ABSTRACT
Background and purpose:Studies have proved that the serine/threonine kinases 31 (STK31) gene plays important roles in human cancers. TheSTK31 gene expression was demonstrated to be regulated by the methylation status of its promoter/exon1 region. Viral infection was revealed to be associated with the hypermethylation of some tumor suppressor genes in some tumor samples. The purposes of this paper were to study the roles ofHPV16E6,E7, orE6/E7 oncogenes in methylation status and expression of theSTK31 gene, and potential effects of DNA methyltransferases (DNMTs) onSTK31 gene methylation status.Methods:Ectopically-expressed HPV16 E6, E7, or E6/E7 cells were estab-lished by transfectingHPV16E6,E7, orE6/E7 oncogenes with lentivirus vectors into HPV-negative cervical cancer cell lines HT-3 and C33A. Bisulfite genomic sequencing PCR (BGS) combined with TA clone and MSP (methylation-specific PCR) were used to analyze methylation status of theSTK31 gene promoter/exon1 region in HPV-positive cervical cancer cell lines (HeLa, SiHa, CaSki), HPV-negative cervical carcinoma cell lines (C33A, HT-3) and the transfected cells. The mRNA and protein expression of STK31, DNMT1, DNMT2, DNMT3a, DNMT3b and DNMT3L were detected by RT-PCR and Western blot.Results:Transfection efficiency was tested by Western blot, which showed that the transfected cells successfully expressed E6, E7, or E6/E7 proteins, respectively. TheSTK31 gene promoter/exon1 was hypomethylated in HPV-positive cell lines HeLa, SiHa and CasKi resulting in detection of mRNA and protein expression.STK31 gene promoter/exon1 showed hypermethylation leading to silenced expression in the two HPV-negative cervical cancer cells HT-3 and C33A. Compared with primary HT-3 and C33A cells, the methylation status ofSTK31 promoter/exon1 was down-regulated that led to expression of STK31 in the ectopically-expressed HPV16 E7 and E6/E7 cells. Expressions of DNMT1,DNMT3a andDNMT3b genes at the level of transcription were higher in C33AE6/E7 and HT-3E6/E7 cells than those in C33A-vector and HT-3 vector cells, respectively (P0.05, data not shown).Conclusion:HPV infection leads to the down-regulated methylation status ofSTK31 promoter/exon1 that results in the expression of STK31.STK31 gene expression is regulated by methylation status of its promoter/exon1 region. HPV16E7 andE6/E7 oncogenes may influence the methylation status ofSTK31 gene promoter/exon1 region by regulating the expression of DNMT2.
ABSTRACT
Picrotoxin is an anatagninst of gamma-aminobutyric acid, which is an internal inhibition-transmitter in the central nervous system, Picrotoxin exerts a biphasic action on the blood pressure and heart rate in rats and cats in vivo. That is to say, in the initial stage, picrotoxin can lower the blood pressure and heart rate, and then an elevation of these two even above the original level can be observed, up to the present, from the authors limited literature, there has been no report dealing with the problem whether picrotoxin can act on an isolated heart directly.In this study, the heart of a frog was isolated and routine intubation of the heart was done for its perfusion. Physiological polyconduction instrument was inserted through a mechanical transducer to record the heart rale and myocardial contractility. A suspending glass microelectrode coupling with a microamplifier is used to record the action potential of the ventricular myocardium. Real time analysis of all the data was accomplished with a microcomputer. The dosages of picrotoxin used were 1,5, 3.0, 6.0, and 12.0 mg per kilogram of body weight.It was found that picrotcxin can directly act on the isolated frog heart. The results were as follows.1 ) Picrotoxin exerts inhibition on the special conduction system of the heart,and the A-V node and venous sinus are very sensitive. Complete or partial transmission block can be induced.2 ) It can elicit clearly a fall of the heart rate but no biphasic action can berevealed. 3) It can reduce the myocardial contractility, suggesting that the calciuminflow during the functioning period of the action potential is effected. 4 ) It can reduce the amplitude of the action potential but no effect on themaximal depolarization speed is observed, suggesting that picrotoxin islikely to affect the level of resting potential but not the action potentialin the depolarized period.
ABSTRACT
The effects of intravcntricular administration of ?-amino - butyric acid (GABA) and its antagonist picrotoxin(PTX) on the discharges of dorsal hippocampal nocieeptive neurons are investigated The noxious discharge of 28 (67%) nociccptive exciting neurons (NENS) are depressed (p