Epigenetic modification of histone 3 at lysine 9 in sheep zygotes and its relationship with DNA methylation.

BACKGROUND: Previous studies indicated that, unlike mouse zygotes, sheep zygotes lacked the paternal DNA demethylation event. Another epigenetic mark, histone modification, especially at lysine 9 of histone 3 (H3K9), has been suggested to be mechanically linked to DNA methylation. In mouse zygotes, the absence of methylated H3K9 from the paternal pronucleus has been thought to attribute to the paternal DNA demethylation. RESULTS: By using the immunofluorescence staining approach, we show that, despite the difference in DNA methylation, modification of H3K9 is similar between the sheep and mouse zygotes. In both species, H3K9 is hyperacetylated or hypomethylated in paternal pronucleus relative to maternal pronucleus. In fact, sheep zygotes can also undergo paternal DNA demethylation, although to a less extent than the mouse. Further examinations of individual zygotes by double immunostaining revealed that, the paternal levels of DNA methylation were not closely associated with that of H3K9 acetylation or tri-methylation. Treatment of either 5-azacytidine or Trichostatin A did not induce a significant decrease of paternal DNA methylation levels. CONCLUSION: Our results suggest that in sheep lower DNA demethylation of paternal genomes is not due to the H3K9 modification and the methylated DNA sustaining in paternal pronucleus does not come from DNA de novo methylation.

MAPK pathway mediates the protective effects of onychin on oxidative stress-induced apoptosis in ECV304 endothelial cells.

Our recent studies have shown that onychin could protect rabbit aortic rings from lysophosphatidylcholine-induced injury by preserving endothelium-dependent relaxation and alleviating acute endothelial damage mediated by oxidative stress. However, the effect of onychin on apoptosis of endothelial cells induced by oxidative stress was not evaluated. In the present study, we investigated the effect of onychin on Hydrogen Peroxide (H2O2) induced apoptosis of ECV304 endothelial cells. Cultured human umbilical vein endothelial cell line (ECV304) was pretreated with vehicle (DMSO), genistein, or different concentrations of onychin (0.1, 0.3, 1, 3, and 10 micromol/L) for 30 minutes and then exposed to 1 mmol/L H2O2 for 24 hours. Cell apoptosis was determined by TUNEL and flow cytometric analysis. Meanwhile, Western-blot was used to measure the expression of phospho-ERK1/2, phospho-p38 and caspase-3. Our data showed that onychin treatment exhibited a protective effect on ECV304 endothelial cells from H2O2-induced apoptosis in a concentration-dependent manner. Moreover, onychin attenuated H2O2-induced phosphorylation of p38MAPK and increased H2O2-induced phosphorylation of ERK1/2. Furthermore, onychin decreased the activation of caspase-3. The opposing effects of onychin on phosphorylation levels of p38MAPK and ERK1/2, and its caspase-3 inhibition might play a role in the beneficial effect of onychin on endothelial injury.

[Cyclosporin A inhibits the adhesion of neutrophil with ECV-304 induced by hypoxia/reoxygenation via ROS-Cyclophilin A-ERK1/2 pathway].

To investigate the inhibition of cyclosporin A (CsA) on neutrophil adhesion to human umbilical vein endothelial cells (HUVECs, ECV-304) induced by hypoxia/reoxygenation and further explore its mechanism, a 1 h hypoxia/4 h reoxygenation model was reproduced using ECV-304. The adhesion rate of neutrophils to ECV-304 was determined by measuring the activity of endogenous hexosaminidase. The expression of endothelial cell adhesion molecules of E-selectin and ICAM-1 was measured by flow cytometry. The expression of cyclophilin A (CyPA) and the activation of ERK1/2 was compared among experimental groups by Western blot. The content of reactive oxygen species (ROS) was measured by Fenton reaction. After being stimulated with 1 h hypoxia/4 h reoxygenation, ECV-304 showed an enhanced neutrophil adhensiveness in association with an increased surface expression of E-selectin and ICAM-1. In parallel, the content of ROS was also increased. These effects were significantly suppressed by the addition of CsA. Most importantly, the expression of CyPA was significantly increased following 1 h hypoxia/4 h reoxygenation, which was accompanied with an increased activation of ERK1/2. Treatment with CyPA inhibitor CsA and CyPA antisense oligonucleotides significantly inhibited the activation of ERK1/2 and decreased the adhesion of neutrophils to ECV-304. The specific ERK1/2 inhibitor PD98059 caused an inhibition of neutrophil adhesion to hypoxia/reoxygenation-stimulated ECV-304. Our data confirm that CsA inhibits neutrophil adhesion to hypoxia/reoxygenation stimulated ECV-304 by a mechanism involving inhibition of the signal transduction of ROS, CyPA and ERK1/2.

Anti-proliferative and pro-apoptotic effect of carvacrol on human hepatocellular carcinoma cell line HepG-2.

Carvacrol is one of the members of monoterpene phenol and is present in the volatile oils of Thymus vulgaris, Carum copticum, origanum and oregano. It is a safe food additive commonly used in our daily life, and few studies have indicated that carvacrol has anti-hepatocarcinogenic activities. The rationale of the study was to examine whether carvacrol affects apoptosis of human hepatoma HepG2 cells. In this study, we showed that carvacrol inhibited HepG2 cell growth by inducing apoptosis as evidenced by Hoechst 33258 stain and Flow cytometric (FCM) analysis. Incubation of HepG2 cells with carvacrol for 24 h induced apoptosis by the activation of caspase-3, cleavage of PARP and decreased Bcl-2 gene expression. These results demonstrated that a significant fraction of carvacrol treated cells died by an apoptotic pathway in HepG2 cells. Moreover, carvacrol selectively altered the phosphorylation state of members of the MAPK superfamily, decreasing phosphorylation of ERK1/2 significantly in a dose-dependent manner, and activated phosphorylation of p38 but not affecting JNK MAPK phosphorylation. These results suggest that carvacrol may induce apoptosis by direct activation of the mitochondrial pathway, and the mitogen-activated protein kinase pathway may play an important role in the antitumor effect of carvacrol. These results have identified, for the first time, the biological activity of carvacrol in HepG2 cells and should lead to further development of carvacrol for liver disease therapy.

Field evaluation of juvenile in vitro embryo transfer (JIVET) in sheep.

The practicality of using juvenile in vitro embryo transfer (JIVET) on a field scale in China was evaluated in each of three seasons (summer, autumn and winter) from 2006 to 2007. A total of 102 donor Merino lambs (18 summer, 69 autumn and 15 winter) aged 4-8 weeks were stimulated with 4 x 40 mg FSH administered at 12h intervals plus 400 IU PMSG given at the time of the first FSH treatment. Overall, 89.2% (91/102) of the lambs exhibited follicle development and 79.1+/-65.5 (mean+/-S.D.) cumulus-oocyte complexes were recovered per donor lamb. Compared with the groups of summer (84.9+/-55.3) and autumn (83.6+/-70.8) lambs, the number of recovered cumulus-oocyte complexes was significantly decreased in winter (51.4+/-43.7; p<0.05). After recovery, the cumulus-oocyte complexes were matured and fertilized in vitro using frozen-thawed semen and culture in synthetic oviduct fluid medium to the 2-4-c stage of development, when they were transferred surgically in groups of 3-8 (5.33+/-1.47) to the ipsilateral uterine horn of a total of 603 synchronized recipients. The overall mean proportion of cumulus-oocyte complexes developing to 2-c embryos was 61.4% (4308/7013) and differed significantly between seasons (summer 38.5%, autumn 66.1%, winter 74.6%; p<0.01). Pregnancy rate assessed by ultrasound examination approximately 60 days after embryo transfer was 54.4% (328/603) overall, and 36.7% (221/603) of the recipients maintained their pregnancy to full-term, producing an average 1.49 (330/221) offspring, of which 1.21 (267/221) were viable and healthy lambs, per pregnant recipient. Pregnancy rate at day 60 was affected by season (summer 40.5%, autumn 56.7%, winter 55.7%; p<0.05), but did not differ significantly between seasons at full-term (summer 34.2%, autumn 38.9%, winter 30.4%; p>0.05). Based on the number of donors stimulated, the total number of offspring and viable progeny produced per donor lamb in autumn (5.81 and 4.87) was significantly (p<0.01) higher than that of summer (2.79 and 1.94) and winter (4.24 and 3.31). This study showed that each donor lamb after stimulation produced an average of 48.6 transferable embryos that resulted in 4.04 viable and healthy progeny. These results indicate that JIVET is a cost-effective method of multiplying desirable sheep genotypes in China.

Identification of peptides inhibiting adhesion of monocytes to the injured vascular endothelial cells through phage-displaying screening.

Using oxidized low-density lipoprotein (LDL)-injured vascular endothelial cells (ECs) as target cells, peptides specifically binding to the injured ECs were screened from a phage-displaying peptide library by using the whole-cell screening technique after three cycles of the adsorption-elution-amplification procedure. Positive phage clones were identified by ELISA, and the inserted amino acid sequences in the displaying peptides were deduced from confirmation with DNA sequencing. The adhesion rate of ECs to monocytes was evaluated by cell counting. The activity of endothelial nitric oxide synthase (eNOS), and the expression levels of caveolin-1 and intercellular adhesion molecule-1 (ICAM-1) were determined by Western blotting. Six positive clones specifically binding to injured ECV304 endothelial cells were selected from fourteen clones. Interestingly, four phages had peptides with tandem leucine, and two of these even shared an identical sequence. Functional analysis demonstrated that the YCPRYVRRKLENELLVL peptide shared by two clones inhibited the expression of ICAM-1, increased nitric oxide concentration in the culture media, and upregulated the expression of caveolin-1 and eNOS. As a result, the adhesion rate of monocytes to ECV304 cells was significantly reduced by 12.1%. These data suggest that the anti-adhesion effect of these novel peptides is related to the regulation of the caveolin-1/nitric oxide signal transduction pathway, and could be of use in potential therapeutic agents against certain cardiovascular diseases initiated by vascular endothelial cell damage.

Serine 331 is major site of phosphorylation and desensitization induced by protein kinase C in thromboxane receptor alpha.

AIM: To identify the specific serine/threonine residues in the C-terminal tail of thromboxane receptor alpha (TPalpha) being phosphorylated and desensitized, and various alanine mutants of these serine/threonine residues were checked for their ability to serve as substrates. METHODS: To facilitate the identification of the intracellular domains involved in phosphorylation, glutathione S-transferase (GST)-intracellular domain fusion proteins were used as substrates for the purified PKC, and then the cDNA of phosphorylated protein was mutagenized to localize the major site of receptor phosphorylation induced by protein kinase C. Human embryonic kidney (HEK) 293 cells stably transfected with the His-tagged wild type or mutant TPalpha were used to study the phosphorylation and desensitization. RESULTS: Only the C-terminal tail can be used as a substrate for the purified PKC. Ser-331 (mP4) was demonstrated to be heavily phosphorylated, Ser-324 (mP1) was shown to be slightly phosphorylated, Ser-329 was illustrated to be faintly phosphorylated, and other Ser/Thr residues were not found to be phosphorylated. Phorbol-12-myristate-13-acetate (PMA) induced receptor phosphorylation in HEK 293 cells expressing the wild type TPalpha. However, PMA did not significantly trigger receptor phosphorylation in HEK 293 cells expressing the S331A mutant receptor. Pretreatment of the cells expressing the wild type with PMA inhibited I-BOP induced Ca2+ release, however, pretreatment of the cells expressing the S331A mutant receptor with PMA did not abolish I-BOP induced Ca2+ release. CONCLUSION: Ser-331 is the major and crucial site of receptor phosphorylation and desensitization.

CYP2D6, GST-M1 and GST-T1 enzymes: expression in parathyroid gland and association with the parathyroid hormone concentration during early renal replacement therapy.

AIMS: The purpose of this research was to characterize CYP2D6, GST-M1 and GST-T1 enzyme expression in human parathyroid tissue, and to determine whether or not there is any association between deficiencies in these enzymes and serum parathyroid hormone concentrations in patients with end-stage renal disease. METHODS: Surgical human parathyroid tissue was obtained and evaluated by immunohistochemistry for cellular localization of CYP2D6, GST-M1 and GST-T1 and colocalization of CYP2D6 with parathyroid hormone. Blood samples were collected from 328 Caucasian patients with end-stage renal disease for genetic testing of CYP2D6*3, *4, *5, *6, *7 and GST-M1*0 and GST-T1*0 alleles. Clinical chemistry data and serum intact parathyroid hormone (iPTH) concentrations were obtained from patient medical records. In 277 of the patients, the same laboratory performed all clinical tests. RESULTS: CYP2D6, GST-M1 and GST-T1 were present in human parathyroid tissue. CYP2D6 was colocalized with parathyroid hormone in parathyroid chief cells. Within the end-stage renal disease population, a nonfunctional CYP2D6 genotype was present in 18.2%[95% confidence interval (CI) 8.0, 28.4] of patients in the 1st iPTH concentration quintile (iPTH < 64 pg x mL(-1)), in 0% (95% CI 0, 7.5) of those in the 2nd quintile, in 1.8% (95% CI 0, 9.3) of those in the 3rd quintile, in 9.1% (95% CI 1.5, 16.7) of those in the 4th quintile, and in 16.7% (95% CI 6.8, 26.5) of those in the 5th quintile (iPTH > 347 pg x mL(-1)) (P = 0.001). Out of 12 CYP2D6-deficient females, seven were in the 1st iPTH concentration quintile and the remaining five were in the 5th quintile. Patients deficient in the GST-M1 and GST-T1 enzymes displayed a far more uniform frequency distribution relative to serum iPTH concentrations. CONCLUSIONS: The presence of CYP2D6, GST-M1 and GST-T1 in parathyroid cells was observed. An association is reported between a lack of CYP2D6 and iPTH concentrations in newly diagnosed end-stage renal disease patients. Gender and concomitant deficiency in GST-M1 and/or GST-T1 appear to define this association further. It remains to be established whether these associations reflect a cause-effect relationship between deficient expression of metabolizing enzymes and severity of secondary manifestation of renal failure.

CYP2C19 genotype and S-mephenytoin 4'-hydroxylation phenotype in a Chinese Dai population.

AIMS: To investigate the incidence of the CYP2C19 polymorphism in the Chinese Dai population. METHODS: One hundred and ninety-three healthy Chinese Dai volunteers were identified with respect to CYP2C19 by genotype and phenotype analyses. A polymerase chain reaction-restriction fragment length polymorphism method was performed for genotyping procedures. The 4'-hydroxymephenytoin (4'-OH-MP) and S/R-mephenytoin ( S/R-MP) excreted in the urine were determined by high-performance liquid chromatography and gas chromatography, respectively. RESULTS: Eighteen subjects were identified as poor metabolisers (PMs). The frequency of PMs in the Chinese Dai subjects was 9.3% (95% confidence interval 5.2, 13.4), which is lower than that in the Chinese Han population ( P<0.05). Chinese Dai subjects had a higher frequency of the mutant CYP2C19*2 allele (0.303) and a lower frequency of the mutant CYP2C19*3 allele (0.034). These two mutant alleles could explain all deficiencies of CYP2C19 activity in the Chinese Dai subjects. The frequency of the CYP2C19*3 allele is significantly lower than that in the Chinese Han population ( P<0.05). The mean S/R ratio was lower in the homozygous extensive metabolisers (EMs) compared with that in heterozygous EMs ( P<0.01), and the latter was lower than that in the PMs ( P<0.01). Furthermore, the mean S/R ratio in CYP2C19*3/ CYP2C19*2 heterozygous PMs was possibly lower than that in the CYP2C19*2/ CYP2C19*2 homozygous PMs ( P<0.05). CONCLUSION: The frequencies of PMs and CYP2C19*3 allele in the Chinese Dai population are significantly lower than those in the Han population. The CYP2C19 genotype analysis is largely consistent with the mephenytoin phenotype analysis. The variability of S/R ratios in EMs and PMs shows a gene-dosage effect.

Comparison of two CYP2D6 genotyping methods and assessment of genotype-phenotype relationships.

BACKGROUND: There have been no published reports comparing the CYP450 GeneChip microarray assay with more standard methods of genetic testing. METHODS: We collected 20-mL blood samples from 236 volunteers for DNA isolation and testing before each individual ingested 60 mg of dextromethorphan, and collected their urine. CYP2D6 alleles *3 to *7, *9, *17, and *41, and multiple CYP2D6 gene copies were tested by allele-specific PCR (AS-PCR), whereas alleles *2 to *4 and *6 to *11 were tested by the Affymetrix CYP450 GeneChip assay. Five of the CYP2D6 alleles (*3, *4, *6, *7, and *9) were tested by both AS-PCR and the CYP450 GeneChip assay in an independent and blinded fashion in 232 of the 236 healthy volunteers. The combined CYP2D6 genotype from both methods was used to divide the population into four subgroups, poor metabolizers (PMs), intermediate metabolizers (IMs), extensive metabolizers (EMs), and ultrarapid metabolizers (UMs), based on their relative function and ability to express the CYP2D6 gene. The urinary elimination of dextromethorphan was assessed in each of these CYP2D6 subgroups. RESULTS: The CYP2D6*3, *4, *6, *7, and *9 alleles showed a high degree of concordance between the CYP450 GeneChip and AS-PCR methods (>99% concordance). The mean (SD) of the log[dextromethorphan metabolic ratio (MR)] in the four CYP2D6 subgroups was PM = 0.49 (0.38); IM = -1.24 (0.53); EM = -2.35 (0.61); and UM = -2.43 (0.38). CONCLUSIONS: Oligonucleotide microarray technology is an efficient and reliable way to test for CYP2D6 gene variation based on five alleles compared by separate methods. The methodology is influenced by the quality and amount of DNA present. The log(dextromethorphan MR) is a highly variable index that appears to reflect the crude nature of the dextromethorphan MR as an indicator of CYP2D6 in vivo enzyme activity.