The lipase labeled with the fluorescein isothiocyanat (FITC) was immobilized on the derivatives of the polyethylene glycol. The article discussed the effect of factors on the characters of lipase and analyzed the relationships among the activity of lipase, conformation, and fluorescence spectrum while the activity and the fluorescence spectrum of immobilized lipase were determined. The results demonstrated that polyethylene glycol 400-diacrylate could form appropriate network to improve the activity of enzyme. Adding ligand induced the lipase's catalytic conformation to increase the activity twice more than before. The active centre of lipase could be released by the extraction of ligand thus increasing the activity. After immobilization, the stability of labeled lipase improved greatly: immobilized lipases retained more than 70% and 60% of initial activity under conditions of 90 degrees C and strong acid or alkali, respectively. After immersing immobilized lipases into guanidine hydrochloride or urea for 15 days, the lipases retained upwards of 70% activity. The fluorescence spectrum could obviously reflect the changes of the activity and conformation of lipase. The fluorescence intensity was the minimum in the optimal pH and temperature. In the denaturing agent it declined as time passed. These results indicated that the unfolded processes of immobilized lipases are different under different conditions.
Dextrans ; chemistry ; Enzyme Stability ; Enzymes, Immobilized ; chemistry ; metabolism ; Fluorescein-5-isothiocyanate ; analogs & derivatives ; chemistry ; Fluorescent Dyes ; chemistry ; Lipase ; chemistry ; metabolism ; Polyethylene Glycols ; chemistry ; Protein Unfolding ; drug effects

Astragalus polysaccharides was lounded to 4-(2-aminoethylphenol), followed by labeling the APS-Tyr with fluorescein-5-isothiocyanate (FITC) at the secondary amino group. The absorption enhancement effects of low molecular weight chitosan and protamine on astragalus polysaccharides were evaluated via Caco-2 cell culture model. The results show that the fluorecent labeling compound has good stability and high sensitivity. On the other hand low molecular weight chitosan and protamine also can promoted absorption of the astragalus polysaccharides without any cytotoxity, and the absorption increase was more significant with increasing the amount of low molecular weight chitosan and protamine. At the same time, the low molecular weight chitosan has slightly better effect. The transepithelial electric resistance (TEER) of Caco-2 cells show that absorption enhancers could improve its membrane transport permeability by opening tight junctions between cells and increasing the cell membrane fluidity.
Absorption ; Astragalus Plant ; chemistry ; Biological Transport ; Caco-2 Cells ; Fluorescein-5-isothiocyanate ; chemistry ; Fluorescent Dyes ; chemistry ; Humans ; Plant Extracts ; chemistry ; pharmacokinetics ; Polysaccharides ; chemistry ; pharmacokinetics

OBJECTIVETo investigate the distribution of Ophiopogon Japonicas polysaccharide (OJP) in hearts of normal rats and myocardial ischemic rats, and obtain the further evidences for determining the mechanism of its anti-myocardial ischemia.

METHODHearts of normal and myocardial ischemic groups were obtained at 5, 10, 30 and 60 min after once intravenous administration of OJP with the dose of 50 mg/kg. The concentration of OJP in samples was subsequently determined by HPGPC with a fluorescence detector.

RESULTThe distribution of OJP in hearts of myocardial ischemic rats was much more than that in normal groups at all the sampling times. Moreover, the differences were proved to be significant by SPSS software with the exception of the situation in the 30 min sampling time.

CONCLUSIONThe enhanced permeability and retention effect caused by ischemia can overcome the negative effect of the reduction of blood flow on the distribution of OJP from blood into myocardial ischemic zones and actually help OJP target myocardial ischemic zones where it can exert its pharmacological effect directly.

Animals ; Calibration ; Fluorescein-5-isothiocyanate ; metabolism ; Male ; Myocardial Ischemia ; metabolism ; Myocardium ; metabolism ; Ophiopogon ; chemistry ; Permeability ; Polysaccharides ; metabolism ; Rats ; Sensitivity and Specificity ; Software ; Time Factors

BACKGROUND: We developed a single-color multitarget flow cytometry (SM-FC) assay, a single-tube assay with graded mean fluorescence intensities (MFIs). We evaluated the repeatability of SM-FC, and its correlation with multicolor flow cytometry (MFC), to assess its application as a routine FC assay. METHODS: We selected CD19, CD3, CD4, and CD8 as antigen targets to analyze a lymphocyte subset. MFIs were graded by adjusting monoclonal antibody (mAb) volumes to detect several cell populations. Dimly labeled mAb was prepared by decreasing mAb volume and the optimum diluted volume was determined by serial dilution. SM-FC repeatability was analyzed 10 times in 2 normal controls. The correlation between SM-FC and MFC was evaluated in 20 normal and 23 patient samples. RESULTS: CV values (0.8-5.0% and 1.3-4.1% in samples 1 and 2, respectively) acquired by SM-FC with CD3-fluorescein alpha-isothyocyanate (FITC)dim+CD4-FITCbright and with CD19-FITCdim+CD3-FITCbright showed good repeatability, comparable to that acquired by MFC (1.6-3.7% and 1.0-4.8% in samples 1 and 2, respectively). Excellent correlation was observed between the 2 methods in the 20 normal samples (B cells, T cells, non-Thelper cells, and Thelper cells; r2=0.87, 0.97, 0.97, and 0.98, respectively; P<0.05). There were also linear relationships between SM-FC with CD19-FITCdim+CD3-FITCbright and CD8-PEdim+CD4-PEbright, and MFC, in the 23 patient samples (B cells, T cells, Tcytotoxic cells, and Thelper cells; r2> or =0.98, 0.99, 0.99, and 0.99, respectively; P<0.05). CONCLUSIONS: The multicolor, single-tube SM-FC technique is a potential alternative tool for identifying a lymphocyte subset.
Antibodies, Monoclonal/chemistry/*immunology ; Antigens, CD19/chemistry/metabolism ; Antigens, CD3/chemistry/metabolism ; Antigens, CD4/chemistry/metabolism ; Antigens, CD8/chemistry/metabolism ; B-Lymphocyte Subsets/immunology/metabolism ; Color ; Flow Cytometry/*methods ; Fluorescein-5-isothiocyanate/*chemistry ; Humans ; T-Lymphocyte Subsets/immunology/metabolism

In this work, polyelectrolyte microcapsules containing gold nanoparticles were prepared via layer by layer assembly. Gold nanoparticles and poly (allyamine hydrochloride) (PAH) were coated on the CaCO3 microparticles. And then EDTA was used to remove the CaCO3 core. Scanning electron microscopy (SEM) was used to characterize the surface of microcapsules. SEM images indicate that the microcapsules and the polyelectrolyte multilayer were deposited on the surface of CaCO3 microparticles. FITC-bovine serum albumin (FITC-BSA, 2 mg) was incorporated in the CaCO3 microparticles by co-precipitation. Fluorescence microscopy was used to observe the fluorescence intensity of microcapsules. The encapsulation efficiency was (34.31 +/- 2.44) %. The drug loading was (43.75 +/- 3.12) mg g(-1).
Calcium Carbonate ; chemistry ; Capsules ; Drug Carriers ; Drug Delivery Systems ; methods ; Electrolytes ; chemistry ; Fluorescein-5-isothiocyanate ; analogs & derivatives ; chemistry ; Gold ; chemistry ; Microscopy, Electron, Scanning ; Microscopy, Fluorescence ; Nanoparticles ; Particle Size ; Serum Albumin, Bovine ; chemistry

The aimed of this study was to prepare stabilized thiomers to overcome the poor stability character of traditional thiomers. Poly(acrylic acid)-cysteine (PAA-Cys) was synthesized by conjugating cysteine with poly(acrylic acid) and poly(acrylic acid)-cysteine-6-mercaptonicotinic acid (PAA-Cys-6MNA, stabilized thiomers) was synthesized by grafting a protecting group 6-mercaptonicotinic acid (6MNA) with PAA-Cys. The free thiol of PAA-Cys was determined by Ellmann's reagent method and the ratio of 6MNA coupled was determined by glutathione reduction method. The study of permeation enhancement and stabilized function was conducted by using Franz diffusion cell method, with fluorescein isothiocyanate dextran (FD4) used as model drug. The influence of polymers on tight junctions of Caco-2 cell monolayer was detected with laser scanning confocal fluorescence microscope. The results indicated that both PAA-Cys and PAA-Cys-6MNA could promote the permeation of FD4 across excised rat intestine, and the permeation function of PAA-Cys-6MNA was not influence by the pH of the storage environment and the oxidation of air after the protecting group 6MNA was grafted. The distribution of tight junction protein of Caco-2 cell monolayer F-actin was influenced after incubation with PAA-Cys and PAA-Cys-6MNA. In conclusion, stabilized thiomers (PAA-Cys-6MNA) maintained the permeation function compared with the traditional thiomers (PAA-Cys) and its stability was improved. The mechanism of the permeation enhancement function of the polymers might be related to their influence on tight junction relating proteins of cells.
Acrylic Resins ; chemistry ; Actins ; metabolism ; Animals ; Caco-2 Cells ; Cysteine ; chemistry ; Dextrans ; Fluorescein-5-isothiocyanate ; analogs & derivatives ; Glutathione ; Humans ; Intestinal Absorption ; Intestinal Mucosa ; drug effects ; Nicotinic Acids ; chemistry ; Rats ; Sulfhydryl Compounds ; chemistry

An in vitro detection method of the gastrointestinal absorption of Pilose Antler protein was established for mixed protein activity. Five bands of protein with molecular weight of 17.8-160 kD derived from the Pilose Antler were extracted and sufficiently labeled with FITC (FITC-PE). The stability and variation of FITC-PE in gastrointestinal circumstances were detected by native polyacrylamide gel electrophoresis and confocal laser scanning microscope. Results showed that the main component of FITC-PE kept invariant after being reacted with artificial gastric fluid and artificial intestinal fluid. The fluorescence signal was detected 20 min after administration in the valgus intestinal purse experiment, and three kinds of protein, with molecular weight of 45, 25, and 17.8 kD, were detected in the mixture of absorbent protein. The research laid the foundation for the further in vivo study of Pilose Antler protein. Meanwhile, it would be an in vitro screening method for the absorption, distribution and metabolism of mixed protein from traditional Chinese medicine.
Animals ; Antlers ; chemistry ; Deer ; Fluorescein-5-isothiocyanate ; Intestinal Absorption ; Intestinal Mucosa ; metabolism ; Male ; Materia Medica ; chemistry ; isolation & purification ; pharmacokinetics ; Microscopy, Confocal ; Molecular Weight ; Native Polyacrylamide Gel Electrophoresis ; Proteins ; chemistry ; isolation & purification ; pharmacokinetics ; Rats ; Rats, Wistar ; Stomach ; metabolism

The aim of this study was to ascertain the folate receptor (FR) targetability by an in vitro study and to acquire FR-targeted images in vivo models by using synthetic folate conjugates. PEG-folate was synthesized and labeled with (99m)Tc and fluorescein isothiocynate (FITC). Cell uptake studies were carried out in KB cells (FR-positive) and A549 cells (FR-negative) using FITC- and the (99m)Tc-labeled conjugates. The radiolabeled conjugate was intravenously injected to KB tumor xenografted mice. After it was injected, gamma images were recorded at 30 min, 1, 2, 3 and 4 hr. Cell uptake studies showed a difference between the KB cells and the A549 cells by flow cytometry analysis and gamma counting. On in vivo images, the tumor-tonormal muscle ratio was greater than 4. It ascertained that the PEG-folate conjugate specifically binds to the FR expressed on tumor cells in vitro. Moreover, it was possible to acquire the FR-targeted gamma images using PEG-folate conjugates in tumor models.
Animals ; Carrier Proteins/*metabolism ; Cell Line, Tumor ; Female ; Fluorescein-5-isothiocyanate/pharmacology ; Folic Acid/*metabolism ; Humans ; Image Processing, Computer-Assisted ; Mice ; Mice, Nude ; Microscopy, Fluorescence ; Neoplasm Transplantation ; Polyethylene Glycols/*chemistry ; Receptors, Cell Surface/*metabolism ; Technetium/*chemistry ; Time Factors

OBJECTIVETo compare the characteristics of three- and two-color flow cytometry for immunologic surveillance of toll-like receptor 4 (TLR-4) after renal transplantation.

METHODThe expressions of CD14, TLR4 and CD80 in peripheral blood monocytes were measured in 10 patients after renal transplantation by three- and two-color fluorescent staining flow cytometry, respectively. The percentages of TLR4 and CD80 in CD14-positive monocytes were calculated, and the characteristics of two methods of flow cytometry were compared.

RESULTSThe two detection methods did not significantly differ in their detection of TLR4 (P=0.198) and CD80 (P=0.872) expressions in CD14-positive monocytes, and the measurements of the two methods for the same sample showed positive correlation (r=1, P=0.000 for TLR4 and r=0.999, P=0.000 for CD80). Three-color flow cytometry was also capable of detecting monocytes positive for both TLR4 and CD80. Compared with two-color flow cytometry, three-color flow cytometry provided more information and required less blood sample and monoclonal antibody.

CONCLUSIONSThree- and two-color flow cytometry have almost the same accuracy in immunologic surveillance of TLR4 after renal transplantation. The former method also provides information of the simultaneous expression of TLR4 and its down stream molecule CD80, which could be critical for imploring the mechanisms by which TLR4 takes part in alloimmunity through modulating CD80 expression, and requires less blood sample and monoclonal antibody.

Adult ; Antibodies, Monoclonal ; chemistry ; immunology ; B7-1 Antigen ; blood ; immunology ; Female ; Flow Cytometry ; methods ; Fluorescein-5-isothiocyanate ; chemistry ; Humans ; Kidney Transplantation ; immunology ; Lipopolysaccharide Receptors ; blood ; immunology ; Male ; Middle Aged ; Monocytes ; immunology ; Reproducibility of Results ; Toll-Like Receptor 4 ; blood ; immunology

The preparation method, serum stability, efficiency of cellular uptake and apoptosis induction of the cell penetrating peptide TAT and cleavable PEG co-modified liposomes loaded with paclitaxel (C-TAT-Lipo) were investigated. The best preparation procedure was performed by orthogonal test based on single factor screening method. First, the paclitaxel (PTX)-loaded liposomes were prepared by filming-rehydration method, evaluated with entrapment efficiency and polydispersity index. The morphology of C-TAT-Lipo was characterized by transmission electron microscopy. Turbidity variations were monitored in the presence of fetal bovine serum (FBS) to evaluate the serum stability of the liposomes developed here. Next, the efficiency of cellular uptake of different Rho-PE-labeled liposomes on B16F1 cells in vitro was evaluated by confocal laser scanning microscopy (CLSM) and flow cytometry. The quantitative analysis of apoptosis induced by different PTX-loaded liposomes was performed by Annexin V-FITC/PI double staining. The optimal formulation was as follows: Chol : lipid: 1 : 8 (molar ratio); drug : lipid: 1 : 40 (mass ratio); lipid concentration: 3 mmol x L(-1); temperature of hydration: 25 degrees C. The mean size and polydispersity index of C-TAT-Lipo were about (97.97 +/- 3.68) nm and 0.196 +/- 0.037, the zeta potential was (-0.89 +/- 0.45) mV, the entrapment efficiency of paclitaxel was (90.16 +/- 1.53)%. The particle sizes did not exhibit significant variations in 50% FBS over 24 h at 37 degrees C. The efficiency of cellular uptake of the C-TAT-Lipo increased 1.40 fold following the cleavage of PEG. Apoptosis analysis showed 59.3% increase of the apoptosis and necrosis profile of C-TAT-Lipo after the detachment of PEG shells, which was markedly higher than that of N-TAT-LP with or without glutathione and SL, respectively. The results indicate that the C-TAT-Lipo is successfully prepared by filming-rehydration method and shows significant antitumor activities.
Animals ; Annexin A5 ; Apoptosis ; Cell Line, Tumor ; Cell-Penetrating Peptides ; pharmacology ; Fluorescein-5-isothiocyanate ; analogs & derivatives ; Liposomes ; chemistry ; Melanoma, Experimental ; Mice ; Microscopy, Confocal ; Paclitaxel ; pharmacology ; Particle Size ; Polyethylene Glycols ; chemistry