BACKGROUND AND OBJECTIVES: Controversy exists about the characteristics of the lipid-oxidizing process, and the molecules in oxidized lipids that are involved in the binding and uptake to macrophages, in atherosclerosis. The aim of this study was to find answers to these questions using oxidized red blood cells (ox-RBCs). MATERIALS AND METHODS: The RBCs were oxidized in the presence of various concentrations of CuSO4, and the degree of oxidation evaluated by the semiquantitative measurement of the thiobarbituric acid reactive substance (TBARS). The ox-RBC was characterized using annexin-V and flow cytometry. The relationships between the CuSO4 concentration, the degree of oxidation, characteristics of the ox-RBC and it's binding to macrophages transformed from THP-1 cells, were evaluated. RESULTS: The RBCs were oxidized, not by their gradual changes, but by the sudden transformation of a proportion of the RBCs in relation to the CuSO4 concentration. There were few RBCs between oxidized and non-oxidized groups. The annexin-V bound only to the ox-RBC, with a similar degree of binding in all ox-RBCs. The binding of ox-RBC to macrophages was completely inhibited by oxidized low density lipoprotein, which was directly related to the CuSO4 concentration, the TBARS and the proportion of ox-RBC. CONCLUSION: These results suggest that the oxidation of lipids might be an on-off phenomenon process. Molecules that have the ability to bind annexin-V, presumptively phosphatidyl serine, may be involved in the process of binding the ox-lipids to macrophages. Further study will be needed to clarify these molecules.
Atherosclerosis ; Binding, Competitive ; Erythrocytes* ; Flow Cytometry ; Lipid Peroxidation ; Lipoproteins ; Macrophages* ; Serine ; Thiobarbituric Acid Reactive Substances

The preparation of a novel amphiphilic dietary fiber LDL adsorbent with sulfonic group and laurylamine group was studied. The effects of reaction time and reaction temperature on the adsorption rate were studied. The results show that the adsorption rates for the removal of Total cholesterol (TC), Low-density lipoprotein cholesterol (LDL-C) and High-density lipoprotein cholesterol (HDL-C) are 40.8%, 50.8% and 23.6%, respectively. The amphiphilic adsorbent has better selectivity in removing LDL-C.
Adsorption ; Binding, Competitive ; Cholesterol ; blood ; isolation & purification ; Dietary Fiber ; pharmacology ; Lipoproteins, LDL ; blood ; isolation & purification

Kinases play crucial roles in the life of cell. Their functional abnormity usually leads to many human major diseases including tumors. The prospecting of ATP-competitive small-molecule kinase inhibitors targeting kinases of therapeutic interest has become the focus of researches. Due to the high conservation of the catalytic domain of kinases, the selectivity of kinase inhibitors is poor in general. However, along with the development of structural biology and computer-aided drug design, great progress in the research of selective, ATP-competitive kinase inhibitors has been achieved in recent years. In this account, the review has been made on the development of the design of selective kinase inhibitors.
Adenosine Triphosphate ; chemistry ; Binding, Competitive ; Drug Design ; Molecular Structure ; Protein Binding ; Protein Kinase Inhibitors ; chemical synthesis ; chemistry ; pharmacology

OBJECTIVETo determine the affinity of new opioid receptor ligands to cloned mu opioid receptors stably expressed in CHO cell.

METHODSThe binding characteristics of the opioid ligand [3H] diprenorphine (3H-dip) were studied by cellular biological techniques and radioligands binding in cloned mu opioid receptors stably expressed in CHO cells in saturation binding experiments, and were followed by competition binding experiments with a variety of new synthesized opioid receptor ligands.

RESULTSThe Kd and Bmax of [3H] diprenorphine bound to mu receptors were 1.06 nmol/L and 930 fmol/mg protein, respectively. Competition binding experiments revealed that ligand 3# and 12# displayed much higher affinity than DAMGO and Morphine for the cloned mu opioid receptor. However, the affinities of ligands 2#, 6#, 8# and 9# were lower than DAMGO and Morphine.

CONCLUSIONThe present results suggest that the new ligands 3# and 12# have higher affinity to mu opioid receptors. However, ligands 2#, 6#, 8# and 9# have lower affinity to mu opioid receptors.

Animals ; Binding Sites ; Binding, Competitive ; CHO Cells ; metabolism ; Cloning, Molecular ; Cricetinae ; Diprenorphine ; pharmacology ; Ligands ; Receptors, Opioid, mu ; biosynthesis ; genetics ; metabolism

Competition ELISA test using sparganum specific monoclonal antibodies (Mab) was investigated to improve the diagnostic specificity of sparganosis. By cell fusion, one hybridoma clone secreting anti-sparganum specific Mab was selected (Sp-20), which reacted on bands of 32 kDa and 38 kDa. Sp-20 reacted on calcium corpuscles on IFA. By micro-ELISA, 16 of 17 sparganosis cases (95%) were found positive, but 1 of 18 clonorchiasis cases (5%), 4 of 16 cysticercosis cases (25%) and 2 of 16 normal controls (11%) showed false positive reactions. On the other hand, by competition ELISA using a sparganum specific Mab (Sp-20), 16 out of 17 (95%) of sparganosis cases were found positive, but 2 of 18 clonorchiasis cases (10%), 2 of 16 cysticercosis cases (12%), 3 of 16 paragonimiasis cases (18%) and 1 of 16 normal controls (6%) showed false positive reactions.
Animal ; Antibodies, Helminth ; Binding, Competitive ; Enzyme-Linked Immunosorbent Assay ; Human ; Sensitivity and Specificity ; Sparganosis/*diagnosis ; Sparganum/immunology ; Support, Non-U.S. Gov't

AIMTo develop a fluorescence polarization-based high throughput screening and identify ligands for human Lectin-like oxidized low-density lipoprotein receptor-1 (hLOX-1).

METHODSSequential ultracentrifugation at 4 degrees C from normolipidemic fasting volunteers to obtain low density lipoprotein (LDL), which was modified by CuSO4 (5 micromol x L(-1)) at 37 degrees C for 24 h. The assay was based on the interaction between receptor and ligand, and hLOX-1 was labeled by FITC and bound to its specific ligand, oxLDL. Different reaction time and DMSO concentration were optimized to determine the stability and tolerance of fluorescence polarization (FP) assay. 3 200 compounds were screened in black 384-well microplate by FP-based competitive displacement assay, at excitation filter of 485 nm and emission filter of 530 nm. Z' was used to assess the assay quality.

RESULTSThe FP-based HTS was formatted in a 384-well microplate with a Z' factor of 0. 75, and three active compounds for hLOX-1 were identified with IC50 below 40 micromol x L(-1) from total 3 200 compounds.

CONCLUSIONThe results indicated that the fluorescence polarization assay is stable, sensitive, reproducible and well suited for high throughput screening efforts.

Binding, Competitive ; Drug Evaluation, Preclinical ; methods ; Fluorescence Polarization ; methods ; Humans ; Ligands ; Lipoproteins, LDL ; metabolism ; Scavenger Receptors, Class E ; metabolism

OBJECTIVETo search alpha-glucosidase inhibitors from Rubia cordifolia.

METHODThe alpha-glucosidase inhibitors were isolated by the column chromatographic techniques and the bioassay-guided method in vitro. A combination of MS and NMR spectroscopy was used to identify the chemical structures. The inhibitory kinetics of the inhibitors were also investigated.

RESULTThe chloroform extract showed high inhibitory activity, and three active compounds were isolated and identified as 1,3-dihydroxy-2-methylanthraquinone (1), 1-hydroxy-2-methylanthraquinone (2) and 1,2-dihydroxyanthraquinone (3). The IC5o values of compound 1-3 were all lower than that of acarbose. Compound 1 and 2 shown competitive type manner on alpha-glucosidase, whereas compound 3 shown noncompetitive type model.

CONCLUSIONCompounds 1-3 as strong inhibitors of alpha-glucosidase were reported for the first time.

Binding, Competitive ; Enzyme Inhibitors ; analysis ; metabolism ; pharmacology ; Glycoside Hydrolase Inhibitors ; Inhibitory Concentration 50 ; Kinetics ; Rubia ; chemistry ; alpha-Glucosidases ; metabolism

OBJECTIVETo develop a more reliable and stable method for determining monoclonal antibody (mAb) affinity constant based on competitive antibody/antigen binding.

METHODSThe Kd value was calculated based on the relationship between the binding proportion of the antigen and the original concentration of the mAb that competed for the binding site of the antigen.

RESULTSThe Kd values measured with this improved method under two different conditions were 2.61x10(-12) mol/L and 2.39x10(-12) mol/L, and those with Friguent method in these two conditions were 5.57x10(-10) mol/L and 1.41x10(-10) mol/L, respectively.

CONCLUSIONCompared with Friguent method, the Kd values measured with this improved method are closer to the actual value, and the measurement results under different experiment conditions are more stable.

Antibodies, Monoclonal ; immunology ; Antibody Affinity ; immunology ; Antigen-Antibody Complex ; immunology ; Binding, Competitive ; Enzyme-Linked Immunosorbent Assay ; Humans

OBJECTIVETo screen the polypeptides specifically binding to human large intestinal cancer LoVo cells from a phage-displayed peptide library for potential use as targeting vectors for large intestinal cancer therapy.

METHODSWith the LoVo cells as the target cells and human normal large intestinal mucosal epithelial cells as the absorber cells for subtraction biopanning from a c7c phage-display peptide library, the positive phage clones were identified by enzyme-linked immunosorbent assay (ELISA) and immunofluorescence detection. The amino acid sequences of the identified peptides were deduced by DNA sequencing.

RESULTSAfter 3 rounds of screening, 5 positive phage clones showing specific binding to LoVo cells and containing conserved motif RPMP were obtained from the 20 randomly selected clones.

CONCLUSIONSpecific peptide against large intestinal cancer cells can be obtained from a phage-display peptide library for use as potential vectors for targeting therapy of large intestinal cancer.

Amino Acid Sequence ; Base Sequence ; Binding, Competitive ; Cell Line, Tumor ; Colorectal Neoplasms ; genetics ; metabolism ; pathology ; Humans ; Molecular Sequence Data ; Peptide Library ; Peptides ; genetics ; isolation & purification ; metabolism ; Protein Binding

OBJECTIVETo determine the plasma protein binding rate of arctiin and arctigenin.

METHODThe ultrafiltration combined with HPLC was employed to determine the plasma protein binding rate of arctiin and arctigenin as well as rat plasma and healthy human plasma proteins.

RESULTThe plasma protein binding rate of arctiin with rat plasma at the concentrations of 64. 29, 32.14, 16.07 mg x L(-1) were (71.2 +/- 2.0)%, (73.4 +/- 0.61)%, (78.2 +/- 1.9)%, respectively; while the plasma protein binding rate of arctiin with healthy human plasma at the above concentrations were (64.8 +/- 3.1)%, (64.5 +/- 2.5)%, (77.5 +/- 1.7)%, respectively. The plasma protein binding rate of arctigenin with rat plasma at the concentrations of 77.42, 38.71, 19.36 mg x L(-1) were (96.7 +/- 0.41)%, (96.8 +/- 1.6)%, (97.3 +/- 0.46)%, respectively; while the plasma protein binding rate of arctigenin with normal human plasma at the above concentrations were (94.7 +/- 3.1)%, (96.8 +/- 1.6)%, (97.9 +/- 1.3)%, respectively.

CONCLUSIONThe binding rate of arctiin with rat plasma protein was moderate, which is slightly higher than the binding rate of arctiin with healthy human plasma protein. The plasma protein binding rates of arctigenin with both rat plasma and healthy human plasma are very high.

Animals ; Binding, Competitive ; Blood Proteins ; metabolism ; Chromatography, High Pressure Liquid ; Furans ; metabolism ; Glucosides ; metabolism ; Humans ; Lignans ; metabolism ; Male ; Protein Binding ; Rats ; Rats, Sprague-Dawley ; Ultrafiltration ; methods