To evaluate the effects of pesticides to parasite eggs, Ascaris suum eggs were incubated with 5 different pesticides (1:1,500-1:2,000 dilutions of 2% emamectin benzoate, 5% spinetoram, 5% indoxacarb, 1% deltamethrin, and 5% flufenoxuron; all v/v) at 20degrees C for 6 weeks, and microscopically evaluated the egg survival and development on a weekly basis. The survival rate of A. suum eggs incubated in normal saline (control eggs) was 90+/-3% at 6 weeks. However, the survival rates of eggs treated with pesticides were 75-85% at this time, thus significantly lower than the control value. Larval development in control eggs commenced at 3 weeks, and 73+/-3% of eggs had internal larvae at 6 weeks. Larvae were evident in pesticide-treated eggs at 3-4 weeks, and the proportions of eggs carrying larvae at 6 weeks (36+/-3%-54+/-3%) were significantly lower than that of the control group. Thus, pesticides tested at levels similar to those used in agricultural practices exhibited low-level ovicidal activity and delayed embryogenesis of A. suum eggs, although some differences were evident among the tested pesticides.
Animals ; Ascaris suum/*drug effects/growth & development ; Female ; Larva/drug effects/growth & development ; Microscopy ; Pesticides/*pharmacology ; Survival Analysis ; Temperature ; Time ; Zygote/*drug effects/growth & development

To further test whether protein kinase A (PKA) can affect the mitotic cell cycle, one-cell stage mouse embryos at S phase (22 h after hCG injection) were incubated in M16 medium containing various concentrations of H-89, a PKA inhibitor. With increasing concentrations of H-89 (0-50 μmol/L), the G(2) phase of eggs was decreased and the cleavage rate was accelerated. A concentration of 40 μmol/L H-89 led to all of the mouse eggs entering the M phase of mitosis. Furthermore, to study the role of PKA in regulating the phosphorylation status of S149 and S321 sites of cell division cycle 25B (CDC25B) on one-cell stage fertilized mouse eggs, pBSK-CDC25B-WT, pBSK-CDC25B-S149A, pBSK-CDC25B-S321A and pBSK-CDC25B-S149A/S321A were transcribed into mRNAs in vitro, then mRNAs were microinjected into S phase of mouse fertilized eggs and cultured in M16 medium pretreated with H-89. Then, the cleavage of fertilized eggs, maturation promoting factor (MPF) activity and phosphorylation status of CDC2-Tyr15 were observed. In the presence of 40 μmol/L H-89, the cleavage rate of fertilized eggs in CDC25B-S/A-mRNAs and CDC25B-WT-mRNA injected groups was significantly higher than that in the control groups, and the peak of MPF activity appeared in the CDC25B-S/A-mRNAs and CDC25B-WT-mRNA injected groups earlier than that in the control groups. CDC2-Tyr15 phosphorylation state was consistent with MPF activity. In conclusion, the present study suggests that PKA regulates the early development of mouse embryos by phosphorylation of S149 and S321 of CDC25B, which plays an important role in the regulation of G(2)/M transition in the mitotic cell cycle of fertilized mouse eggs.
Animals ; Cyclic AMP-Dependent Protein Kinases ; genetics ; physiology ; Embryonic Development ; physiology ; Female ; Male ; Mice ; Microinjections ; Mitosis ; drug effects ; Phosphorylation ; Serine ; genetics ; metabolism ; Zygote ; cytology ; growth & development ; cdc25 Phosphatases ; genetics ; metabolism

OBJECTIVETo evaluate the inhibitory effects of HCV antisense oligonucleotide drugs in vivo.

METHODSTransgenic mice were generated by microinjection. The construct of luciferase controlled by HCV 5'NCR that contains CMV promotor was injected into the male pronuclus of fertilized eggs of ICR mice.

RESULTSSixty-eight survival birth transgenic mice were identified by PCR amplification with tail DNA, 13 of whom were positive with an integration ratio of 19.2% (13/68). Transgenic mRNA was detected by RT-PCR in the tissue of three mice's offspring that contain transgenic DNA. Luciferase expression was detected in a line (35#) using the luciferase assay system and the expression persisted in the F2 generation. The phenotype of the mice in this line was normal and there was no significant difference in physiology from normal mice.

CONCLUSIONSThis line of transgenic mice will provide a useful animal model for the study of function of HCV 5'NCR and the evaluation of HCV antisense drugs in vivo.

Animals ; Artificial Gene Fusion ; methods ; Drug Evaluation ; Hepacivirus ; drug effects ; genetics ; Luciferases ; biosynthesis ; genetics ; Mice ; Mice, Inbred ICR ; Mice, Transgenic ; Microinjections ; methods ; Models, Animal ; Promoter Regions, Genetic ; genetics ; RNA, Antisense ; pharmacology ; RNA, Messenger ; biosynthesis ; Reverse Transcriptase Polymerase Chain Reaction ; Zygote ; cytology ; growth & development