AIMTo confirm the stability of exogenous genes in the generation of transgenic chickens using ejaculated chicken sperm, the deoxyribonuclease (DNase) activity was evaluated in the seminal plasma of ejaculated semen and the stability of DNA was examined by adding lipofection reagents.

METHODSA PCR fragment (249 bp) of pEGFPN-1 vector was used as the DNA substrate and was incubated with the seminal plasma at 40 degree C for 30 min. Then, the whole reaction solution was subjected to agarose gel electrophoresis and the DNA size was evaluated under UV light.

RESULTSThe DNA substrate was completely diminished after incubation with seminal plasma. However, the substrate was intact after incubation with heat-treated seminal plasma or incubation with seminal plasma in the presence of 0.5 mmol/L approximately 5 mmol/L EDTA. The substrate was stabilized in the seminal plasma by the addition of commercially available lipofection reagents.

CONCLUSIONThe DNase activity is present in the seminal plasma of ejaculated chicken semen. However, DNA is stable in the liposomal-DNA complex.

Animals ; Cations, Divalent ; pharmacology ; Chickens ; physiology ; DNA ; analysis ; DNA Primers ; Deoxyribonucleases ; analysis ; Edetic Acid ; pharmacology ; Hot Temperature ; Indicators and Reagents ; Male ; Reverse Transcriptase Polymerase Chain Reaction ; Semen ; enzymology

AIMTo investigate whether estrogen was involved in relaxation of rabbit cavernous smooth muscle.

METHODSRelaxation response of the rabbit cavernous smooth muscles to 17beta-estradiol (0.3, 3, 30 and 300 nmol/L) were observed in vitro. The response of the muscle strips to estrogen after incubation with either actinomycin D (10 micromol/L) or L-NAME (10 micromol/L) were also evaluated. Inside-out mode of patch clamp in a single smooth muscle cell was applied to investigate the Maxi-K channel activities.

RESULTSEstrogen caused a dose-dependent relaxation of the strips precontracted with norepinephrine. The maximal response was noted about 10 minutes after treatment. The estrogen-induced relaxation was prevented by neither actinomycin D nor L-NAME, suggesting that the response was not mediated by gene transcription or nitric oxide (NO). Application of 17beta-estradiol increased the Maxi-K channel activities.

CONCLUSION17beta-estradiol may be involved in relaxation of rabbit cavernous smooth muscles via a non-genomic and NO independent mechanism. 17beta-estradiol stimulates Maxi-K channel of the rabbit cavernous myocyte.

Animals ; Dactinomycin ; pharmacology ; Dose-Response Relationship, Drug ; Electrophysiology ; Enzyme Inhibitors ; pharmacology ; Estradiol ; pharmacology ; In Vitro Techniques ; Indicators and Reagents ; Male ; Membrane Potentials ; drug effects ; Muscle Relaxation ; drug effects ; Muscle, Smooth ; drug effects ; NG-Nitroarginine Methyl Ester ; pharmacology ; Nitric Oxide Synthase ; antagonists & inhibitors ; Nitric Oxide Synthase Type III ; Norepinephrine ; pharmacology ; Patch-Clamp Techniques ; Penis ; drug effects ; Peptides ; pharmacology ; Potassium Channel Blockers ; pharmacology ; Rabbits ; Vasoconstrictor Agents ; pharmacology

Apoptosis is a programmed, physiologic mode of cell death that plays an important role in tissue homeostasis. As for the central nervous system, ischemic insults can induce pathophysiologic cascade of apoptosis in neurophils. Impairment of astroctye functions during brain ischemia can critically influence neuron survival by neuronglia interactions. We aimed to elucidate the protective effect of ketamine on apoptosis by energy deprivation in astrocytes. Ischemic insults was induced with iodoacetate/ carbonylcyanide mchlorophenylhydrazone (IAA/CCCP) 1.5 mM/ 20 micrometer or 150 micrometer/2 micrometer for 1 hr in the HTB-15 and CRL-1690 astrocytoma cells. Then these cells were reperfused with normal media or ketamine (0.1 mM) containing media for 1 hr or 24 hr. FITC-annexin-V staining and propidium iodide binding were determined by using flow cytometry. Cell size and granularity were measured by forward and side light scattering properties of flow cytometry system, respectively. An addition of keta-mine during reperfusion increased the proportion of viable cells. Ketamine alleviated cell shrinkage and increased granularity during the early period, and ameliorated cell swelling during the late reperfusion period. Ketamine may have a valuable effect on amelioration of early and late apoptosis in the astrocytoma cells, even though the exact mechanism remains to be verified.
Anesthetics, Dissociative/*pharmacology ; Annexin A5/pharmacology ; Apoptosis ; Astrocytes/metabolism ; Astrocytoma/*drug therapy/pathology ; Brain/pathology ; Carbonyl Cyanide m-Chlorophenyl Hydrazone/pharmacology ; Cell Line, Tumor ; Cell Size ; Cell Survival ; Central Nervous System/drug effects/pathology ; Enzyme Inhibitors/pharmacology ; Flow Cytometry/*methods ; Humans ; Indicators and Reagents/pharmacology ; Iodoacetates/pharmacology ; Ischemia/pathology ; Ketamine/metabolism/*pharmacology ; Light ; Neurons/metabolism/pathology ; Neutrophils/metabolism ; Perfusion ; Propidium/pharmacology ; Scattering, Radiation ; Time Factors ; Uncoupling Agents/pharmacology

OBJECTIVETo investigate the effect of RNA interference mediated angiopoietin-2 (ANG-2) gene silencing on human endometrial carcinoma cell line Ishikawa.

METHODSShort hairpin RNA (shRNA) targeting ANG-2 gene was designed and transfected into Ishikawa cells with Lipofectamine 2000. The mRNA and protein expression level of ANG-2, proliferation, morphological changes, apoptosis, cell cycle and invasive ability of the cells after transfection were analyzed.

RESULTSThe shRNA targeting the human ANG-2 gene effectively decreased the expression of ANG-2 on both mRNA and protein level, the proliferation inhibition rate of the Ishikawa cells was 63.11%, cell apoptosis was induced, and the cell cycle was arrested in G1 phase. The apoptotic rate of the Ishikawa cells in the transfected group was significantly higher, and the invasive ability was decreased markedly, than that of control groups.

CONCLUSIONThe shRNA targeting human ANG-2 gene could reduce ANG-2 expression, inhibit cellular growth and invasion in Ishikawa cells in vitro.

Angiopoietin-2 ; antagonists & inhibitors ; genetics ; Apoptosis ; drug effects ; genetics ; Cell Cycle ; drug effects ; genetics ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Endometrial Neoplasms ; genetics ; pathology ; Female ; Gene Expression ; drug effects ; Gene Expression Regulation, Neoplastic ; Gene Silencing ; drug effects ; physiology ; Humans ; Indicators and Reagents ; Lipids ; pharmacology ; RNA Interference ; RNA, Small Interfering ; genetics ; pharmacology ; Transfection ; Tumor Cells, Cultured

Nitric oxide (NO) is a non-adrenergic, non-cholinergic neurotransmitter found in the enteric nervous system that plays a role in a variety of enteropathies, including inflammatory bowel disease. Alteration of nitrergic neurons has been reported to be dependent on the manner by which inflammation is caused. However, this observed alteration has not been reported with acetic acid-induced colitis. Therefore, the purpose of the current study was to investigate changes in nitrergic neuromuscular transmission in experimental colitis in a rat model. Distal colitis was induced by intracolonic administration of 4% acetic acid in the rat. Animals were sacrificed at 4 h and 48 h postacetic acid treatment. Myeloperoxidase activity was significantly increased in the acetic acid-treated groups. However, the response to 60 mM KCl was not significantly different in the three groups studied. The amplitude of phasic contractions was increased by Nomega-nitro-L-arginine methyl ester (L-NAME) in the normal control group, but not in the acetic acid-treated groups. Spontaneous contractions disappeared during electrical field stimulation (EFS) in normal group. However, for the colitis groups, these contractions initially disappeared, and then reappeared during EFS. Moreover, the observed disappearance was diminished by L-NAME; this suggests that these responses were NO-mediated. In addition, the number of NADPH-diaphorase positive nerve cell bodies, in the myenteric plexus, was not altered in the distal colon; whereas the area of NADPH-diaphorase positive fibers, in the circular muscle layer, was decreased in the acetic acidtreated groups. These results suggest that NO-mediated inhibitory neural input, to the circular muscle, was decreased in the acetic acid-treated groups.
Acetic Acid/toxicity ; Animals ; Colitis/chemically induced/*pathology/*physiopathology ; Colon/drug effects/enzymology/*innervation/pathology ; Indicators and Reagents/toxicity ; Male ; Muscle Contraction/drug effects ; Muscle, Smooth/drug effects/metabolism ; Myenteric Plexus/pathology ; NADPH Dehydrogenase/metabolism ; NG-Nitroarginine Methyl Ester/pharmacology ; Neuromuscular Junction/drug effects/*metabolism ; Nitrergic Neurons/drug effects/*metabolism ; Nitric Oxide/*metabolism ; Peroxidase/metabolism ; Potassium Chloride/pharmacology ; Rats ; Rats, Sprague-Dawley