Chi-Hun Park

Eugenol inhibits calcium currents in dental afferent neurons.

Eugenol is a topical analgesic agent widely used in the dental clinic. To elucidate the molecular mechanism underlying its analgesic action, we investigated the effect of eugenol on high-voltage-activated calcium channel (HVACC) currents in dental primary afferent neurons, and with a heterologous expression system. Dental primary afferent neurons were identified by retrograde labeling with a fluorescent dye, DiI. Eugenol inhibited HVACC currents in both capsaicin-sensitive and capsaicin-insensitive dental primary afferent neurons. The HVACC inhibition by eugenol was not blocked by capsazepine, a competitive transient receptor potential vanilloid 1 (TRPV1) antagonist. Eugenol inhibited N-type calcium currents in the cell line C2D7, stably expressing the human N-type calcium channels, where TRPV1 was not endogenously expressed. Our results suggest that the HVACC inhibition by eugenol in dental primary afferent neurons, which is not mediated by TRPV1 activation, might contribute to eugenol’s analgesic effect. Abbreviations: high-voltage-activated calcium channel, HVACC; transient receptor potential vanilloid 1, TRPV1; trigeminal ganglion, TG; dorsal root ganglion, DRG; capsazepine, CZP.

Methylation status of differentially methylated regions at Igf2/H19 locus in porcine gametes and preimplantation embryos

The aim of this study was to demonstrate how differential methylation imprints are established during porcine preimplantation embryo development. For the methylation analysis, the primers for the three Igf2/H19 DMRs were designed and based upon previously published sequences. The methylation marks of Igf2/H19 DMRs were analysed in sperm and MII oocytes with our results showing that these regions are fully methylated in sperm but remain unmethylated in MII oocytes. In order to identify the methylation pattern at the pronuclear stage, we indirectly compared the methylation profile of Igf2/H19 DMR3 in each zygote derived by in vitro fertilization, parthenogenesis, and androgenesis. Interestingly, this region was found to be differently methylated according to parental origins; DMR3 was hemimethylated in in vitro fertilized zygotes, fully methylated in parthenogenetic zygotes, and demethylated in androgenetic zygotes. These results indicate that the methylation mark of the paternal allele is erased by active demethylation, and that of the maternal one is de novo methylated. We further examined the methylation imprints of Igf2/H19 DMR3 during early embryonic development. The hemimethylated pattern as seen in zygotes fertilized in vitro was observed up to the 4-cell embryo stage. However, this mark was exclusively demethylated at the 8-cell stage and then restored at the morula stage. These results suggest that methylation imprints are established via dynamic changes during early embryonic development in porcine embryos.

Analysis of Imprinted Gene Expression in Normal Fertilized and Uniparental Preimplantation Porcine Embryos

In the present study quantitative real-time PCR was used to determine the expression status of eight imprinted genes (GRB10, H19, IGF2R, XIST, IGF2, NNAT, PEG1 and PEG10) during preimplantation development, in normal fertilized and uniparental porcine embryos. The results demonstrated that, in all observed embryo samples, a non imprinted gene expression pattern up to the 16-cell stage of development was common for most genes. This was true for all classes of embryo, regardless of parental-origins and the direction of imprint. However, several differentially expressed genes (H19, IGF2, XIST and PEG10) were detected amongst the classes at the blastocyst stage of development. Most interestingly and despite the fact that maternally and paternally expressed genes should not be expressed in androgenones and parthenogenones, respectively, both uniparental embryos expressed these genes when tested for in this study. In order to account for this phenomenon, we compared the expression patterns of eight imprinted genes along with the methylation status of the IGF2/H19 DMR3 in haploid and diploid parthenogenetic embryos. Our findings revealed that IGF2, NNAT and PEG10 were silenced in haploid but not diploid parthenogenetic blastocysts and differential methylation of the IGF2/H19 DMR3 was consistently observed between haploid and diploid parthenogenetic blastocysts. These results appear to suggest that there exists a process to adjust the expression status of imprinted genes in diploid parthenogenetic embryos and that this phenomenon may be associated with altered methylation at an imprinting control region. In addition we believe that imprinted expression occurs in at least four genes, namely H19, IGF2, XIST and PEG10 in porcine blastocyst stage embryos.

Eugenol Inhibits K+ Currents in Trigeminal Ganglion Neurons

Eugenol, a natural capsaicin congener, is widely used in dentistry. Eugenol inhibits voltage-activated Na+ and Ca2+ channels in a transient receptor potential vanilloid 1 (TRPV1)-independent manner. We hypothesized that eugenol also inhibits voltage-gated K+ currents, and investigated this in rat trigeminal ganglion neurons and in a heterologous system using whole-cell patch clamping. Eugenol inhibited voltage-gated K+ currents, and the inhibitory effects of eugenol were observed in both capsaicin-sensitive and capsaicin-insensitive neurons. Pre-treatment with capsazepine, a well-known antagonist of TRPV1, failed to block the inhibitory effects of eugenol on K+ currents, suggesting no involvement of TRPV1. Eugenol inhibited human Kv1.5 currents stably expressed in Ltk − cells, where TRPV1 is not endogenously expressed. We conclude that eugenol inhibits voltage-gated K+ currents in a TRPV1-independent manner. The inhibition of voltage-gated K+ currents is likely to contribute to the irritable action of eugenol. Abbreviations: human Kv1.5 channel, hKv1.5; transient receptor potential vanilloid 1, TRPV1.

Substance P Sensitizes P2X3 in Nociceptive Trigeminal Neurons

Peripheral inflammation produces pain hypersensitivity by sensitizing nociceptors. Potentiation of P2X3 receptor activity in nociceptors may play an important role in this peripheral sensitization. However, we do not fully understand how P2X3 activity is elevated in inflammation. Thus, we investigated whether P2X3 activity in trigeminal nociceptive neurons is regulated by the neurokinin-1 (NK-1) receptor that is activated by an inflammatory mediator, substance P. Single-cell RT-PCR and immunohistochemistry revealed that NK-1 in nociceptive neurons was mainly co-expressed with P2X3. Ca2+ imaging and whole-cell patch-clamp recordings indicated that both substance P and Sar-substance P, a selective NK-1 agonist, significantly potentiated α,β-meATP-induced currents and [Ca2+]i responses in nociceptive neurons. These potentiating effects were completely blocked by GR82334, a specific NK-1 antagonist. Our results demonstrate that substance P sensitizes P2X3 receptor through the activation of NK-1, thus warranting these receptors as possible targets for pain therapy in the orofacial region. Abbreviations: α,β-methylene adenosine 5′-triphosphate (ATP), α,β-meATP; neurokinin-1, NK-1; single-cell reverse-transcription polymerase chain-reaction, single-cell RT-PCR; [Sar9,Met(O2)11]-substance P, Sar-substance P.

Production of Multiple Transgenic Yucatan Miniature Pigs Expressing Human Complement Regulatory Factors, Human CD55, CD59, and H-Transferase Genes

The present study was conducted to generate transgenic pigs coexpressing human CD55, CD59, and H-transferase (HT) using an IRES-mediated polycistronic vector. The study focused on hyperacute rejection (HAR) when considering clinical xenotransplantation as an alternative source for human organ transplants. In total, 35 transgenic cloned piglets were produced by somatic cell nuclear transfer (SCNT) and were confirmed for genomic integration of the transgenes from umbilical cord samples by PCR analysis. Eighteen swine umbilical vein endothelial cells (SUVEC) were isolated from umbilical cord veins freshly obtained from the piglets. We observed a higher expression of transgenes in the transgenic SUVEC (Tg SUVEC) compared with the human umbilical vein endothelial cells (HUVEC). Among these genes, HT and hCD59 were expressed at a higher level in the tested Tg organs compared with non-Tg control organs, but there was no difference in hCD55 expression between them. The transgenes in various organs of the Tg clones revealed organ-specific and spatial expression patterns. Using from 0 to 50% human serum solutions, we performed human complement-mediated cytolysis assays. The results showed that, overall, the Tg SUVEC tested had greater survival rates than did the non-Tg SUVEC, and the Tg SUVEC with higher HT expression levels tended to have more down-regulated α-Gal epitope expression, resulting in greater protection against cytotoxicity. By contrast, several Tg SUVEC with low CD55 expression exhibited a decreased resistance response to cytolysis. These results indicated that the levels of HT expression were inversely correlated with the levels of α-Gal epitope expression and that the combined expression of hCD55, hCD59, and HT proteins in SUVECs markedly enhances a protective response to human serum-mediated cytolysis. Taken together, these results suggest that combining a polycistronic vector system with SCNT methods provides a fast and efficient alternative for the generation of transgenic large animals with multiple genetic modifications.

Control of nuclear remodelling and subsequent in vitro development and methylation status of porcine nuclear transfer embryos

We attempted to control the nuclear remodelling of somatic cell nuclear transfer embryos (NTs) and examined their subsequent development and DNA methylation patterns in pigs. Porcine foetal fibroblasts were fused to enucleated oocytes treated with either 5 mM caffeine for 2.5 h or 0.5 mM vanadate for 0.5 h. After activation, NTs were cultured in vitro for 6 days to examine their development. The nuclear remodelling type of the reconstituted embryos was evaluated 1 h after fusion. Methylated DNA of in vitro-fertilised (IVF) embryos and NTs at various developmental stages and of donor cells was detected using a 5-methylcytosine (5-MeC) antibody. Caffeine-treated NTs induced premature chromosome condensation at a high rate (P<0.05), whereas most vanadate-treated NTs formed a pronucleus-like structure. Although cleavage rates to the two-cell stage did not differ among groups, delayed cleavage was observed in the vanadate-treated group. The blastocyst formation rate was significantly reduced by vanadate treatment compared with caffeine-treated and non-treated (control) NT groups (P<0.05). The apoptotic cell index of NT blastocysts was lower in the caffeine-treated group than in other groups (P<0.05). The methylation patterns were similar among NTs, but more hypermethylated DNA was observed at the four-cell stage of control and vanadate-treated NTs when compared with that in IVF embryos (P<0.05). Thus, the nuclear remodelling type controlled by caffeine or vanadate treatment can affect in vitro development and the methylation status of NTs in relation to nuclear reprogramming.

A modified swim-up method reduces polyspermy during in vitro fertilization of porcine oocytes

The general method of porcine in vitro fertilization (IVF), involving the co-culture of both gametes in a medium drop, is thought to be the main reason for the high incidence of polyspermy. The aim of this study was to reduce the polyspermic fertilization of porcine embryos during IVF by the modified swim-up method, based on general sperm swim-up technique. Within this design, a 70 microm pore sized cell strainer was used to separate the sperm pellet placed at the bottom of a tube from the mature oocytes placed within the upper region. The separation of gametes using this permeable barrier was to ensure that only motile sperm gained access to the oocytes. It was found that the rate of polyspermy was significantly lowered for the sperm preparations from three boar breeds in modified swim-up method when compared with that of the general microdrop method (p<0.05). However, the penetration rates were found to be similar in both methods for two boar breeds. The average occurrence of blastocysts with more total cell number was higher in the modified swim-up method, while no significant difference in blastocyst rates between the two IVF methods was observed. The frequency of normal diploid embryos was also significantly higher in the modified swim-up method and polyploidy was more frequently observed in microdrop method (p<0.05). Our results demonstrated that the modified swim-up IVF method could reduce polyspermic penetration, and consequently produce better quality and karyotypically normal embryos in porcine IVF.

X-linked gene transcription patterns in female and male in vivo, in vitro and cloned porcine individual blastocysts.

To determine the presence of sexual dimorphic transcription and how in vitro culture environments influence X-linked gene transcription patterns in preimplantation embryos, we analyzed mRNA expression levels in in vivo-derived, in vitro-fertilized (IVF), and cloned porcine blastocysts. Our results clearly show that sex-biased expression occurred between female and male in vivo blastocysts in X-linked genes. The expression levels of XIST, G6PD, HPRT1, PGK1, and BEX1 were significantly higher in female than in male blastocysts, but ZXDA displayed higher levels in male than in female blastocysts. Although we found aberrant expression patterns for several genes in IVF and cloned blastocysts, similar sex-biased expression patterns (on average) were observed between the sexes. The transcript levels of BEX1 and XIST were upregulated and PGK1 was downregulated in both IVF and cloned blastocysts compared with in vivo counterparts. Moreover, a remarkable degree of expression heterogeneity was observed among individual cloned embryos (the level of heterogeneity was similar in both sexes) but only a small proportion of female IVF embryos exhibited variability, indicating that this phenomenon may be primarily caused by faulty reprogramming by the somatic cell nuclear transfer (SCNT) process rather than in vitro conditions. Aberrant expression patterns in cloned embryos of both sexes were not ameliorated by treatment with Scriptaid as a potent HDACi, although the blastocyst rate increased remarkably after this treatment. Taken together, these results indicate that female and male porcine blastocysts produced in vivo and in vitro transcriptional sexual dimorphisms in the selected X-linked genes and compensation of X-linked gene dosage may not occur at the blastocyst stage. Moreover, altered X-linked gene expression frequently occurred in porcine IVF and cloned embryos, indicating that X-linked gene regulation is susceptible to in vitro culture and the SCNT process, which may eventually lead to problems with embryonic or placental defects.

Cerebral blood volume calculated by dynamic susceptibility contrast-enhanced perfusion MR imaging: preliminary correlation study with glioblastoma genetic profiles.

Purpose To evaluate the usefulness of dynamic susceptibility contrast (DSC) enhanced perfusion MR imaging in predicting major genetic alterations in glioblastomas. Materials and Methods Twenty-five patients (M:F = 13∶12, mean age: 52.1±15.2 years) with pathologically proven glioblastoma who underwent DSC MR imaging before surgery were included. On DSC MR imaging, the normalized relative tumor blood volume (nTBV) of the enhancing solid portion of each tumor was calculated by using dedicated software (Nordic TumorEX, NordicNeuroLab, Bergen, Norway) that enabled semi-automatic segmentation for each tumor. Five major glioblastoma genetic alterations (epidermal growth factor receptor (EGFR), phosphatase and tensin homologue (PTEN), Ki-67, O6-methylguanine-DNA methyltransferase (MGMT) and p53) were confirmed by immunohistochemistry and analyzed for correlation with the nTBV of each tumor. Statistical analysis was performed using the unpaired Student t test, ROC (receiver operating characteristic) curve analysis and Pearson correlation analysis. Results The nTBVs of the MGMT methylation-negative group (mean 9.5±7.5) were significantly higher than those of the MGMT methylation-positive group (mean 5.4±1.8) (p = .046). In the analysis of EGFR expression-positive group, the nTBVs of the subgroup with loss of PTEN gene expression (mean: 10.3±8.1) were also significantly higher than those of the subgroup without loss of PTEN gene expression (mean: 5.6±2.3) (p = .046). Ki-67 labeling index indicated significant positive correlation with the nTBV of the tumor (p = .01). Conclusion We found that glioblastomas with aggressive genetic alterations tended to have a high nTBV in the present study. Thus, we believe that DSC-enhanced perfusion MR imaging could be helpful in predicting genetic alterations that are crucial in predicting the prognosis of and selecting tailored treatment for glioblastoma patients.