The bioactive protein-phycocyanin and all the proteins of Spirulina Platensis were isolated and purified. Photo-reactive proteins were synthesized by coupling the proteins with (N-(4-azidobenzoyloxy)succinimide) and were spread onto the 24-well cell culture polystyrene plate. Then the coated surface was exposed to ultraviolet irradiation for chemical fixation of proteins via the conversion of the phenylazido group to the highly reactive phenyl-nitrene which spontaneously formed covalent bonds with neighboring hydrocarbons. On these proteins-immobilized polystyrene plates, the liver cancer cells 7402 were cultured under the serum-free conditions, and the inhibition activity on proliferation of liver cancer cells was investigated and analyzed.
Animals ; Bacterial Proteins ; chemistry ; pharmacology ; Biocompatible Materials ; pharmacology ; Cell Division ; drug effects ; Cyanobacteria ; chemistry ; Liver Neoplasms ; pathology ; Photochemistry ; Polystyrenes ; chemistry ; Spirulina ; Succinimides ; chemistry ; Tumor Cells, Cultured

OBJECTIVE: Authors aimed to determine the targeting ability of vascular endothelial growth factor receptor 2 (VEGFR2)-conjugated quantum dots (QDs) in vitro, and apply it for a xenograft prostate cancer mouse model. MATERIALS AND METHODS: Conjugation reaction of QDs was performed by using the N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide (EDC) and sulfo-(N-hydroxysulfosuccinimide) (Sulfo-NHS). The human umbilical vein cord endothelial cells (HUVECs) were incubated with QDs, conjugated with antiVGFR2, to see a specific binding in vitro. Fluorescent cell images were taken by a confocal microscope. The human prostate cancer cells (PC3) were injected to five nude mice on hind limbs to make the xenograft tumor model. QD-antiVEGFR2 antibody complex was injected into the tumor model and fluorescence measurements were performed at 1, 4, 9, 12, 15, and 24 hours after the injection. RESULTS: The specific interaction between HUVECs and QD-antiVEGFR2 antibody was clearly shown in vitro. The in vivo fluorescence image disclosed that there was an increased signal of tumor, 12 hours after the injection of QDs. CONCLUSION: By showing endothelial cells binding with QDs-antiVEGFR2 antibodyand an experimental application of the antibody for VEGFR2 imaging in the prostate cancer xenograft mouse model, we suggests that the antibody-conjugated QDs can be a potential imaging tool for angiogenesis of the cancer.
Animals ; Carbodiimides/pharmacology ; Cell Line, Tumor ; Disease Models, Animal ; Electrophoresis, Agar Gel ; Fluorescence ; Male ; Mice ; Mice, Nude ; Microscopy, Confocal ; Neovascularization, Pathologic/*pathology ; Prostatic Neoplasms/*pathology ; *Quantum Dots ; Succinimides/pharmacology ; Transplantation, Heterologous ; Vascular Endothelial Growth Factor Receptor-2/*antagonists & inhibitors

OBJECTIVETo establish an assay method for detecting the migration of transferred apoptotic cells into the recipient using flow cytometry.

METHODSSpleen lymphocytes were isolated and labeled with an intracellular amine dye, carboxyfluorescein diacetate succinimidyl ester (CFSE), to allow discrimination. The labeled cells were induced with dexamethasone to undergo apoptosis and transferred into recipient mice via tail venous transfusion. Flow cytometry and histological examination of different tissues were performed at different time points. The stability of CFSE labeling for apoptotic cells was also tested.

RESULTSThe CFSE-labeled apoptotic cells were highly fluorescent with a positive labeling rate of (98.0+/-1.9)%. The stability of CFSE-labeling was testified, and the CFSE-labeled apoptotic cells entering different tissues at different time points were detected by flow cytometry and verified by histological examination.

CONCLUSIONFlow cytometry using CFSE labeling is reliable, sensitive, precise and convenient for apoptotic cell tracing in vivo and in vitro.

Adoptive Transfer ; methods ; Animals ; Apoptosis ; Dexamethasone ; pharmacology ; Female ; Flow Cytometry ; methods ; Fluoresceins ; chemistry ; pharmacokinetics ; Fluorescent Dyes ; chemistry ; pharmacokinetics ; Lymphocytes ; chemistry ; cytology ; drug effects ; Mice ; Mice, Inbred BALB C ; Reproducibility of Results ; Spleen ; cytology ; Succinimides ; chemistry ; pharmacokinetics

OBJECTIVETo evaluate the antiandrogenic effect of heterocyclic fungicide dimethachlon and its mechanism.

METHODSA combination of in vivo and in vitro assays was selected. Hershberger assay was used to determine the antiandrogenic potential of dimethachlon in vivo. Six-week-old castrated male SD rats were administrated once daily for 7 days with testosterone propionate (TP, 100 micro g/d, sc) plus gavage doses of dimethachlon (50, 100 or 200 mg x kg(-1) x d(-1)), or procymidone (150 or 300 mg x kg(-1) x d(-1), positive control), or iprodione (100 mg x kg(-1) x d(-1), positive control), or flutamide (50 mg x kg(-1) x d(-1), positive control). Transcriptional activation assay in vitro was employed to determine the mechanism of antiandrogenic activity of dimethachlon. Human hepatoma liver cells HepG2 were transiently cotransfected with human androgen receptor (AR) expression plasmid and AR-dependent luciferase report plasmid. Transfected cells were exposed to various concentrations of dimethachlon or flutamide with or without dihydrotestosterone to induce the expression of luciferase gene.

RESULTSIn Hershberger assay, dimethachlon, as well as other known antiandrogens, caused decrease in weight of androgen dependent organs or tissues. In 200 mg/kg group, the weight of seminal vesicle, ventral prostate, dorsolateral prostate, Cowper's gland, and levator ani plus bulbocavernosus muscles decreased by 57.8%, 44.8%, 43.9%, 30.1%, and 34.1% respectively, but did not decrease in the vehicle control group. The order of their antiandrogenic potencies was: flutamide > procymidone > dimethachlon > iprodione. In transcriptional activation assay, dimethachlon could inhibit dihydrotestosterone-dependent AR activity in transfected HepG2 cells in dose-effect relationship. The inhibiting potency of dimethachlon was about 1/100 of that of flutamide.

CONCLUSIONDimethachlon has antiandrogenic effect, and acts as an AR antagonist. Its antiandrogenic potency is lower than flutamide and procymidone, but higher than iprodione.

Aminoimidazole Carboxamide ; analogs & derivatives ; pharmacology ; toxicity ; Androgen Antagonists ; pharmacology ; toxicity ; Androgens ; blood ; metabolism ; Animals ; Body Weight ; drug effects ; Bridged Bicyclo Compounds ; pharmacology ; toxicity ; Cell Line, Tumor ; Chlorobenzenes ; pharmacology ; toxicity ; Dose-Response Relationship, Drug ; Flutamide ; pharmacology ; toxicity ; Fungicides, Industrial ; pharmacology ; toxicity ; Humans ; Hydantoins ; Luciferases ; genetics ; metabolism ; Male ; Pesticides ; pharmacology ; toxicity ; Plasmids ; genetics ; Rats ; Rats, Sprague-Dawley ; Receptors, Androgen ; drug effects ; genetics ; metabolism ; Succinimides ; pharmacology ; toxicity ; Transfection

BACKGROUNDThe common γ chain (γc) plays a critical role in regulating proliferation, differentiation, and apoptosis of peripheral T-cells. It was previously confirmed that blocking the γc signal can successfully induce transplant tolerance in a murine model. Here we investigated the potential mechanism.

METHODSSplenocytes from C57BL/6 mice were transfused into T-cell deficient Balb/c nude mice that were reconstituted with syngeneic wild-type T-cells labeled with 5-carboxyfluorescein diacetate succinimidyl ester (CFSE). After 24 hours, recipients received i.p. injection of mixture of anti-γc mAbs, or with isotype control IgG2a. The labeled T-cells were harvested from recipient spleens after 12 and 48 hours. T-cell proliferation and apoptosis were detected by flow cytometry.

RESULTST-cell proliferation was markedly inhibited and apoptotic T cells could be detected at 12 hours after the mAbs injection. Proliferation was inhibited at 48 hours, but the proportion of apoptotic T-cells was not more than at 12 hours. In the control group, however, T-cells actively proliferated and no significant apoptosis was detected at either time point.

CONCLUSIONSThe results suggested that blockade of γc signals can synergize with donor splenocyte transfusion and lead to inhibition of antigen-specific T-cell proliferation and induction of apoptotic T-cell death. This protocol may develop a novel approach to induce donor-specific tolerance.

Animals ; Antibodies, Monoclonal ; pharmacology ; Apoptosis ; drug effects ; Cells, Cultured ; Flow Cytometry ; Fluoresceins ; Immune Tolerance ; drug effects ; Interleukin Receptor Common gamma Subunit ; antagonists & inhibitors ; metabolism ; Lymphocyte Activation ; drug effects ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Mice, Nude ; Signal Transduction ; drug effects ; Spleen ; cytology ; Succinimides ; T-Lymphocytes ; cytology ; drug effects