of SSP.Abbreviations 1PI -1-proteinase inhibitor - NE crude extract of human neutrophil elastase - PE porcine pancreatic elastase - PBS phosphate buffered saline - SSP staphylococcal serine proteinase     

Soft textural and emulsifiable gelatin formed by conjugating with fatty-acylated saccharide

Ossein gelatin (OG) and low-molecular-weight ossein gelatin (LOG) were conjugated with a glucose/fructose stearic acid monoester (GE/FE), which had been prepared from the hydrolysate of a sucrose stearic acid diester, by the Maillard reaction to improve their physical properties. The molar ratio of each conjugate (GE/FE-OG or GE/FE-LOG) was about 1:1, and the conjugation resulted in a decreased isoelectric point. The GE/FE-OG gel showed a lower storage modulus, melting temperature, and enthalpy change than those for the OG gel at both the early and late stages of gelation, and each gel at pH 3.0 showed somewhat lower characteristics than those at pH 7.0. The conjugates acquired superior emulsifying ability, GE/FE-LOG in particular exhibiting markedly higher emulsifying ability in the acidic pH range, in the presence of NaCl, and over a wide temperature range. It is thus expected that conjugation with GE/FE could be effective for providing a new type of gelatin with a soft texture, easy melting, and emulsifying ability.     

Identification of the structural and functional human homolog of the yeast ubiquitin conjugating enzyme UBC9.

Ubiquitin conjugating enzymes (UBCs) are a family of proteins directly involved in ubiquitination of proteins. Ubiquitination is known to be involved in control of a variety of cellular processes, including cell proliferation, through the targeting of key regulatory proteins for degradation. The ubc9 gene of the yeast Saccharomyces cerevisiae (Scubc9) is an essential gene which is required for cell cycle progression and is involved in the degradation of S phase and M phase cyclins. We have identified a human homolog of Scubc9 (termed hubc9) using the two hybrid screen for proteins that interact with the human papillomavirus type 16 E1 replication protein. The hubc9 encoded protein shares a very high degree of amino acid sequence similarity with ScUBC9 and with the homologous hus5+ gene product of Schizosaccharomyces pombe. Genetic complementation experiments in a S.cerevisiae ubc9ts mutant reveal that hUBC9 can substitute for the function of ScUBC9 required for cell cycle progression.     

Water-soluble galactomannan from the seeds of Lotus corniculatus L.: structure and properties

Galactomannan, a water-soluble heteropolysaccharide, was isolated from the seed of Far-Eastern population of ground honeysuckle Lotus corniculatus L. (yield, 1.65%). Analysis of this galactomannan showed that is consists of D-mannose and D-galactose residues (molar ratio, 1.22:1). Its aqueous solutions were characterized by specific rotation [alpha]D = +84.10 and characteristic viscosity [eta] = 559 ml/g. Analysis of this heteropolysaccharide using chemical and enzymatic procedures, as well as IR- and 13C-NMR spectroscopy, showed that its main chain comprises 1,4-beta-D-mannopyranose residues, 95.5% of which are substituted at C-6 with single residues of alpha-D-galactopyranose.     

Characterization of proteinase activation dynamics by capillary electrophoresis conjugating with fluorescent protein-based probe

In this paper, a novel strategy was reported to characterize dynamics of proteinase activation based on capillary electrophoresis (CE), using caspase-2 as the model system. A fusion protein conjugating an amino acid sequence VDVAD with two fluorescent proteins enhanced cyan fluorescence protein (ECFP) and red fluorescence protein (DsRed), ECFP-VDVAD-DsRed, was specially designed and expressed in HeLa cells as the substrate of proteinase caspase-2. In this substrate, the sequence VDVAD could be specifically recognized and cleaved by caspase-2 as soon as its activation was initiated with treatment of a certain dose of cisplatin to HeLa cells, which led to a break of the substrate into two fragments. Analyses of the cell lysates using CE in a time course of the apoptosis illustrated the dynamics of caspase-2 activation. It showed that the employment of fusion fluorescent protein greatly facilitated both CE separation and identification of the analytes. This result from cell colony by CE was compared with that from single cell achieved by optical imaging.     

Structure elucidation and properties of a non-ionic galactomannan derived from the Cassia pleurocarpa seeds

A non-ionic water soluble galactomannan made up of d-galactose and d-mannose was isolated from the seed endosperm of Cassia pleurocarpa. Acid catalyzed fragmentation, periodate oxidation, methylation and selective enzymatic hydrolysis revealed that the repeating unit of the heteropolysaccharide had a backbone of beta(1-->4) linked d-mannopyranosyl units to which D-galactopyranosyl units are linked as side chains through alpha(1-->6) linkages. The properties such as viscosity, water/saline retention, gelling behavior, and shelf life of the galactomannan indicated that the material may be exploited in biomedical applications for example drug delivery and tissue engineering fields.     

Protection from drug-induced hepatocellular changes by pretreatment with conjugating enzyme inhibitors in rats

The present paper describes the role of conjugating enzymes in the development of hepatotoxicity after administration of repeated doses of a novel monoamine oxidase type-A (MAO-A) inhibitor, (5R)-3-[2-(( 1S)-3-cyano-1-hydroxypropyl)benzothiazol-6-yl]-5-methoxymethyl-2-oxazolidinone (E2011). The effects of pretreatment with three kinds of conjugating enzyme inhibitors on hepatic lesions induced by E2011 were evaluated in female Sprague-Dawley rats. The inhibitors used were 2,6-dichloro-4-nitrophenol (DCNP; inhibitor of sulfotransferase (ST)), pentachlorophenol (PCP; inhibitor of both ST and acetyltransferase (AT)) or ranitidine (inhibitor of UDP-glucuronosyltransferase (UDP-GT)). Two weeks treatment of E2011 alone at an oral dosage of 150 mg/kg induced hepatocellular changes characterized by nuclear enlargement. Daily pretreatment with DCNP (10 mg/kg, i.p.) enhanced the E2011-induced hepatocellular changes accompanied by single cell necrosis. On the other hand, the hepatotoxicity was clearly diminished by PCP (5 mg/kg, i.p.). Ranitidine pretreatment had no effect. Protection by PCP was attributed to the inhibitory effects of AT in addition to ST; it was considered that the hepatocellular changes caused by E2011 were largely dependent on the formation of acetyl conjugate(s).     

Depalmitylation with hydroxylamine alters the functional properties of rhodopsin

Rhodopsin, the photosensitive protein found in rod photoreceptors, has two covalently attached palmitates that are thought to anchor a portion of the C terminus to the disc membrane, forming a fourth cytoplasmic loop. Using hydroxylamine (NH2OH) to cleave the thioester linkage, we have characterized the effect of depalmitylation on certain functional properties of rhodopsin. Treatment of rod outer segment membranes (prepared from rat retinas previously labeled in vivo with [3H]palmitate) with 1 M NH2OH typically removed greater than or equal to 75% of the [3H]palmitate initially bound to rhodopsin. Spectrophotometry of rod outer segment membranes that had been treated with 1 M NH2OH indicated preservation of 85% of the native rhodopsin and no effect on the shape of the absorbance spectrum of rhodopsin. In vivo labeled rhodopsin that had been treated with 1 M NH2OH did not reincorporate free endogenous [3H] palmitate over a 2-h incubation period. Both NH2OH-treated and untreated rhodopsin incorporated [14C]palmitate from exogenously added [14C]palmitoyl-CoA. This incorporation was substantially greater in the NH2OH-treated sample. The removal of palmitate by NH2OH inhibited rhodopsin regeneration by 44% and increased the ability of rhodopsin to activate transducin's light-dependent GTPase activity by 61%. However, the removal of palmitate from rhodopsin did not affect the light-dependent binding of transducin (T alpha and T beta gamma).     

Altered functional properties of tritordeum by transformation with HMW glutenin subunit genes

The high-molecular-weight (HMW) subunits of wheat glutenin are the major determinants of the gluten visco-elasticity that allows wheat doughs to be used to make bread, pasta and other food products. In order to increase the proportions of the HMW subunits, and hence improve breadmaking performance, particle bombardment was used to transform tritordeum, a fertile amphiploid between wild barley and pasta wheat, with genes encoding two HMW glutenin subunits (1Ax1 and 1Dx5). Of the 13 independent transgenic lines recovered (a transformation frequency of 1.4%) six express the novel HMW subunits at levels similar to, or higher than, those of the endogenous subunits encoded on chromosome 1B. Small-scale mixograph analysis of T₂ seeds from a line expressing the transgene for 1Dx5 indicated that the addition of novel HMW subunits can result in significant improvements in dough strength and stability, thus demonstrating that transformation can be used to modify the functional properties of tritordeum for improved breadmaking. Received: 15 January 1999 / Accepted: 5 February 1999     

Role of galactomannan composition on the binary gel formation with xanthan

The influence of the galactomannan characteristic ratios (M/G) on the temperature of gelation (Tg) and the gel strength of mixtures of galactomannan with xanthan is reported. Two galactomannans were investigated: one highly substituted from the seeds of Mimosa scabrella (M/G = 11), and the other, less substituted, from the endosperm of Schizolobium parahybae, with (M/G = 30) [Ganter JLMS, Zawadzki-Baggio SF, Leitner SC, Sierakowski MR, Reicher F. J Carbohydr Chem 1993;12:753]. The xanthan:galactomannan systems (4:2 g l(-1), in 5 mM NaCl) showed a temperature of gel formation (Tg) of 24 degrees C for that of S. parahybae [Bresolin TMB, Milas M, Rinaudo M and Ganter JLMS. Int J Biol Macromol 1998;23:263] and 20 degrees C for the galactomannan of M. scabrella, determined by viscoelastic measurements and microcalorimetry. A Tg of 40-50 degrees C was found by Shatwell et al. [Shatwell KP, Sutherland IW, Ross-Murphy SB, Dea ICM. Carbohydr Polym 1991;14:29] for locust bean gum-LBG (M/G = 43). Lundin and Hermansson [Lundin L, Hermansson AM. Carbohydr Polym 1995;26:129] reported a difference of 13 degrees C for Tg of two LBG samples with M/G = 3 (40 degrees C) and 5 (53 degrees C), in mixtures with xanthan. It appears that the more substituted galactomannans have lower temperatures of gelation in the presence of xanthan. The mechanism of gelation depends also on the M/G ratio. For the lower values it involves only disordered xanthan chains in contrast to M/G ratios higher than 3. In addition, the presence of the galactomannan from M. scabrella increased slightly the temperature of the conformational change (Tm) of xanthan probably due to the ionic strength contribution of proteins (3.9%) present in the galactomannan. On the other hand, the galactomannans from S. parahybae, with 1.5% of proteins and M. scabrella, with 2.4% of protein, did not show this effect, the Tm of xanthan alone or in a mixture being practically unchanged.     

Cyclization of polyubiquitin by the E2-25K ubiquitin conjugating enzyme

For most substrates of ubiquitin (Ub)-dependent degradation, recognition by the proteasome is mediated by a covalently attached signal assembled from multiple ubiquitins linked to each other via the C terminus of one Ub and the epsilon-amine of Lys(48) of another Ub. Among Ub-conjugating enzymes, E2-25K is unique in its ability to synthesize in vitro unanchored Lys(48)-linked poly-Ub chains from mono- or poly-Ub, E1, and ATP; thus, E2-25K has distinct binding sites for donor and acceptor (poly)Ub. During studies of chain assembly by E2-25K, we observed that Lys(48)-linked tri-Ub was efficiently converted to a new species that upon SDS-polyacrylamide gel electrophoresis migrated between linear di-Ub and tri-Ub. Analysis of this product by mass spectrometry and tryptic digestion showed that it was a cyclic form of tri-Ub. Cyclization of tri-Ub requires E1, E2-25K, ATP, and that the linear substrate has a free Gly(76) C terminus on the proximal end Ub and a Lys(48) side chain available on the distal end Ub. E2-25K similarly can catalyze the cyclization of longer poly-Ub chains, including tetra- and penta-Ub. Although cyclic tri-Ub resists hydrolysis by the PA700 or isopeptidase T deubiquitinating enzymes, it can be disassembled to Ub monomers by isopeptidase(s) in a red blood cell extract. Thus, if cyclic poly-Ub forms in vivo, it will not accumulate as a dead-end product.     

Improved production of two expansin-like proteins in Pichia pastoris and investigation of their functional properties

The genes encoding expanin-like proteins from Trichoderma reesei (TrSwo1) and Bacillus subtilis (BsEXLX1) were successfully expressed in Pichia pastoris. The yields of two recombinant proteins were significantly improved by the use of PMSF (phenylmethylsulfonyl fluoride) and a commercial protease inhibitor cocktail. Under the optimum culture conditions, the highest TrSwo1/BsEXLX1 expression level reached was approximately 120/860 mg l⁻¹, which was almost 2.4/86-fold as much as the highest expression level in other host cells. Purified BsEXLX1/TrSwo1 displayed synergism in cellulose hydrolysis with endoglucanase, and the maximum amount of reducing sugars released was almost 2.0/2.5-fold as high as those in reaction mixtures without expansin-like proteins. The synergistic effect reached the maximum level when 1 mg of target protein per g of filter paper was loaded. Both proteins exhibited relatively high thermal stability at temperatures of 50, 70 and 90 °C, and retained more than 45% residual activities after 1 h of pre-incubation at 100 °C, suggesting remarkable heat tolerance. They also showed resistance to denaturation by urea and SDS. Under several enzymatic hydrolysis conditions, the synergistic activity of TrSwo1 was higher than that of BsEXLX1, indicating stronger disrupting activity of TrSwo1 on cellulose than BsEXLX1. This is the first study to report high-efficient expression and unreported properties of BsEXLX1/TrSwo1.     

Rational design of a thrombin electrochemical aptasensor by conjugating two DNA aptamers with G-quadruplex halves

A novel strategy for the fabrication of an electrochemical aptasensor is proposed; this strategy has been employed in this work to assay thrombin concentration. Two well-designed oligonucleotides were used as the core element. G-quadruplex–hemin complexes can be formed on the surface of the electrode to give a detectable signal only when thrombin is not bound to the aptamers. The detection limit of the biosensor has been lowered to 10 nM. Moreover, since the electroactive probe is not required to be bound to the oligonucleotide, this strategy may integrate the advantages of being both label-free and cost-effective.     

Depolymerization of legume seed galactomannan by Celloviridin G20x

The depolymerization of legume galactomannans by the commercial preparation Celloviridin G20x was studied with the aim of obtaining macromolecular fragments of a constant composition. Four galactomannans, representative of the entire range of monomer ratios characteristic of this class of phytopolysaccharides, were hydrolyzed. The galactomannans were isolated from oriental goat’s rue (Galega orientalis Lam., Man: Gal 1.1), guar (Cyamopsis tetragonoloba L., Man: Gal 1.6), honeylocust (Gleditsia triacanthos f. inermis L., Man: Gal 2.4), and sophora (Styphnolobium japonicum (L.) Schott., Man: Gal 5.1). Fragments with a monomer ratio close to that in the original polysaccharides were obtained with a high yield (75–80%). The degree of substitution of the 1,4-β-D-mannopyranose chain at position C-6 with α-galactose residues influenced the molecular weight of the final reaction product.