OBJECTIVETo establish an ELISA approach to study the interaction of polysaccharides with cytokine in vitro.

METHODSThe heparin BSA complexes (HBC) were synthesized with a chemistry method and separated using a 1 X 90 cm column of Separose 4B. After identification of the complex via SDS-PAGE,the wells of ELISA plates were coated with HBC and the interaction of HBC with interferon-gamma (IFN-gamma) was detected. The effects of heparin, low molecular weight heparin (LMW heparin), chondroitin sulfate (CS), hyaluronic acid (HA) and carrageenans on the binding of HBC to IFN-gamma were tested in this system.

RESULTHuman recombinant IFN-gamma bound to heparin in a concentration dependent manner, the binding of IFN-gamma to HBC was detected at the concentration of 0.25 ng, and saturated at around 2 ng. Free heparin, LMW heparin, CS,HA and carrageenans competed for the binding of IFN-gamma to HBC with significant different ability. The IC(50)concentrations of heparin and LMW heparin were 2.40 microg/ml and 18.60 microg/ml respectively.

CONCLUSIONIFN-gamma is a cytokine with high binding affinity to heparin and carrageenans family but poor to CS-A and CS-C. ELISA is a simple, sensitive approach to detect the interaction of polysaccharides with cytokine in vitro.

Enzyme-Linked Immunosorbent Assay ; methods ; Heparin ; metabolism ; Interferon-gamma ; metabolism ; Polysaccharides ; metabolism ; Serum Albumin, Bovine ; metabolism

OBJECTIVETo establish a direct and fast method separating calcium levofolinate and calcium dextrofolinate in a bovine serum albumin stationary phase chiral column.

METHODSUsing EC150/4 RESOLVOSIL BSA-7(150 mm x 4 mm) chiral separation column, with 0.20 mol/L, pH=5.0 phosphate buffer as mobile phase HPLC method was performed to separate calcium folinate enantiomers.

RESULTThe capacity factor and resolution of the two calcium folinate enantiomers were greatly affected by mobile phase buffer concentration,pH and the column temperature. And the retention time of calcium levofolinate and calcium dextrofolinate were 18.5 min and 22.6 min, respectively. The resolution, R(s)=1.49.

CONCLUSIONCalcium folinate enantiomers are separated successfully using this method.

Chromatography, High Pressure Liquid ; Hydrogen-Ion Concentration ; Leucovorin ; chemistry ; metabolism ; Serum Albumin, Bovine ; metabolism ; Stereoisomerism

AIMTo study the binding of sibutramine hydrochloride (SH) and bovine serum albumin (BSA) in physiological condition by spectroscopic method.

METHODSThe quenching mechanism of the fluorescence of bovine serum albumin by sibutramine hydrochloride was studied with the fluorescence and the absorption spectroscopy. The binding constants K and the number of binding sites were determined at different temperatures according to Scatchard equation and the main binding force was discussed by thermodynamic equations. The effect of the drug on bovine serum albumin conformation was also studied by using synchronous fluorescence spectroscopy.

RESULTSThe quenching mechanism of sibutramine hydrochloride to bovine serum albumin was static quenching. The binding constants K at 8 degrees C, 25 degrees C, 37 degrees C were 1.21 x 10(5), 8.31 x 10(4), 6.97 x 10(4) L x mol(-1) with one binding site, respectively. The thermodynamic parameters of the reaction were deltaH = -9.70 kJ x mol(-1), deltaS = 56.41 J x mol(-1) x K(-1).

CONCLUSIONThe binding force is electrostatic interaction. Sibutramine hydrochloride can be deposited and transported by serum protein in vivo. Sibutramine hydrochloride has nearly no effect on the serum protein conformation.

Animals ; Binding Sites ; Cattle ; Cyclobutanes ; metabolism ; Protein Binding ; Serum Albumin, Bovine ; metabolism ; Spectrometry, Fluorescence ; methods ; Spectrophotometry, Ultraviolet ; methods

Attenuated total reflection (ATR) FT-IR spectroscopy was used to quantitatively characterize the extent of bovine serum albumin (BSA) adsorbed on the surface-coating-modified poly(ether urethane) (PEU) matrix. The two surface modifying additives (SMA) were respectively a tri-block-coupling-polymer of stearyl poly (ethylene oxide)-4,4'-methylene diphenyl diisocyanate-stearyl poly(ethylene oxide), for short MSPEO, and another similar block-coupling polymer with the Cibacron Blue F3G-A endgroups, for short cibaMPEO. The experiments of static BSA adsorption were composed by two parts. One was static isothermal adsorption, and the other was static adsorption kinetics. The quantitative characterization was based on the optical principles of FT-IR, method of experiment and index of the apparatus, by which the enhancement of BSA adsorption on the SMA-modified surfaces was confirmed.
Adsorption ; Animals ; Biocompatible Materials ; Cattle ; Polyethylene Glycols ; Polyurethanes ; Serum Albumin, Bovine ; metabolism ; Spectroscopy, Fourier Transform Infrared ; Stearates

OBJECTIVETo investigate the different velocity of polymorphonuclear neutrophil (PMN) infiltration, erythrocyte diapedesis and plasma exudation into the pulmonary tissue of the rats inflicted with smoke inhalation injury, so as to explore the different mechanisms of their existence in rat pulmonary tissue after inhalation injury.

METHODSThe rat smoke inhalation injury model was employed in the study. Wistar rats were inflicted with smoke inhalation injury, and then sacrificed at 1, 3, 6, 12 and 24 post injury hours (PIH). 131I-BSA, 99mTc-PMN or 99mTc-erythrocytes (RBC) were injected into rat pulmonary tissue 1 hour before sacrifice. Isotonic saline was infused into blood vessel to wash out circulation blood. Then the pulmonary tissue samples were harvested for gamma-value counting and then weighed. The infiltration of 131I-BSA, 99mTc-PMN or 99mTc-RBC in pulmonary tissue per gram and per minute was calculated, and MPO content was measured by phosphate T-tolidine method.

RESULTSThe amount of RBC diapedesis in rat lung tissue peaked at 1 PIH, decreased thereafter and approached to normal level at 24 PIH. The amount of PMN infiltration increased at 3 PIH, slightly decreased at 6 PIH but still higher than that in normal tissue, and increased again at 24 PIH. The pulmonary tissue content of MPO gradually increased from 1 PIH to 24 PIH. The pulmonary tissue content of 131I-BSA began to increase at 1 PIH and peaked at 6 PIH, and remained higher than that in normal tissue till 24 PIH.

CONCLUSIONEven though there was remarkable postburn increase in the erythrocyte diapedesis, neutrophil infiltration and albumin exudation with different peak time points (1, 3 and 6 PIH, respectively), Inflammation seemed not to be the premise of erythrocyte diapedesis, while the secondary inflammatory reaction might be the main cause of pulmonary edema.

Animals ; Capillary Permeability ; Exudates and Transudates ; metabolism ; Hemorrhage ; etiology ; Neutrophil Infiltration ; Rats ; Rats, Wistar ; Serum Albumin, Bovine ; metabolism ; Smoke Inhalation Injury ; complications ; metabolism

OBJECTIVETo observe the effect of bombesin noncytoskeleton form and intracellular free calcium ([Ca2+]i) concentration in PC-3 prostate cancer cell line.

METHODSImmunofluorescent histochemistry (IH) combined with laser scanning confocal microscopy (LSCM) was used to examine the expression of cytokeratin (CK) in PC-3 cells treated with definite concentrations of BBS and observe its effect on cytoskeleton form. Fluo-3/AM fluorescence technique and LSCM were adopted to measure the [Ca2+]i concentration after different concentrations (10(-9), 10(-7) and 10(-5) mol/L) of BBS were added in PC-3 cells.

RESULTSBBS (10(-5) mol/L) stimulated the expression of CK in PC-3 cells and the formation of lamellipodium, and increased the [Ca2+]i concentration, with concentration dependence.

CONCLUSIONDefinite concentrations of BBS could obviously enhance the [Ca2+] i concentration, CK expression and cytoskeleton morphology of PC-3 cells. The results provide a basis for further studies on the role of BBS in tumour researches as well as in intracellular signal transmission.

Bombesin ; pharmacology ; Calcium ; analysis ; Cytoskeleton ; drug effects ; metabolism ; Fluoroimmunoassay ; Humans ; Keratins ; biosynthesis ; Male ; Microscopy, Confocal ; Prostatic Neoplasms ; metabolism ; Serum Albumin, Bovine ; Tumor Cells, Cultured

OBJECTIVE: To evaluate the effectiveness of IL-10 in suppressing 18F-FDG uptake in the inflammatory granuloma of SD rats. METHODS: Eight SD rats were killed, and their blood was collected sterily. After centrifugion, the white blood cells were incubated in PRMI 1640 for 3 days. Then each culture flask of white blood cells was divided into two equal parts. To one group was added 0.2 mL IL-10 solution (0.1 mg/mL); to the control was added with 0.2 mL of 0.9% sodium chloride solution. All cells were then incubated for 120 minutes at 37 degree, after which 18F-FDG (1.85 MBq) was added. Sixty minutes later, the cells were washed twice with PBS and the extent of uptake 18F-FDG determined. In vivo, an inflammatory granuloma was produced by hypodermic injection of rats with a mixture of Freund's complete adjuvant, bovine serum albumin and talcum powder. Each rat was maintained for 8 weeks. Imaging of the inflammatory granulomas was performed using the 18F-FDG signal. IL-10 was injected into SD rats at 10 μg/kg of body weight. Sixty minutes later, 7.4 MBq of 18F-FDG were injected, and, after a further 60 minutes, the rats underwent a PET-CT scan. The region of interest (ROI) of the inflammatory granuloma was delineated and the standard uptake value (SUV) calculated. A second PET-CT scan was done without IL-10 on the next day. The granulomatous tissue underwent pathological examination. RESULTS: In the intro test, the with blood cell uptaking ratio of 18F-FDG was (50.3±6.7)% without IL-10, and (34.6±3.5)% with IL-10(t=8.9, P<0.01). IL-10 suppressed the rat white blood cell uptaking 18F-FDG. In the PET-CT scan, the SUV of ROI on inflammatory granuloma was 1.7±0.4 with IL-10 and 2.1±0.3 without IL-10 (t=20.6, P<0.01). IL-10 suppressed the inflammatory granuloma uptaking 18F-FDG. CONCLUSION IL-10 can suppress the inflammatory granuloma of SD rats uptaking 18F-FDG.
Animals ; Female ; Fluorodeoxyglucose F18 ; pharmacokinetics ; Freund's Adjuvant ; Granuloma ; chemically induced ; metabolism ; Interleukin-10 ; pharmacology ; Lung Diseases ; chemically induced ; metabolism ; Male ; Radiopharmaceuticals ; pharmacokinetics ; Rats ; Serum Albumin, Bovine

OBJECTIVETo study the incorporation rate and release behavior of bovine serum albumin (BSA) incorporated into the calcium phosphate coating by biomimetic deposition, as well as the physical and chemical properties of the hybrid coating, and to provide experimental basis for the fabrication of growth factor/biomimetic calcium phosphate coating and exploration for the loading/release behavior of growth factors.

METHODSPure titanium specimens were immersed into saturated calcium phosphate solutions(SCP) containing no BSA (controlled group) and 3 different concentrations of BSA (experimental groups) : 1, 10 and 100 mg/L. Biomimetic calcium phosphate coating was formed on titanium surface and BSA was incorporated into the coating through co-deposition. The topography of the specimen was observed using scanning electron microscopy (SEM). Chemical structure and phase composition of coatings were detected by Fourier infrared spectroscopy (FTIR) analysis and X-ray diffraction (XRD) respectively. BSA incorporation rate and release profile were determined by bicinchoninic acid protein assay kit.

RESULTSThe biomimetic calcium phosphate coating was mainly composed of hydroxyapatite and octacalcium phosphate. BSA was successfully incorporated into the calcium phosphate coatings in all the 3 experimental groups. With the increase of BSA concentration, plate-like units of the coatings were turned into small grid structure. BSA incorporation rates of the three experimental groups were (72.4 ± 2.4)%, (62.3 ± 0.9)% and (42.2 ± 1.7)% respectively. The in vitro release test showed that all three BSA release profiles could be divided into two significant different stages: early burst release stage and later sustained release stage. The amount of BSA release of the 3 experimental groups in 24 h and 30 d were (1.57 ± 0.09), (8.82 ± 0.93), (140.24 ± 3.12) µg, and (2.39 ± 0.29), (14.39 ± 0.70), (151.06 ± 2.00) µg respectively.

CONCLUSIONSBiomimetic calcium phosphate coating can be used as an effective carrier for protein. BSA concentration has an impact on the incorporation rate and release speed of BSA from the calcium phosphate coating. Favorable BSA incorporation rate and release behavior can be obtained at BSA concentration of 10 mg/L.

Biomimetic Materials ; Calcium Phosphates ; chemistry ; Durapatite ; In Vitro Techniques ; Microscopy, Electron, Scanning ; Serum Albumin, Bovine ; metabolism ; Spectrophotometry, Infrared ; Surface Properties ; Titanium ; X-Ray Diffraction

AIMTo prepare cells scaffolds with the characteristics of sustained release of proteins.

METHODSChitosan scaffolds was prepared by freeze-drying. Porosity and water content of scaffolds were determined. Bovine serum album (BSA) was selected as a model protein. Poly (lactic-co-glycolic acid) (PLGA) microspheres were prepared by double emulsion solvent evaporation and encapsulated into chitosan scaffolds. The morphology of PLGA microspheres and various scaffolds were observed using scanning electron microscope. Release behavior of BSA from various chitosan scaffolds was investigated.

RESULTSThe chitosan scaffold represents porous. At the -70 degrees C of quenching temperature, the porosity and water content of chitosan scaffolds were 78.6% +/- 1.5% and 85.1% +/- 6.2%, respectively. PLGA microspheres can be uniformly encapsulated into scaffolds without any morphology change. Significant sustained release of BSA from PLGA microspheres encapsulated into scaffolds was obtained. The cumulative release at 168 h was only 33.5%, while that of BSA from chitosan scaffolds at 24 h was above 90%. The release behavior can be controlled by adjusting the amount of chitosan in scaffolds and the type of PLGA.

CONCLUSIONThe novel chitosan scaffolds encapsulating PLGA microspheres proved to be a promising cells scaffolds with controlling the release of growth factors in tissue engineering.

Chitosan ; administration & dosage ; chemistry ; Drug Carriers ; Freeze Drying ; methods ; Lactic Acid ; chemistry ; Microspheres ; Polyglycolic Acid ; chemistry ; Polymers ; chemistry ; Serum Albumin, Bovine ; metabolism ; Tissue Engineering ; methods

AIMTo obtain Clenbuterol monoclonal antibodies.

METHODSClenbuterol complete antigen was prepared with diazotization method. BALB/c mice was immunized with subtractive immunization, Clenbuterol monoclonal antibody was prepared with rule hybridoma technique.

RESULTSThe mice obtained tolerance to BSA by subtractive immunization. The rate of the hybridoma cell with positive reaction which had obtained was 8.2%, and the specific clenbuterol monoclonal antibody was obtained at last.

CONCLUSIONMonoclonal antibodies to micromolecule contaminant be prepared by subtractive immunization, could decrease the workload in the bolting of monoclonal antibodies, and increase the chance to obtain the antibody of expected.

Animals ; Antibodies, Monoclonal ; biosynthesis ; immunology ; Clenbuterol ; immunology ; Female ; Hybridomas ; metabolism ; Immunization ; methods ; Male ; Mice ; Mice, Inbred BALB C ; Serum Albumin, Bovine ; immunology