Effect of chitosan degradation on its interaction with β-lactoglobulin

Complexes between chitosan and β-lactoglobulin (β-Lg) were investigated, and their formation was found to depend on pH and ionic strength. The electrostatic attraction between the cationic polysaccharide and the negatively charged protein above its isoelectric point has been identified as the main driving force in the molecular recognition process. At low protein concentration, soluble complexes were shown to be formed, and their structural features were characterized by circular dichroism (CD) and steady-state fluorescence. Both the overall secondary structure of the protein and the local environment probed by its tryptophan residues are not affected by the presence of chitosan in the complex. Furthermore, the formation of the complex does not lead to a net stabilization of the native state of the protein over its denatured state due to formation of a similarly stable complex between the polyelectrolyte and the denatured state of the protein. The formation of coacervates between β-Lg and chitosan was also characterized as a function of average molecular weight of chitosan (subjected to ultrasonication for different periods of time: 0, 5, 15, and 30 min) by means of both turbidimetric and calorimetric techniques. The combination of turbidimetric as well as isothermal calorimetric titrations have allowed the deconvolution of two processes usually coupled in the formation of protein-polyelectrolyte coacervates: the formation of complex coacervates as the protein sites become saturated by polyelectrolyte molecules and the redissolution of the coacervates as the polyelectrolyte-to-protein ratio increases.     

Effects of estradiol-17β and hCG supplementation on superovulatory responses and embryo quality in swamp buffalo (Bubalus bubalis) implanted with norgestomet

Twenty-four cycling swamp buffaloes with normal reproductive histories and 2–3 months postpartum were used to investigate the effect of addition of estradiol-17β and human chorionic gonadotrophin (hCG) to the superovulation regime on the level of ovarian stimulation and embryo production.The estrous cycles of buffaloes were synchronized by prostaglandin injection and then divided into two groups for superovulatory treatment. Those in Group 1 (n = 12) received a implant containing 3 mg norgestomet (Syncro-Mate-B) for 9 days (insertion day is Day 0), with 4000 IU of equine chorionic gonadotrophin (eCG) and 500 μg cloprostenol i.m. given at Day 7. Group 2 (n = 12) received the same regime as Group 1, together with 7.5 mg estradiol-17β given in three intramuscular injections on Days 3, 5 and 7 in decreasing doses (4.0, 2.5 and 1.0 mg, respectively) and 5000 I.U hCG i.v. coincidentally with the first insemination. Estrus was monitored visually and by placing treated animals with bulls. Each animal was inseminated twice with frozen sperm after standing estrus. The numbers of corpora lutea (CL) and follicles greater than 8 mm in diameter were recorded via palpation per rectum at 6 days after implant removal. Two days later 11 animals from Group 2 and two from Group 1 were slaughtered for direct observation of ovarian responses and for embryo collection.The mean number of CL were 0.91 ± 0.66 and 9.08 ± 5.0 for Groups 1 and 2, respectively. The average recovery rate based on CL counts at slaughter was 60% in Group 2. No embryos were recovered from the two animals in Group 1. Seventy-nine percent of the collected ova were fertilized and more than 60% of them had developed into hatched blastocysts. The percentages of buffalo with excellent and good estrus were 41.6 and 91.6% for Groups 1 and 2, respectively.These results showed that the supplementation of estradiol-17β and the hCG treatment significantly improved the level of ovarian stimulation in swamp buffalo.     

α-Tocopherol and l-ascorbic acid increase the in vitro development of IVM/IVF swamp buffalo ( Bubalus bubalis) embryos

This study was conducted to investigate the effects of capacitating agents added at in vitro fertilization (IVF) and antioxidants supplemented during in vitro culture (IVC) on the development of buffalo embryos. In experiment I, in vitro embryo development of buffalo embryos was compared when the IVF medium was supplemented with heparin, caffeine and calcium ionophore A23187 either alone or in combination. There was no significant difference (P > 0.05) in the cleavage rates of oocytes among the treatment groups but the development rate to the blastocyst stage and the cell numbers of blastocyst in the heparin-treated group were significantly higher (P < 0.05) than that of other treatments. In experiment II, in vitro embryo development of buffalo embryos was compared when IVC medium was supplemented with either α-tocopherol (250 and 500 μM) or l-ascorbic acid (250 and 500 μM). The rate of development to the blastocyst stage of embryos cultured in medium supplemented with 250 μM α-tocopherol (33%, 41/123) and 250 μM l-ascorbic acid (31%, 38/123) was significantly higher (P < 0.05) than that of those cultured in medium alone (19%, 20/108) but not significantly different (P < 0.05) from medium supplemented with either 500 μM α-tocopherol (24%, 30/123) or 500 μM l-ascorbic acid (25%, 33/133). These results suggest that buffalo spermatozoa treated with heparin were suitable for IVF and that α-tocopherol and l-ascorbic acid added during IVC increased the rate of buffalo embryo development.     

Effect of different seasons on concentration of plasma luteinizing hormone and seminal quality vis-à-vis freezability of buffalo bulls (Bubalus bubalis)

Seasonal variations in semen quality, freezability and plasma luteinizing hormone (LH) levels were studied between summer and spring. Semen volume, density and initial sperm motility did not differ significantly between different seasons. Plasma LH decreased between summer and spring but the differences were, however, not significant. Pre-freezing motility did not differ significantly but post-freezing motility varied significantly (P<0.01) between seasons. Post-freezing motility was lowest during summer and highest during winter. It can be concluded that summer spermatozoa may be fragile and cannot withstand freezing stress. To increase reproductive efficiency in buffalo during summer, semen should be frozen during winter and spring and used during hot weather conditions. Seasonal variations in plasma LH levels were insignificant.     

Chemico-biological interaction of Etravirine and its β-Cyclodextrin complex with macromolecular targets

The interaction of etravirine with β-cyclodextrin is analyzed by UV–visible absorption, infrared, fluorescence, nuclear magnetic resonance, two-dimensional rotational frame nuclear Overhauser effect spectroscopy, and molecular modeling studies. The 4-hydroxy-3, 5-dimethylbenzonitrile moiety is found to take part in the binding. The stoichiometry of the inclusion complex of ET with β-CD is 1:1 with the binding constant of 2.03 × 10³ mol^?1 dm³. The binding of ET with calf thymus DNA (ctDNA) and bovine serum albumin (BSA) protein is investigated in the presence and the absence of β-CD. Fluorescence enhancement is observed during the binding of ET with ctDNA in the absence of β-CD, whereas in the presence of β-CD, fluorescence quenching is observed. The binding constants of the binding of ET and ET–β-CD to ctDNA are 7.84 × 10⁴ and 4.38 × 10⁴ mol^?1 dm³, respectively. The binding constant of the binding of ET and ET–β-CD to BSA are 3.14 × 10⁴ and 1.6396 × 10⁴ mol^?1 dm³, respectively. The apparent binding constants between ET–β-CD complex and ctDNA or BSA protein decreases significantly. The numbers of binding sites of interaction of ET with BSA protein and the binding distance between BSA protein and ET the absence and the presence of β-CD differ. β-CD modulates the binding of ET with the macromolecular targets.     

Interactions of β-lactoglobulin with serotonin and arachidonyl serotonin

β‐Lactoglobulin (β‐LG) is a lipocalin, which is the major whey protein of cow's milk and the milk of other mammals. However, it is absent from human milk. The biological function of β‐LG is not clear, but its potential role in carrying fatty acids through the digestive tract has been suggested. β‐LG has been found in complexes with lipids such as butyric and oleic acids and has a high affinity for a wide variety of compounds. Serotonin (5‐hydroxytryptamine, 5‐HT), an important compound found in animals and plants, has various functions, including the regulation of mood, appetite, sleep, muscle contraction, and some cognitive functions such as memory and learning. In this study, the interaction of serotonin and one of its derivatives, arachidonyl serotonin (AA‐5HT), with β‐LG was investigated using circular dichroism (CD) and fluorescence intensity measurements. These two ligands interact with β‐LG forming equimolar complexes. The binding constant for the serotonin/β‐LG interaction is between 10⁵ and 10⁶ M⁻¹, whereas for the AA‐5HT/β‐LG complex it is between 10⁴ and 10⁵ M⁻¹ as determined by measurements of either protein or ligand fluorescence. The observed binding affinities were higher in hydroethanolic media (25% EtOH). The interactions between serotonin/β‐LG and AA‐5HT/β‐LG may compete with self‐association (micellization) of both the ligand and the protein. According to far‐ and near‐UV CD results, these ligands have no apparent influence on β‐LG secondary structure, however they partially destabilize its tertiary structure. Their binding by β‐LG may be one of the peripheral mechanisms of the regulation of the content of serotonin and its derivatives in the bowel of milk‐fed animals. © 2011 Wiley Periodicals, Inc. Biopolymers 95: 871–880, 2011.     

Biophysical characterization of genistein–membrane interaction and its correlation with biological effect on cells — The case of EYPC liposomes and human erythrocyte membranes

With application of EPR and ¹H NMR techniques genistein interaction with liposomes formed with egg yolk lecithin and with erythrocyte membranes was assessed. The present study addressed the problem of genistein localization and its effects on lipid membrane fluidity and protein conformation. The range of microscopic techniques was employed to study genistein effects on HeLa cells and human erythrocytes. Moreover, DPPH bioassay, superoxide anion radical test and enzymatic measurements were performed in HeLa cells subjected to genistein. The gathered results from both EPR and NMR techniques indicated strong ordering effect of genistein on the motional freedom of lipids in the head group region and the adjacent hydrophobic zone in liposomal as well as in red blood cell membranes. EPR study of human ghost showed also the changes in the erythrocyte membrane protein conformation. The membrane effects of genistein were correlated with the changes in internal membranes arrangement of HeLa cells as it was noticed using transmission electron microscopic and fluorescent techniques. Scanning electron and light microscopy methods showed that one of the aftermaths of genistein incorporation into membranes was creation of echinocytic form of the red blood cells with reduced diameter. Genistein improved redox status of HeLa cells treated with H₂O₂ by lowering radicals' level.     

Development of a sequential multicolor-FISH approach with 13 chromosome-specific painting probes for the rapid identification of river buffalo (Bubalus bubalis, 2n = 50) chromosomes

The development of new molecular techniques (array CGH, M-FISH, SKY-FISH, etc.) has led to great advancements in the entire field of molecular cytogenetics. However, the application of these methods is still very limited in farm animals. In the present study, we report, for the first time, the production of 13 river buffalo (Bubalus bubalis, 2n?=?50) chromosome-specific painting probes, generated via chromosome microdissection and the DOP-PCR procedure. A sequential multicolor-FISH approach is also proposed on the same slide for the rapid identification of river buffalo chromosome/arms, namely, 1p-1q, 2p-2q, 3p-3q, 4p-4q, 5p-5q, 18, X, and Y, using both conventional and late-replicating banded chromosome preparations counterstained by DAPI. The provided ‘bank’ of chromosome-specific painting probes is useful for any further cytogenetic investigation not only for the buffalo breeds, but also for other species of the family Bovidae, such as cattle, sheep, and goats, for chromosome abnormality diagnosis, and, more generally, for evolutionary studies.     

In-silico modeling of a novel OXA-51 from β-lactam-resistant Acinetobacter baumannii and its interaction with various antibiotics

Acinetobacter baumannii, one of the major Gram negative bacteria, causes nosocomial infections such as pneumonia, urinary tract infection, meningitis, etc. β-lactam-based antibiotics like penicillin are used conventionally to treat infections of A. baumannii; however, they are becoming progressively less effective as the bacterium produces diverse types of β-lactamases to inactivate the antibiotics. We have recently identified a novel β-lactamase, OXA-51 from clinical strains of A. baumannii from our hospital. In the present study, we generated the structure of OXA-51 using MODELLER9v7 and studied the interaction of OXA-51 with a number of β-lactams (penicillin, oxacillin, ceftazidime, aztreonam and imipenem) using two independent programs: GLIDE and GOLD. Based on the results of different binding parameters and number of hydrogen bonds, interaction of OXA-51 was found to be maximum with ceftazidime and lowest with imipenem. Further, molecular dynamics simulation results also support this fact. The lowest binding affinity of imipenem to OXA-51 indicates clearly that it is not efficiently cleaved by OXA-51, thus explaining its high potency against resistant A. baumannii. This finding is supported by experimental results from minimum inhibitory concentration analysis and transmission electron microscopy. It can be concluded that carbapenems (imipenem) are presently effective β-lactam antibiotics against resistant strains of A. baumannii harbouring OXA-51. The results presented here could be useful in designing more effective derivatives of carbapenem.     

The coordination of copper(II) with β-casomorphin and its fragments

The synthesis of β-casomorphin-5 (Tyr-Pro-Phe-Pro-Gly, H₂L) and a number of its peptide fragments is described. Complexes formed between these peptides and Cu(II) have been investigated spectrophotometrically, using CD and EPR spectroscopy, and potentiometrically. Results show that, with tyrosine as the N-terminal residue, the major complex formed at physiological pH is the dimeric species, [Cu₂L₂], bonded through the phenolic O^? of the Tyr residue of one ligand and the N-terminal amine nitrogen of the second ligand molecule. There is no evidence for coordination through the peptide nitrogens unless the terminal Tyr group is removed.     

Purification and Characterization of β-d-Galactosidase and α-d-Mannosidase from Papaya (Carica papaya) Seeds

β-D-Galactosidase was purified 115-fold from a saline extract of papaya seeds by fractionation with ammonium sulfate, DEAE-Sephadex chromatography and gel-filtration on Sephadex G-75, G-150, and G-100. The purified β-D-galactosidase (MW, 56,000 daltons) had an isoelectric point (pI) at pH 8.4 and the optimal pH for its activity was 3.5 to 4.5. The enzyme activity was inhibited by Cu²⁺,Ag⁺,Hg²⁺,Pb²⁺,NaAsO₂ and р-chloromercuribenzoate at concentrations of 1x10^-3 M. Among the various mono- and oligosaccharides tested, D-galactose, D-galacturonic acid, D-galactono-γ-lactone and melibiose significantly inhibited the enzyme activities at concentrations of 2xl0^-3 to 1X10^-2M. The purified enzyme hydrolyzed β-nitrophenyl β-D-galactoside (Km = 1.0X10^-3M), methyl β-D-galactoside (Km=1.6x10^-2M), aminoethyl β-D-galactoside (Km =3.3X10^-2M) and lactose (Km = 9.1X10^-2M). β-(l→3)-Linked galactotetraosyl-eryth itol and asialo-glycopeptide isolated from fetuin were also hydrolyzed to the extent of 78 and 75%, 4respectively, on the basis of their galactose contents.

∝-D-Mannosidase from papaya seeds was also purified 130-fold by ammonium sulfate fractionation, DEAE-Sephadex chromatography, gel-filtration on Sephadex G-150 and hydroxylapatite chromatography. The purified enzyme (MW, 156,000 daltons), consisting of two subunits (78,000x2), was inhibited by Hg²⁺,Ag⁺,Cu²⁺, р-chloromercuribenzoate, D-glucose, D-glucosamine and D-mannose at concentrations of lx10^-3 to 1x10^-2M. The ∝-D-mannosidase hydrolyzed р-nitrophenyl ∝-D-mannoside (Km=5.6x10^-3M), methyl ∝-D-mannoside (Km=2.8X10^-2M), ∝-D-mannosyl-D-mannitol (Km=2.2X10^-2M), ∝-(l→2)linked D-mannobiosyl-D-mannitol (Km=6.3x10^-3M) and D-mannotriosyl-D-mannitol (Km=5.3x10^-3 M).     

Enzymatic modification of β-lactoglobulin with N-fatty-acyl-dipeptide by transglutaminase from Streptomyces mobaraense

-Lactoglobulin was enzymatically acylated with N-lauroyl-l-glutaminyl-glycine and N-lauroyl-l-glutamyl-l-lysine using transglutaminase from Streptomyces mobaraense. The modification of the protein with N-fatty-acyl-dipeptide was confirmed by SDS-PAGE, gel chromatography, HPLC, amino acid analysis, and TOF-MS. The degrees of the protein modification with N-lauroyl-l-glutaminyl-glycine and N-lauroyl-l-glutamyl-l-lysine were estimated to be 2–4 and 1.5 residues per molecule, respectively.     

Characterization of PSII–LHCII supercomplexes isolated from pea thylakoid membrane by one-step treatment with α- and β-dodecyl-d-maltoside

It was the work of Jan Anderson, together with Keith Boardman, that showed it was possible to physically separate photosystem I (PSI) from photosystem II (PSII), and it was Jan Anderson who realized the importance of this work in terms of the fluid-mosaic model as applied to the thylakoid membrane. Since then, there has been a steady progress in the development of biochemical procedures to isolate PSII and PSI both for physical and structural studies. Dodecylmaltoside (DM) has emerged as an effective mild detergent for this purpose. DM is a glucoside-based surfactant with a bulky hydrophilic head group composed of two sugar rings and a non-charged alkyl glycoside chain. Two isomers of this molecule exist, differing only in the configuration of the alkyl chain around the anomeric centre of the carbohydrate head group, axial in α-DM and equatorial in β-DM. We have compared the use of α-DM and β-DM for the isolation of supramolecular complexes of PSII by a single-step solubilization of stacked thylakoid membranes isolated from peas. As a result, we have optimized conditions to obtain homogeneous preparations of the C₂S₂M₂ and C₂S₂ supercomplexes following the nomenclature of Dekker & Boekema (2005 Biochim. Biophys. Acta 1706, 12–39). These PSII–LHCII supercomplexes were subjected to biochemical and structural analyses.     

Purification and Characterization of Extracellular β-Xylosidase and β-Glucosidase from Aspergillus fumigatus

A β-xylosidase (β-d-xyloside xylohydrolase, EC 3.2.1.37) and β-glucosidase (β-d-glucoside glucohydrolase, EC 3.2.1.21) extracted from a wheat bran culture of Aspergillus fumigatus were purified up to 90-fold and 131-fold, respectively, by ammonium sulfate precipitation, gel filtration, ion exchange chromatography, and hydroxylapatite chromatography. Molecular weights of the β-xylosidase and β-glucosidase were 360,000 and 380,000, respectively, each consisting of four identical subunits. The isoelectric points of β-xylosidase and β-glucosidase were at pH 5.4 and 4.5, respectively. The optimum temperature for the β-xylosidase was 75°C, being stable up to 65°C for 20 min and for the β-glucosidase was 65°C, being stable up to 60°C for 20 min. The optimum pH for both enzymes was about 4.5, being stable between 2 and 8 at 50°C for 20 min. Both enzymes were inhibited by Fe³⁺, Cu²⁺, Hg²⁺, SDS, and p-chloromercuribenzoate. The apparent Michaelis constants of the β-xylosidase were 2.0 and 23.8 mM for p-nitrophenyl-β-xyloside and xylobiose, respectively, and those of the β-glucosidase were 1.4, 11.4, and 24.8 mM for p-nitrophenyl-β-glucoside, gentiobiose, and cellobiose, respectively. To produce xylose from crude xylooligosac-charides prepared by steam-explosion of cotton seed waste (DP ≤10, 53%, total sugars = 150 g/ liter), the crude enzyme from A. fumigatus (β-xylosidase activity = 14.7 units/ml, xylanase activity = 20 units/ml) could hydrolyze the substrate at 55°C and pH 4.5 resulting in almost complete conversion to xylose (160 g/liter).     

Characterization of glycoprotein E C-End of West Nile virus and evaluation of its interaction force with αVβ3 integrin as putative cellular receptor

Recombinant polypeptide containing the 260–466 amino acid sequence of West Nile virus (WNV) strain LEIV-Vlg99-27889-human glycoprotein E (gpE, E_260–466) was constructed. Immunochemical similarity between the E_260–466 and gpE of WNV was proven by enzyme immunoassay (EIA), immunoblot, competitive EIA, hemagglutination inhibition, and neutralization tests using polyclonal and monoclonal antibodies against the viral gpE and recombinant E_260–466. Polypeptide E_260–466 induced formation of virus neutralizing and cross-reactive antibodies that were interactive with various epitopes of this recombinant protein. It is shown by evaluation of the interaction of E_260–466 with one of the proposed cell receptors of WNV that average E_260–466-αVβ3 integrin-specific interaction force measured using atomic force spectroscopy was 80 and 140 pN for single and double interactions, correspondingly. Taken together with previously described interaction between laminin-binding protein (LBP) and WNV gpE domain II, it is proposed that WNV gpE can interact specifically with two cellular proteins (LBP and αVβ3 integrin) during virus entry.     

Characterization and optimization of β-galactosidase immobilization process on a mixed-matrix membrane

β-Galactosidase is an important enzyme catalyzing not only the hydrolysis of lactose to the monosaccharides glucose and galactose but also the transgalactosylation reaction to produce galacto-oligosaccharides (GOS). In this study, β-galactosidase was immobilized by adsorption on a mixed-matrix membrane containing zirconium dioxide. The maximum β-galactosidase adsorbed on these membranes was 1.6 g/m², however, maximal activity was achieved at an enzyme concentration of around 0.5 g/m². The tests conducted to investigate the optimal immobilization parameters suggested that higher immobilization can be achieved under extreme parameters (pH and temperature) but the activity was not retained at such extreme operational parameters. The investigations on immobilized enzymes indicated that no real shift occurred in its optimal temperature after immobilization though the activity in case of immobilized enzyme was better retained at lower temperature (5 °C). A shift of 0.5 unit was observed in optimal pH after immobilization (pH 6.5 to 7). Perhaps the most striking results are the kinetic parameters of the immobilized enzyme; while the Michaelis constant (K_m) value increased almost eight times compared to the free enzyme, the maximum enzyme velocity (V_max) remained almost constant.     

Characterization of a β-xylosidase from Aspergillus terreus (IJIRA 6.2)

Aspergillus terreus (IJIRA 6.2), a common soil microorganism, produces an extracellular β-xylosidase during its growth on wheat bran. The enzyme has been purified 328 fold (with a sp act of 4233 units/mg protein) by chromatography on DEAE-Sephadex A-25, hydroxyapatite, ConA-Sepharose and gel filtration on Sephacryl-S-300. Molecular mass of β-xylosidase by gel filtration was estimated to be about 95,000 and sedimentation coefficient of 5.6S was determined by glycerol density gradient centrifugation. The enzyme displayed maximum activity at pH 5.0 and 40°C; and in the absence of substrate, the β-xylosidase was stable up to 50°C and between pH 4.5 to 6.5. The purified enzyme hydrolysed p-nitrophenyl-β-Dxylopyranoside (PNPX) and xylooligosaccharides but not xylan, carboxymethyl cellulose or cellobiose. With PNPX as the substrate, the purified β-xylosidase exhibited a Km of 1.0 mM and D(+) xylose served as a competitive inhibitor with a K₁ of 10.5 mM.