The first report of polymorphisms of the prion protein gene (PRNP) in Pekin ducks (Anas platyrhynchos domestica).

Background: Prion diseases have been extensively reported in various mammalian species and are caused by a pathogenic prion protein (PrPSc), which is a misfolded version of cellular prion protein (PrPC). Notably, no cases of prion disease have been reported in birds. Single nucleotide polymorphisms (SNPs) of the prion protein gene (PRNP) that encodes PrP have been associated with susceptibility to prion diseases in several species. However, no studies on PRNP polymorphisms in domestic ducks have been reported thus far. Method: To investigate PRNP polymorphisms in domestic ducks, we isolated genomic DNA from 214 Pekin duck samples and sequenced the coding region of the Pekin duck PRNP gene. We analyzed genotype, allele, and haplotype distributions and linkage disequilibrium (LD) among the SNPs of the Pekin duck PRNP gene. In addition, we evaluated the effects of the one non-synonymous SNP on the function and structure of PrP using the PROVEAN, PANTHER, SNPs & GO, SODA, and AMYCO in silico prediction programs. Results: We found five novel SNPs, c.441 T > C, c.495 T > C, c.582A > G, c.710C > T(P237L), and c.729C > T, in the ORF region of the PRNP gene in 214 Pekin duck samples. We observed strong LD between c.441 T > C and c.582A > G (0.479), and interestingly, the link between c.495 T > C and c.729C > T was in perfect LD, with an r2 value of 1.0. In addition, we identified the five major haplotype frequencies: TTACC, CTGCC, CTACC, CCGCT, and CTATC. Furthermore, we found that the non-synonymous SNP, c.710C > T (P237L), had no detrimental effects on the function or structure of Pekin duck PrP. However, the non-synonymous SNP had deleterious effects on the aggregation propensity and solubility of Pekin duck PrP compared with wildtype Pekin duck PrP. Conclusion: To the best of our knowledge, this study is the first report on the genetic characteristics of PRNP SNPs in Pekin ducks.

Identification of a novel risk factor for chronic wasting disease (CWD) in elk: S100G single nucleotide polymorphism (SNP) of the prion protein gene (PRNP).

Prion diseases are fatal and malignant infectious encephalopathies induced by the pathogenic form of prion protein (PrPSc) originating from benign prion protein (PrPC). A previous study reported that the M132L single nucleotide polymorphism (SNP) of the prion protein gene (PRNP) is associated with susceptibility to chronic wasting disease (CWD) in elk. However, a recent meta-analysis integrated previous studies that did not find an association between the M132L SNP and susceptibility to CWD. Thus, there is controversy about the effect of M132L SNP on susceptibility to CWD. In the present study, we investigated novel risk factors for CWD in elk. We investigated genetic polymorphisms of the PRNP gene by amplicon sequencing and compared genotype, allele, and haplotype frequencies between CWD-positive and CWD-negative elk. In addition, we performed a linkage disequilibrium (LD) analysis by the Haploview version 4.2 program. Furthermore, we evaluated the 3D structure and electrostatic potential of elk prion protein (PrP) according to the S100G SNP using AlphaFold and the Swiss-PdbViewer 4.1 program. Finally, we analyzed the free energy change of elk PrP according to the S100G SNP using I-mutant 3.0 and CUPSAT. We identified 23 novel SNP of the elk PRNP gene in 248 elk. We found a strong association between PRNP SNP and susceptibility to CWD in elk. Among those SNP, S100G is the only non-synonymous SNP. We identified that S100G is predicted to change the electrostatic potential and free energy of elk PrP. To the best of our knowledge, this was the first report of a novel risk factor, the S100G SNP, for CWD.

Development of a chicken interferon-induced transmembrane protein 3 (IFITM3)-specific monoclonal antibody using phage display.

Interferon-induced transmembrane protein 3 (IFITM3) has potent antiviral activity against several viruses. Recent studies have reported that the chicken IFITM3 gene also plays a pivotal role in blocking viral replication, but these studies are considerably limited due to being conducted at the RNA level only. Thus, the development of a chicken IFITM3 protein-specific antibody is needed to validate the function of IFITM3 at the protein level. Epitope prediction was performed with the immune epitope database analysis resource (IEDB-AR) program. The epitope was validated by four in silico programs, Jped4, Clustal Omega, TMpred and SOSUI. Chicken IFITM3 protein-specific monoclonal antibodies were screened by enzyme-linked immunosorbent assay through affinity between recombinant IFITM3 protein and phage-displayed candidate antibodies. Validation of the reactivity of the chicken IFITM3 protein-specific antibody to chicken tissues was carried out using western blotting. We developed a chicken IFITM3 protein-specific monoclonal antibody using phage display. The reactivity of the antibody with peripheral chicken tissues was confirmed using western blotting. To the best of our knowledge, this was the first development of a chicken IFITM3 protein-specific monoclonal antibody using phage display.

Suppressing Halide Segregation in Wide-Band-Gap Mixed-Halide Perovskite Layers through Post-Hot Pressing.

Mixed-halide perovskites (MHPs) have attracted attention as suitable wide-band-gap candidate materials for tandem applications owing to their facile band-gap tuning. However, when smaller bromide ions are incorporated into iodides to tune the band gap, photoinduced halide segregation occurs, which leads to voltage deficit and photoinstability. Here, we propose an original post-hot pressing (PHP) treatment that suppresses halide segregation in MHPs with a band gap of 2.0 eV. The PHP treatment reconstructs open-structured grain boundaries (GBs) as compact GBs through constrained grain growth in the in-plane direction, resulting in the inhibition of defect-mediated ion migration in GBs. The PHP-treated wide-band-gap (2.0 eV) MHP solar cells showed a high efficiency of over 11%, achieving an open-circuit voltage (Voc) of 1.35 V and improving the maintenance of the initial efficiency under the working condition at AM 1.5G. The results reveal that the management of GBs is necessary to secure the stability of wide-band-gap MHP devices in terms of halide segregation.

First report of a strong association between genetic polymorphisms of the prion protein gene (PRNP) and susceptibility to chronic wasting disease in sika deer (Cervus nippon).

Prion diseases are incurable neurodegenerative disorders caused by proteinase K-resistant prion protein (PrPSc ) derived from normal prion protein (PrPC ) encoded by the prion protein gene (PRNP). Although the cervid PRNP gene plays a pivotal role in the pathological mechanism of chronic wasting disease (CWD), there is no existing association analysis between susceptibility to CWD and genetic polymorphisms of the PRNP gene in sika deer. We investigated genetic polymorphisms of the PRNP gene using amplicon sequencing in sika deer. In addition, to identify a genetic susceptibility factor, we compared the genotype, allele and haplotype frequencies of the PRNP gene between CWD-positive and CWD-negative sika deer. Furthermore, to assess the effect of the genetic polymorphisms on sika deer prion protein (PrP), we performed in silico analysis using PolyPhen-2, PROVEAN and AMYCO. Finally, we analysed the tertiary structure and electrostatic potential of sika deer PrP based on single nucleotide polymorphisms (SNPs) using the SWISS-MODEL and Swiss-PdbViewer programs. We found a total of 24 SNPs of the PRNP gene, including 22 novel SNPs (10 synonymous SNPs and 12 nonsynonymous SNPs), in sika deer. Among the nonsynonymous SNPs, we found a strong association of susceptibility to CWD with c.56G > A (Ser19Asn). In addition, we found that c.56G > A (Ser19Asn), c.296A > T (His99Leu) and c.560T > A (Val187Asp) were predicted to have damaging effects on sika deer PrP. Furthermore, we observed significant alterations in the electrostatic potential of sika deer PrP by genetic polymorphisms of the 187Asp allele. To the best of our knowledge, this was the first association study between genetic polymorphisms of the PRNP gene and susceptibility to CWD in sika deer.

Polymorphisms of the prion-related protein gene are strongly associated with cervids' susceptibility to chronic wasting disease.

BACKGROUND: Chronic wasting disease (CWD) is a cervid prion disease that is caused by abnormal prion protein (PrPSc ). Recent studies have reported that prion family genes showed a strong association with the susceptibility of several types of prion diseases. To date, an association study of the prion-related protein gene (PRNT) has not been performed in any type of cervid prion disease. METHODS: In the present study, we investigated PRNT polymorphisms in large deer, including 235 elk, 257 red deer and 150 sika deer. We compared genotype, allele and haplotype frequencies of PRNT polymorphisms between CWD-negative animals and CWD-positive animals to find an association of PRNT polymorphisms with the susceptibility of CWD. RESULTS: We found a total of five novel single nucleotide polymorphisms (SNPs) in the cervid PRNT gene. Interestingly, we observed significantly different distributions of genotypes and allele frequencies of three PRNT SNPs, including c.108C>T, c.159+30C>T and c.159+32A>C, between CWD-negative and CWD-positive red deer. In addition, significant differences of two haplotype frequencies in red deer were found between the CWD-negative and CWD-positive groups. However, the association identified in the red deer was not found in elk and sika deer. CONCLUSION: To the best of our knowledge, this report is the first to describe the strong association of PRNT SNPs with the susceptibility of CWD.

Absence of proteinase K-resistant PrP in Korean Holstein cattle carrying potential bovine spongiform encephalopathy-related E211K somatic mutation.

Bovine spongiform encephalopathy (BSE) is a kind of prion disease caused by proteinase K-resistant prion protein (PrPSc ) in cattle. Although BSE has been reported worldwide, BSE-infected cases have never been reported in Korea. In a previous study, we identified BSE-related somatic mutation E211K in 3 Korean Holstein cattle. In Korea, the BSE surveillance system has been established. However, several genetic factors have not been controlled simultaneously thus far. In the present study, we performed enhanced surveillance of prion disease-related factors in Korean cattle, including Holstein cattle and Hanwoo (Korean native cattle), which is widely raised for meat. We investigated the germline mutation E211K at codon 211 of the PRNP gene and analysed genotype, allele and haplotype frequencies of the 23- and 12-bp insertion/deletion polymorphisms of the PRNP gene using direct DNA sequencing. In addition, we investigated linkage disequilibrium (LD) and compared haplotype distributions of polymorphisms among cattle breeds. Furthermore, we carried out BSE diagnosis in the medulla oblongata (MO) of Korean cattle including 3 Korean Holstein cattle carrying somatic mutation E211K using Western blotting analysis. We did not find the E211K mutation in the PRNP gene in any of the Korean cattle and found significantly different genotype, allele and haplotype distributions of the 23- and 12-bp insertion/deletion polymorphisms of the PRNP gene in male Holstein compared with male Hanwoo, female Hanwoo and total Hanwoo. In addition, only male Holstein showed weak LD between 23- and 12-bp insertion/deletion polymorphisms. Furthermore, the PrPSc bands were not detected in all Korean cattle tested. To the best of our knowledge, the enhanced surveillance system of BSE was conducted for the first time in Korean cattle.

Regulatory Single Nucleotide Polymorphism of the Bovine IFITM3 Gene Induces Differential Transcriptional Capacities of Hanwoo and Holstein Cattle.

Interferon-induced transmembrane protein 3 (IFITM3), a crucial effector of the host's innate immune system, prohibits an extensive range of viruses. Previous studies have reported that single nucleotide polymorphisms (SNPs) of the IFITM3 gene are associated with the expression level and length of the IFITM3 protein and can impact susceptibility to infectious viruses and the severity of infection with these viruses. However, there have been no studies on polymorphisms of the bovine IFITM3 gene. In the present study, we finely mapped the bovine IFITM3 gene and annotated the identified polymorphisms. We investigated polymorphisms of the bovine IFITM3 gene in 108 Hanwoo and 113 Holstein cattle using direct sequencing and analyzed genotype, allele, and haplotype frequencies and linkage disequilibrium (LD) between the IFITM3 genes of the two cattle breeds. In addition, we analyzed transcription factor-binding sites and transcriptional capacity using PROMO and luciferase assays, respectively. Furthermore, we analyzed the effect of a nonsynonymous SNP of the IFITM3 gene using PolyPhen-2, PANTHER, and PROVEAN. We identified 23 polymorphisms in the bovine IFITM3 gene and found significantly different genotype, allele, and haplotype frequency distributions and LD scores between polymorphisms of the bovine IFITM3 gene in Hanwoo and Holstein cattle. In addition, the ability to bind the transcription factor Nkx2-1 and transcriptional capacities were significantly different depending on the c.-193T > C allele. Furthermore, nonsynonymous SNP (F121L) was predicted to be benign. To the best of our knowledge, this is the first genetic study of bovine IFITM3 polymorphisms.

The First Report of the Prion Protein Gene (PRNP) Sequence in Pekin Ducks (Anas platyrhynchos domestica): The Potential Prion Disease Susceptibility in Ducks.

Pathogenic prion protein (PrPSc), converted from normal prion protein (PrPC), causes prion disease. Although prion disease has been reported in several mammalian species, chickens are known to show strong resistance to prion diseases. In addition to chickens, the domestic duck occupies a large proportion in the poultry industry and may be regarded as a potential resistant host against prion disease. However, the DNA sequence of the prion protein gene (PRNP) has not been reported in domestic ducks. Here, we performed amplicon sequencing targeting the duck PRNP gene with the genomic DNA of Pekin ducks. In addition, we aligned the PrP sequence of the Pekin duck with that of various species using ClustalW2 and carried out phylogenetic analysis using Molecular Evolutionary Genetics Analysis X (MEGA X). We also constructed the structural modeling of the tertiary and secondary structures in avian PrP using SWISS-MODEL. Last, we investigated the aggregation propensity on Pekin duck PrP using AMYCO. We first reported the DNA sequence of the PRNP gene in Pekin ducks and found that the PrP sequence of Pekin ducks is more similar to that of geese than to that of chickens and mallards (wild ducks). Interestingly, Pekin duck PrP showed a high proportion of ß-sheets compared to that of chicken PrP, and a high aggregation propensity compared to that of avian PrPs. However, Pekin duck PrP with substitutions of chicken-specific amino acids showed reduced aggregation propensities. To the best of our knowledge, this is the first report on the genetic characteristics of the PRNP sequence in Pekin ducks.

Genetic association between the rs12252 SNP of the interferon-induced transmembrane protein gene and influenza A virus infection in the Korean population.

BACKGROUND: Interferon-induced transmembrane protein 3 (IFITM3) is a potent host antiviral effector protein that blocks the invasion of various viruses, including the influenza A virus (IAV). The C allele of the rs12252 single nucleotide polymorphism (SNP) shows vulnerability to the pandemic 2009 H1N1 IAV in European and Asian populations. OBJECTIVE: Here, we estimated the disease susceptibility of the rs12252 SNP with the pandemic 2009 H1N1 IAV infection in the Korean population. RESULTS: We carried out direct sequencing of the IFITM3 gene and compared the genotype and allele frequencies of the rs12252 SNP of the IFITM3 gene in healthy Koreans and pandemic 2009 H1N1 IAV-infected patients. Notably, we observed that healthy individuals had a similar genotype distribution of the rs12252 SNP (P = 0.140) as patients. The dominant model and recessive model did not find a statistically significant difference in genotype distribution between healthy individuals and patients. In addition, the allele distribution of the rs12252 SNP of in healthy individuals and patients also showed a similar genetic distribution (P = 0.757). However, the genetic distribution of rs12252 SNP in merged patient group (Koreans and Chinese populations) showed significant association with susceptibility of pandemic 2009 IAV (P = 0.0393). CONCLUSION: To the best of our knowledge, this was the first evaluation of the susceptibility of the pandemic 2009 H1N1 IAV in the Korean population.

Identification of the prion-related protein gene (PRNT) sequences in various species of the Cervidae family.

Chronic wasting disease (CWD) is caused by abnormal deleterious prion protein (PrPSc), and transmissible spongiform encephalopathy occurs in the Cervidae family. In recent studies, the susceptibility of prion disease has been affected by polymorphisms of the prion gene family. However, the study of the prion-related protein gene (PRNT) is rare, and the DNA sequence of this gene was not fully reported in all Cervidae families. In the present study, we amplified and first identified PRNT DNA sequences in the Cervidae family, including red deer, elk, sika deer and Korean water deer, using polymerase chain reaction (PCR). We aligned nucleotide sequences of the PRNT gene and the amino acid sequences of prion-related protein (Prt) protein among several species. In addition, we performed phylogenetic analysis to measure the evolutionary relationships of the PRNT gene in the Cervidae family. Furthermore, we performed homology modeling of the Prt protein using SWISS-MODEL and compared the structure of Prt protein between sheep and the Cervidae family using the Swiss-PdbViewer program. We obtained much longer PRNT sequences of red deer compared to the PRNT gene sequence registered in GenBank. Korean water deer denoted more close evolutionary distances with goats and cattle than the Cervidae family. We found 6 Cervidae family-specific amino acids by the alignment of Prt amino acid sequences. There are significantly different distributions of hydrogen bonds and the atomic distance of the N-terminal tail and C-terminal tail between sheep and the Cervidae family. We also detected the mRNA expression of PRNT gene in 3 tissues investigated. To our knowledge, this report is the first genetic study of the PRNT gene in the Cervidae family.

Genetic characteristics and polymorphisms in the chicken interferon-induced transmembrane protein (IFITM3) gene.

The interferon-induced transmembrane protein 3 (IFITM3) gene is classified as a small interferon-stimulated gene and is associated with a broad spectrum of antiviral functions against several fatal enveloped viruses, including influenza A viruses (IAVs). The rs12252 single nucleotide polymorphism (SNP) of the IFITM3 gene in humans was associated with susceptibility to H1N1 influenza in a 2009 pandemic. In addition, overexpression of the IFITM3 protein potently inhibits the highly pathogenic avian influenza H5N1 virus in ducks and chickens. Although chickens are a major host of influenza viruses and the IFITM3 gene participates in the host antiviral system, studies on chicken IFITM3 gene are very rare. To investigate the genetic characteristics of the chicken IFITM3 gene, we performed direct sequencing and alignment in 108 Dekalb White and 72 Ross breeds. We also investigated the genotype and haplotype frequencies and linkage disequilibrium of the IFITM3 gene polymorphisms and evaluated whether the non-synonymous SNPs are deleterious. We found significantly different genotype, allele and haplotypes frequencies between two chicken breeds, Dekalb White and Ross. Furthermore, we compared and analyzed the promoter structure of the chicken IFITM3 gene with that of several species. We found that birds have a long C-terminal domain and inverted topology of the IFITM3 protein compared to mammals. We also identified fourteen genetic polymorphisms in the chicken IFITM3 gene. L100 M and N125H were predicted as 'probably damaging' and L100 M can alter the length of its conserved intracellular loop (CIL). Furthermore, chickens, but not mammals, contain CpG islands (CGIs) in this promoter region.

No polymorphisms in the coding region of the prion-like protein gene in Thoroughbred racehorses.

Prion diseases are fatal neurodegenerative diseases characterised by the accumulation of an abnormal prion protein isoform (PrPSc), which is converted from the normal prion protein (PrPC). Prion diseases have been reported in an extensive number of species but not in horses up to now; therefore, horses are known to be a species resistant to prion diseases. The prion-like protein gene (PRND) is closely located downstream of the prion protein gene (PRNP) and the prion-like protein (Doppel) is a homologue with PrP. Previous studies have shown that an association between prion diseases and polymorphisms of the PRND gene is reported in the main hosts of prion diseases. Hence, we examined the genetic variations of the PRND gene in Thoroughbred horses. Interestingly, polymorphisms of the PRND gene were not detected. In addition, we conducted a comparative analysis of the amino acid sequences of the PRND gene to identify the differences between horses and other species. The amino acid sequence of the horse PRND gene showed the highest identity to that of sheep (83.7%), followed by that of goats, cattle and humans. To the best of our knowledge, this is the first genetic study of the PRND gene in horses.

Prion-like protein gene (PRND) polymorphisms associated with scrapie susceptibility in Korean native black goats.

The polymorphisms of the prion protein (PRNP) gene, which encodes normal prion proteins (PrP), are known to be involved in the susceptibility of prion diseases. The prion-like protein (Doppel) gene (PRND) is the paralog of the PRNP gene and is closely located downstream of the PRNP gene. In addition, the polymorphisms of PRND correlate with disease susceptibility in several animals. We analyzed the genotype and allele frequencies of PRND polymorphisms in 246 Korean native black goats and found a total of six single nucleotide polymorphisms (SNPs) with one novel SNP, c.99C>T. We observed linkage disequilibrium (LD) within and between loci. PRND c.28T>C, c.151A>G, and c.385G>C and PRND c.65C>T and c.286G>A were in perfect LD and we have reported for the first time strong LD between PRND and PRNP or prion-related protein gene (PRNT) loci. Specifically, between the PRND c.28T>C, c.151A>G and c.385G>C and the PRNP codon 143, PRND c.99C>T and the PRNP codon 102 or PRND SNPs (c.28T>C, c.151A>G and c.385G>C) and PRNT SNP (c.321T>C). Furthermore, we confirmed that the genotype distribution of the PRNP p.His143Arg was significantly different according to that of the PRND c.28T>C (P < 0.0001). Finally, using PolyPhen-2 and PROVEAN, we predicted that two non-synonymous SNPs, c.65C>T and c.286G>A, in the PRND gene can have a detrimental effect on Doppel. To the best of our knowledge, this is the first report of genetic characteristics of the PRND gene in Korean native black goats.

The first report of genetic variations in the chicken prion protein gene.

Abnormal structural changes of the prion protein (PrP) are the cause of prion disease in a wide range of mammals. However, spontaneous infected cases have not been reported in chicken. Genetic variations of the prion protein gene (PRNP) may impact susceptibility to prion disease but have not been investigated thus far. Because an investigation of the chicken PRNP can improve the understanding of characteristics related to resistance to prion disease, research on the chicken PRNP is highly desirable. In this study, we investigated the genetic characteristics of the chicken PRNP gene. For this, we performed direct sequencing in 106 Dekalb White chickens and analyzed the genotype and allele frequencies of chicken PRNP gene. We found two insertion and deletion polymorphisms in the chicken PRNP: c.163_180delAACCCAGGGTACCCCCAT and c.268_269insC. The former is a U2 hexapeptide deletion polymorphism. Of the 106 samples, 13 (12.26%) were insertion homozygotes, 89 (83.96%) were heterozygotes, and 4 (3.77%) were deletion homozygotes in c.163_180delAACCCAGGGTACCCCCAT. In the c.268_269insC polymorphism, 102 (96.23%) were deletion homozygotes, and 4 (3.77%) were heterozygotes. Insertion homozygotes of c.268_269insC were not detected. Two polymorphisms were in perfect linkage disequilibrium (LD) with a D' value of 1.0, and three haplotypes were identified. Furthermore, PROVEAN evaluates 163_180delAACCCAGGGTACCCCCAT as 'deleterious' with a score of - 13.173. Furthermore, single nucleotide polymorphisms (SNPs) in the open reading frame (ORF) of the PRNP gene were not found in the chicken. To the best of our knowledge, this was the first report on the genetic variations of the chicken PRNP gene.

Discotic liquid crystalline hydrazone compounds: synthesis and mesomorphic properties.

[reaction: see text] 1,3,5-Trisacetoacetamidobenzene with three 1,3-diketo groups was synthesized by the reaction of 1,3,5-triaminobenzene with diketene. Discotic hydrazone compounds were prepared by the diazo coupling reaction between 1,3,5-trisacetoacetamidobenzene and diazonium salts of 4-alkyloxyphenylamines. The compounds existed in hydrazone forms exclusively, being stabilized by the intramolecular hydrogen bonds, and showed discotic nematic or columnar hexagonal mesophases.

Effects of Oxygen Free Radical on Action Potential in Mouse Atrial Myocardium

BACKGROUND AND OBJECTIVES: Reactive oxygen species are known to be produced when atrial fibrillation develops. This study was performed to investigate the effects of hydrogen peroxide (H2O2) on the action potential parameters of the mouse atrium. SUBJECTS AND METHODS: Mouse (ICR) atrial fibers were excised and immersed in cold bicarbonate-containing Tyrode's solution. The preparations were then perfused with oxygenated (95% O2, 5% CO2) Tyrode's solution and driven by an electrical stimuli 1 ms in duration at a frequency of 1 Hz. The transmembrane potentials were recorded at 0, 2.5, 5, 10, 20 and 30 minute, and compared between groups I (control), II (H2O2 0.1 mM), III (H2O2 0.5 mM) and IV (H2O2 1 mM). RESULTS: In group I, the maximal diastolic potential (MDP), action potential amplitude (APA), maximal slope at phase 0 depolarization (Vmax), action potential duration until 50% and 90% of repolarization (APD50, APD90) were unchanged with increasing time. In group II, the MDP and APA were unchanged, but the Vmax was decreased, and the APD50 and APD90 prolonged. In group III, the MDP was increased and the Vmax decreased; the APD50 and APD90 were prolonged, but the APA unchanged. In group IV, the MDP was increased, the Vmax and APA decreased And the APD50 and APD90 prolonged. After-depolarization was observed in 40% (8/20) and 54.5% (12/22) of groups III and IV, respectively, and asystole occurred in 18.2% (4/22) of group IV. CONCLUSION: Hydrogen peroxide changed the action potential parameters in both time and dose dependent manner, and also elicited after-depolarization at higher concentrations. These results suggest reactive oxygen species are involved in the electrical remodeling and arrhythmogenesis in atrial myocardium.

Influence of Vasoactive Substances on the Regulation of Cardiac ATP-Sensitive Potassium Channel Activity

BACKGROUND AND OBJECTIVES: It has been known that various vasoactive agents are involved in the regulation of cardiac function through the modification of the K+ channel activities, including the ATP-sensitive K+ channel (KATP). We examined the effects of several vasoactive agents on the cardiac KATP currents in isolated cardiac myocytes. MATERIALS AND METHODS: Ventricular myocytes were isolated from the hearts of ICR mice by enzymatic digestion. The channel currents were recorded by the excised inside-out and cell-attached patch clamp configurations. RESULTS: In the excised inside-out patches, bradykinin (BRK; 1-10 micrometer) and prostaglandin I2 (PGI; 10-50 micrometer) did not affect the channel activities, whereas the vasodilators increased the attenuated channel activities in the presence of 100 micrometer ATP. BRK and PGI in parallel shifted the dose-response curves of ATP (1-1,000 micrometer), and this inhibited the KATP currents to the right. Endothelin (ET-1; 0.1-1 nM) and leukotriene D4 (LTD; 3-10 micrometer) decreased the channel activities immediately after making the inside-out patches. However, the vasoconstrictors did not affect the attenuated channel activities by ATP. In the cell-attached patches, both BRK and PGI increased the channel activities and these effects were markedly attenuated by glibenclamide (50 micrometer). ET-1 and LTD did not affect the baseline channel activities in the cell-attached patches, but they markedly attenuated the dinitrophenol-induced activities. CONCLUSION: It was inferred that certain vasoactive substances are involved in the regulation of cardiac KATP channel activities, and that bradykinin and PGI2 enhance the channel activities, and ET-1 and LTD4 inhibit the channel activities.

Effects of Oxygen Free Radicals on KATP Channel Activity in Mouse Cardiac Myocytes

BACKGROUND AND OBJECTIVE: Oxygen-derived free radicals (OFRs), produced as myocardium is reperfused after ischemic injury, contribute to reversible and irreversible cellular injury. KATP channels, activated by ischemia, have been reported to participate in the arrhythmogenic response to acute myocardial ischemia. Therefore, we examined the effects of OFRs on the regulation of KATP channel activity. MATERIALS AND METHODS: Isolated mice (ICR) hearts were perfused with Tyrode's solution on a Langendorff apparatus. Single ventricular myocytes were isolated using enzymatic digestion with collagenase and protease. Single channel currents in the inside-out patch mode were recorded. OFRs were applied by mixing hypoxanthine and xanthine oxidase. The currents were recorded in the patch membrane at a holding potential of -60 mV. RESULTS: OFRs generated by 0.1 U/mL xanthine oxidase and 0.5 mM hypoxanthine had no effects on the activities of KATP channels before and after treatment with 200 micrometer ATP. OFRs generated with 0.2 U/mL xanthine oxidase and 1.0 mM hypoxanthine reactivated the channel activities which had been attenuated by 100 micrometer ATP. In the presence of 100 U/mL superoxide dismutase and 122 U/mL catalase, which are OFRs scavengers, OFRs did not affect the KATP channels activities. CONCLUSION: OFRs generated by the reaction of hypoxanthine and xanthine oxidase increased the KATP channel activities in the inside-out patch.