DNA binding compounds were previously shown to bind to the right-handed DNA forms and hybrid B-Z forms in a highly cooperative manner and indicate that structural specificity plays a key role in a ligand binding to DNA. In this study, the modes of binding and structural specificity of agents to unusual DNA are examined by a variety of fluorescence techniques (intensity, polarization and quenching, etc.) to explore a reliable method to detect the association environment of ligands to deoxyoligonucleotides initially containing a B-Z junction between the left-handed Z-DNA and right-handed B-DNA. The results of fluorescence energy transfer measurement demonstrated that the ligand molecules bind to the allosterically converted DNA structures by intercalation. In the absence of high-resolution structural data, this fluorescence energy transfer measurement allowed reliable measures and infer the binding environment of ligands to the allosteric DNA structures.
Allosteric Regulation ; Circular Dichroism ; DNA/*chemistry/*metabolism ; Energy Transfer ; Ethidium/*metabolism ; Exodeoxyribonucleases/metabolism ; Ligands ; Motion ; Nucleic Acid Conformation ; Oligodeoxyribonucleotides/*metabolism

OBJECTIVEthe main purpose of this study is to develop an assay for detection of skin viability in situ.

METHODSTwo nucleic acid dyes, SYTO 13 and ethidium bromide (EB), are used to assess the viability of skin tissue. The viability of cells can be determined by different colors under fluorescence microscope. In this study, the recovery of skin restored at 4 degrees C for different periods is measured by SYTO/EB dyes and a type of indirect assessment, WST-1. Then both results are compared.

RESULTSThe results measured by two methods showed that the viability of skin tissue was nearly 30% after two weeks storage. At any point of experiment, the data from two methods have not obvious variance.

CONCLUSIONThe assessment of skin viability by SYTO/EB is also quick, sensitive and convenient. At the same time the viability of any type of cells can be observed in situ. So it can be considered as an available method for detection of skin viability.

Animals ; Cell Survival ; physiology ; Cryopreservation ; Ethidium ; Male ; Models, Animal ; Rats ; Skin ; cytology ; metabolism ; Skin Physiological Phenomena ; Tissue Preservation ; methods

The uptake of glucose for metabolism and growth is essential to most animal cells and is mediated by glucose-transporter (GLUT) proteins. The aim of this study was to determine which class of glucose transporter molecules was responsible for uptake of glucose in the mouse early embryo and at which stage the corresponding genes were expressed. In addition, co-culture system with vero cell was used to investigate the effect of the system on GLUT expression. Two-cell stage embryos were collected from the superovulated ICR female and divided into 3 groups. As a control, embryos were cultured in 0.4% BSA-T6 medium which includes glucose. For the experimental groups, embryos were cultured in either co-culture system with vero cells or glucose-free 76 medium supplemented with 0.4% BSA and pyruvate as an energy substrate. 2-cell to blastocyst stage embryos in those groups were respectively collected into microtubes (50 embryos/tube). Total RNA was extracted and RT-PCR was performed. The products were analysed after staining ethidium bromide by 2% agarose gel electrophoresis. Blastocysts were collected from each group at 120hr after hCG injection. They were fixed in 2.5% glutaraldehyde, stained with hoechst, and mounted for observation. In control, GLUT1 was expressed from 4-cell to blastocyst. GLUT2 and GLUT3 were expressed in morula and blastocyst. GLUT4 was expressed in all stages. When embryos were cultured in glucose-free medium, no significant difference was shown in the expression of GLUTI1, 2 and 3, compared to control. However GLUT4 was not expressed until morular stage. When embryos were co-cultured with vero cell, there was no significant difference in the expression of GLUT1, 2, 3 and 4 compared to control. To determine cell growth of embryos, the average cell number of blastocyst was counted. The cell number of co-culture (93.8+/-3.1, n=35) is significantly higher than that of control and glucose-free group (76.6 +/- 3.8, n=35 and 68.2+/-4.3, n=30). This study shows that the GLUT genes are expressed differently according to embryo stage. GLUTs were detectable throughout mouse preimplantation development in control and co-culture groups. However, GLUT4 was not detected from 2- to 8-cell stage but detected from morula stage in glucose-free medium, suggested that GLUT genes are expressed autocrinally in the embryo regardless of the presence of glucose as an energy substrate. In addition, co-culture system can increase the cell count of blastocyst but not improve the expression of GLUT. In conclusion, expression of GLUT is dependent on embryo stage in preimplantation embryo development.
Animals ; Blastocyst ; Cell Count ; Coculture Techniques ; Electrophoresis, Agar Gel ; Embryonic Development ; Embryonic Structures* ; Ethidium ; Female ; Glucose Transport Proteins, Facilitative* ; Glucose* ; Glutaral ; Humans ; Metabolism ; Mice* ; Morula ; Pregnancy ; Pyruvic Acid ; RNA ; Vero Cells

OBJECTIVETo observe the inhibition of flavonoids extract (FE) from Inula britannica on oxidative stress in rat aorta after balloon injury.

METHODThe model of vascular intimal hyperplasia was established by balloon injury. The rats were randomly divided into four groups: control, model, FE and captopril (CAP, positive control). The FE group was treated by intragastric administration with FE in dose of 12.5, 25, 50 mg x kg(-1) x d(-1). The level of malondialdehyde (MDA) and superoxide dismutase (SOD) in serum were detected by thiobarbituric acid (TAB) method and xanthine oxidase method respectively, and superoxide anion (O2-) in vessel was detected by dihydroethidium (DHE) staining. The histochemistry, immunohistochemistry and Western blot were used to observe the changes of appearance and SOD expression in vascular tissues after balloon injury.

RESULTThe best concentration of FE to rats was 50 mg x kg(-1) x d(-1). The neointima thickness in the model group was significantly higher than that in the FE group (50 mg x kg(-1) x d(-1)) and control group at 14 days after balloon injury (P < 0.01). The lever of MDA in serum of FE group was decreased (P < 0.01) and SOD was increased (P < 0.05) in both serum and vascular tissues. The level of O2*- in the drug group was lower than that in the model group.

CONCLUSIONFE can enhance the antioxidation capacities of vessel tissues by suppressing the formation of O2- induced by injury, by which FE inhibites neointima formation after balloon injury in rat.

Animals ; Antioxidants ; administration & dosage ; chemistry ; Aorta ; drug effects ; metabolism ; Blood Vessels ; drug effects ; metabolism ; Catheterization ; adverse effects ; Down-Regulation ; Ethidium ; analogs & derivatives ; blood ; metabolism ; Flavonoids ; administration & dosage ; chemistry ; Inula ; chemistry ; Male ; Oxidative Stress ; drug effects ; Random Allocation ; Rats ; Superoxide Dismutase ; blood ; metabolism

AIMTo determine the cellular distribution of secretory phospholipase A(2) (sPLA(2)) in dependence on the acrosomal state and under the action of elastase released under inflammatory processes from leukocytes.

METHODSAcrosome reaction of spermatozoa was triggered by calcimycin. Human leukocyte elastase was used to simulate inflammatory conditions. To visualize the distribution of sPLA(2) and to determine the acrosomal state, immunofluorescence techniques and lectin binding combined with confocal laser scanning fluorescence microscopy and flow cytometry were used.

RESULTSAlthough sPLA(2) was detected at the acrosome and tail regions in intact spermatozoa, it disappeared from the head region after triggering the acrosome reaction. This release of sPLA(2) was associated with enhanced binding of annexin V-fluoroscein isothiocyanate (FITC) to spermatozoa surfaces, intercalation of ethidium-homodimer I, and binding of FITC-labelled concanavalin A at the acrosomal region. Spermatozoa from healthy subjects treated with elastase were characterized by release of sPLA(2), disturbance of acrosome structure, and loss of vitality.

CONCLUSIONThe ability of spermatozoa to release secretory phospholipase A(2) is related to the acrosomal state. Premature destabilization of the acrosome and loss of sPLA(2) can occur during silent inflammations in the male genital tract. The distribution pattern of sPLA(2) in intact spermatozoa might be an additional parameter for evaluating sperm quality.

Acrosome ; drug effects ; physiology ; Acrosome Reaction ; drug effects ; Annexin A5 ; metabolism ; Anti-Bacterial Agents ; pharmacology ; Calcimycin ; pharmacology ; Ethidium ; Flow Cytometry ; Fluorescent Dyes ; Humans ; In Vitro Techniques ; Male ; Microscopy, Confocal ; Pancreatic Elastase ; metabolism ; Phosphatidylserines ; metabolism ; Phospholipases A2, Secretory ; metabolism ; Semen ; cytology ; drug effects ; Spermatozoa ; drug effects ; enzymology