Protein kinase Cδ (PKCδ) involved in the regulation of pAkt1 (Ser473) on the release of mouse oocytes from diplotene arrest.

Defects in meiotic maturation may lead to chromosome segregation errors and genomic instability. Previous reports indicated that protein kinase C delta (PKCδ) may interact with microtubule organizing center-associated protein to play a role on meiotic spindle organization of mammalian oocytes. In this study, we explored the potential role of protein kinase B alpha (PKBα/Akt1), PKCδ, and their relationship on meiotic maturation of mouse oocytes. We examined the expression and localization of pAkt1 (Ser473) and/or pPKCδ (Thr505) under the treatment of SH-6 or Sotrastaurin. With the increasing of SH-6 concentrations, we observed that the protein levels of pAkt1 (Ser473) and the percentages of GVBD were decreased gradually. And the distribution of pAkt1 (Ser473), Cdc25B, pCdc2 (Tyr15), and α-tubulin around nucleus was also highly disordered under the treatment of 10-µM SH-6, a special inhibitor of pAkt1 (Ser473). In addition, the levels of pAkt1 (Ser473) were decreased with the treatment of Sotrastaurin, an inhibitor of PKCδ, suggesting that Akt1 may be one of the downstream targets of PKCδ. So, we deduced that PKCδ may be involved in regulating the release from diplotene arrest of mouse oocytes by controlling the levels of pAkt1 Ser473. SIGNIFICANCE OF THE STUDY: As a drug target, Akt is still the focus of research in somatic and fertilized eggs. Here, our data depicted a series of molecular events of pAkt1 (Ser473) and PKCδ via their special inhibitor and antibody in order to analyse their role and relationship on the release of mouse oocytes from diplotene arrest. Our results may offer important data for clinic research scientist to recognize the target role of pAkt1 (Ser473) via PKCδ in cell cycle regulation.

Developing a Novel Gene-Delivery Vector System Using the Recombinant Fusion Protein of Pseudomonas Exotoxin A and Hyperthermophilic Archaeal Histone HPhA.

Non-viral gene delivery system with many advantages has a great potential for the future of gene therapy. One inherent obstacle of such approach is the uptake by endocytosis into vesicular compartments. Receptor-mediated gene delivery method holds promise to overcome this obstacle. In this study, we developed a receptor-mediated gene delivery system based on a combination of the Pseudomonas exotoxin A (PE), which has a receptor binding and membrane translocation domain, and the hyperthermophilic archaeal histone (HPhA), which has the DNA binding ability. First, we constructed and expressed the rPE-HPhA fusion protein. We then examined the cytotoxicity and the DNA binding ability of rPE-HPhA. We further assessed the efficiency of transfection of the pEGF-C1 plasmid DNA to CHO cells by the rPE-HPhA system, in comparison to the cationic liposome method. The results showed that the transfection efficiency of rPE-HPhA was higher than that of cationic liposomes. In addition, the rPE-HPhA gene delivery system is non-specific to DNA sequence, topology or targeted cell type. Thus, the rPE-HPhA system can be used for delivering genes of interest into mammalian cells and has great potential to be applied for gene therapy.

Influence of the hypothalamic paraventricular nucleus (PVN) on heart rate variability (HRV) in rat hearts via electronic lesion.

Previous literatures have indicated that hypothalamic paraventricular nucleus (PVN) neurons are important for regulating the level of sympathetic and vagal nervous activity. Sympathovagal balance is closely related to heart rate variability (HRV). However, it still requires further elucidation regarding the effect of PVN on HRV by regulating sympathovagal balance. To detect the influence of the PVN on HRV, we evaluated the changes in time domain (including standard deviation of the R-R intervals (SDNN), and the root mean square of successive differences (RMSSD)) and frequency domain (including low frequency (LFnu), high frequency (HFnu) and the ratio of LF/HF) in HRV upon appropriate electronic stimulation, and lesions on the PVN of the rat in vivo. Cardiac vagal modulation was evaluated by HFnu; sympathetic modulation was evaluated by LFnu. Sympathovagal balance was evaluated by LF/HF and SDNN. Upon electronic stimulating (less than 0.6 mA) to the PVN of rats, we found that LFnu and HFnu changed correspondingly but recovered after the stimulation. When the PVN of the rats was injured, the RR intervals were enhanced with the rats' unilaterally or bilaterally injured PVN, especially the bilateral lesion. Meanwhile, LFnu, LF/HF and SDNN decreased gradually, accompanied with an increase of HFnu levels. So these PVN changes may indicate alterations of the sympathovagal balance.

Identification and detection of the isolated sinus venosus from the Asian toad.

The pacemaker activity of mammalian sinoatrial node (SAN) of the heart plays a fundamental role in the integration of vital functions. Studying factors such as drugs that influence pacemaker activity of SAN has its significance. In this study, we isolated sinus venosus, SAN from toads (Bufo gargarizans), and analysed its electronic signal, histological characteristics and the influence of acetylcholine (ACh) and ivabradine on its pacemaker activity using PowerLab® and Chart® 5.0 software. We found that when isolated sinus venosus was treated with ACh, its histological distribution was disorganized and inter-beat (RR) interval was also broadened. The high frequency normalized unit (HFnu) and Poincaré plot of heart rate variability (HRV) of the isolated sinus venosus was also altered upon ACh treatment in a time-dependent and dose-dependent manner. When treated with ivabradine, these parameters of HRV such as square root of the mean of the squared differences between adjacent NN intervals (RMSSD) and HFnu were in the upward tendency, but low frequency normalized unit and low frequency/high frequency were in the opposite tendency. Taken together, we have developed a new model for studying the influences of drugs on autorhythmicity using isolated sinus venosus of the toad. With this model, we showed that ACh and ivabradine may affect the pacemaker activity by stimulating muscarinic receptor or inhibiting If current, respectively.

[Effect of vagal nerve stimulation on heart rate and heart rate variability in the toads].

OBJECTIVE: To observe the effect of vagal stimulation of different frequencies on heart rate and heart rate variability in the toads. METHODS: The animals were prepared to be models of isolated heart with right vagosympathetic trunks, then the nerves stimulated by different frequencies. Collected the electrocardiogram of isolated hearts and analyzed the data with HRV software. RESULTS: Since the effect of sympathetic nerve had been diminished, vagal stimulation resulted in HR being markedly attenuated (P < 0.01), SDNN and RMSSD being significantly increased (P < 0.01). There was no significant difference in different levels of frequency. Contrast to group control, HR,SDNN and RMSSD each changed clearly. At the frequency of 0.2 Hz, HF increased markedly (P < 0.01), and LF/HF decreased obviously (P < 0.05) in group P + P. While at the frequency of 0.8 Hz, HF and LF/HF were both around the level of pre-stimulation. CONCLUSION: These findings suggest that in the certain electrical stimulation frequency range, the effect of vagal on HR increases along with the frequency. Without the effect of sympathetic nerve, the modulation of vagal to HR and HRV may have different ways.