Changyong Choe

Thermosensitivity of the two‐pore domain K+ channels TREK‐2 and TRAAK

TREK‐1, TREK‐2 and TRAAK are members of the two‐pore domain K+ (K2P) channel family and are activated by membrane stretch and free fatty acids. TREK‐1 has been shown to be sensitive to temperature in expression systems. We studied the temperature‐sensitivity of TREK‐2 and TRAAK in COS‐7 cells and in neuronal cells. In transfected COS‐7 cells, TREK‐2 and TRAAK whole‐cell currents increased ∼20‐fold as the bath temperature was raised from 24°C to 42°C. Similarly, in cell‐attached patches of COS‐7 cells, channel activity was very low, but increased progressively as the bath temperature was raised from 24°C to 42°C. The thresholds for activation of TREK‐2 and TRAAK were ∼25°C and ∼31°C, respectively. Other K2P channels such as TASK‐3 and TRESK‐2 were not significantly affected by an increase in temperature from 24°C to 37°C. When the C‐terminus of TREK‐2 was replaced with that of TASK‐3, its sensitivity to free fatty acids and protons was abolished, but the mutant could still be activated by heat. At 37°C, TREK‐1, TREK‐2 and TRAAK were sensitive to arachidonic acid, pH and membrane stretch in both cell‐attached and inside‐out patches. In cerebellar granule and dorsal root ganglion neurones, TREK‐1, TREK‐2 and TRAAK were generally inactive in the cell‐attached state at 24°C, but became very active at 37°C. In cell‐attached patches of ventricular myocytes, TREK‐1 was also normally closed at 24°C, but was active at 37°C. These results show that TREK‐2 and TRAAK are also temperature‐sensitive channels, are active at physiological body temperature, and therefore would contribute to the background K+ conductance and regulate cell excitability in response to various physical and chemical stimuli.

Expression and localization of two-pore domain K+ channels in bovine germ cells

Two-pore domain K(+) (K(2P)) channels that help set the resting membrane potential of excitable and nonexcitable cells are expressed in many kinds of cells and tissues. However, the expression of K(2P) channels has not yet been reported in bovine germ cells. In this study, we demonstrate for the first time that K(2P) channels are expressed in the reproductive organs and germ cells of Korean cattle. RT-PCR data showed that members of the K(2P) channel family, specifically KCNK3, KCNK9, KCNK2, KCNK10, and KCNK4, were expressed in the ovary, testis, oocytes, embryo, and sperm. Out of these channels, KCNK2 and KCNK4 mRNAs were abundantly expressed in the mature oocytes, eight-cell stage embryos, and blastocysts compared with immature oocytes. KCNK4 and KCNK3 were significantly increased in eight-cell stage embryos. Immunocytochemical data showed that KCNK2, KCNK10, KCNK4, KCNK3, and KCNK9 channel proteins were expressed at the membrane of oocytes and blastocysts. KCNK10 and KCNK4 were strongly expressed and distributed in oocyte membranes. These channel proteins were also localized to the acrosome sperm cap. In particular, KCNK3 and KCNK4 were strongly localized to the post-acrosomal region of the sperm head and the equatorial band within the sperm head respectively. These results suggest that K(2P) channels might contribute to the background K(+) conductance of germ cells and regulate various physiological processes, such as maturation, fertilization, and development.

Dual effects of fluoxetine on mouse early embryonic development

Fluoxetine, a selective serotonin reuptake inhibitor, regulates a variety of physiological processes, such as cell proliferation and apoptosis, in mammalian cells. Little is known about the role of fluoxetine in early embryonic development. This study was undertaken to investigate the effect of fluoxetine during mouse early embryonic development. Late two-cell stage embryos (2-cells) were cultured in the presence of various concentrations of fluoxetine (1 to 50μM) for different durations. When late 2-cells were incubated with 5μM fluoxetine for 6h, the percentage that developed into blastocysts increased compared to the control value. However, late 2-cells exposed to fluoxetine (5μM) over 24h showed a reduction in blastocyst formation. The addition of fluoxetine (5μM) together with KN93 or KN62 (calcium/calmodulin-dependent protein kinase II (CaMKII) inhibitors) failed to increase blastocyst formation. Fluoxetine treatment inhibited TREK-1 and TREK-2, members of the two-pore domain K(+) channel family expressed in mouse embryos, activities, indicating that fluoxetine-induced membrane depolarization in late 2-cells might have resulted from TREK inhibition. In addition, long-term exposure to fluoxetine altered the TREK mRNA expression levels. Furthermore, injection of siRNA targeting TREKs significantly decreased blastocyst formation by ~30% compared to injection of scrambled siRNA. Long-term exposure of fluoxetine had no effect on blastocyst formation of TREK deficient embryos. These results indicate that low-dose and short-term exposures of late 2-cells to fluoxetine probably increase blastocyst formation through activation of CaMKII-dependent signal transduction pathways, whereas long-term exposure decreases mouse early embryonic development through inhibition of TREK channel gating.

K(+) efflux through two-pore domain K(+) channels is required for mouse embryonic development.

Numerous studies have suggested that K(+) channels regulate a wide range of physiological processes in mammalian cells. However, little is known about the specific function of K(+) channels in germ cells. In this study, mouse zygotes were cultured in a medium containing K(+) channel blockers to identify the functional role of K(+) channels in mouse embryonic development. Voltage-dependent K(+) channel blockers, such as tetraethylammonium and BaCl(2), had no effect on embryonic development to the blastocyst stage, whereas K(2P) channel blockers, such as quinine, selective serotonin reuptake inhibitors (fluoxetine, paroxetine, and citalopram), gadolinium trichloride, anandamide, ruthenium red, and zinc chloride, significantly decreased blastocyst formation (P<0.05). RT-PCR data showed that members of the K(2P) channel family, specifically KCNK2, KCNK10, KCNK4, KCNK3, and KCNK9, were expressed in mouse oocytes and embryos. In addition, their mRNA expression levels, except Kcnk3, were up-regulated by above ninefold in morula-stage embryos compared with 2-cell stage embryos (2-cells). Immunocytochemical data showed that KCNK2, KCNK10, KCNK4, KCNK3, and KCNK9 channel proteins were expressed in the membrane of oocytes, 2-cells, and blastocysts. Each siRNA injection targeted at Kcnk2, Kcnk10, Kcnk4, Kcnk3, and Kcnk9 significantly decreased blastocyst formation by ~38% compared with scrambled siRNA injection (P<0.05). The blockade of K(2P) channels acidified the intracellular pH and depolarized the membrane potential. These results suggest that K(2P) channels could improve mouse embryonic development through the modulation of gating by activators.

Proteomic Analysis of Differentially Expressed Proteins in Bovine Endometrium with Endometritis

Endometritis is one of the primary reasons for reproductive failure. In order to investigate endometritis-associated marker proteins, proteomic analysis was performed on bovine endometrium with endometritis. In bovine endometritis, desmin, α-actin-2, heat-shock protein (HSP) 27, peroxiredoxin-6, luteinizing hormone receptor isoform 1, collectin-43 precursor, deoxyribonuclease-I (DNase-I), and MHC class I heavy chain (MHC-Ih) were up-regulated. In contrast, transferrin, interleukin-2 precursor, hemoglobin β subunit, and potassium channel tetramerisation domain-containing 11 (KCTD11) were down-regulated in comparison to normal endometrium. The proteomic results were validated by semiquantitative-PCR and immunoblot analysis. The mRNA levels of desmin, transferrin, α-actin-2, HSP27, KCTD11, and MHC-Ih were up-regulated by over 1.5-fold, and showed a pattern similar to their proteomic profiles. Desmin and α-actin-2 protein showed positive correlations between proteomic analysis and immunoblot analysis. These results suggest that desmin and α-actin-2 may play important roles in endometritis-related function, and could be useful markers for the diagnosis of bovine endometritis.

Identification of Differentially Expressed Genes in Bovine Follicular Cystic Ovaries

Follicular cystic ovary (FCO) is one of the most frequently diagnosed ovarian diseases and is a major cause of reproductive failure in mammalian species. However, the mechanism by which FCO is induced remains unclear. Genetic alterations which affect the functioning of many kinds of cells and/or tissues could be present in cystic ovaries. In this study, we performed a comparison analysis of gene expression in order to identify new molecules useful in discrimination of bovine FCO with follicular cystic follicles (FCFs). Normal follicles and FCFs were classified based on their sizes (5 to 10 mm and ≥25 mm). These follicles had granulosa cell layer and theca interna and the hormone 17β-estradiol (E(2))/ progesterone (P(4)) ratio in follicles was greater than one. Perifollicular regions including follicles were used for the preparation of RNA or protein. Differentially expressed genes (DEG) that showed greater than a 2-fold change in expression were screened by the annealing control primer (ACP)-based PCR method using GeneFishing™ DEG kits in bovine normal follicles and FCFs. We identified two DEGs in the FCFs: ribosomal protein L15 (RPL15) and microtubule-associated protein 1B (MAP1B) based on BLAST searches of the NCBI GenBank. Consistent with the ACP analysis, semi-quantitative PCR data and Western blot analyses revealed an up-regulation of RPL15 and a down-regulation of MAP1B in FCFs. These results suggest that RPL15 and MAP1B may be involved in the regulation of pathological processes in bovine FCOs and may help to establish a bovine gene data-base for the discrimination of FCOs from normal ovaries.

Occurrence of Clostridium perfringens according to Raising Periods in Broilers

Department of Infectious Diseases & Avian Diseases, College Veterinary Medicine and Korea Zoonosis Research Institute,Jeonbuk National University, Jeonju 561-756, KoreaABSTRACT The objective of this study was to investigate occurrence patterns of Clostridium perfringens on different raising periods in broilers. In different raising periods, we investigated the change in the gross lesion and microscopic histological findings of the mucose of the small intestine, colony forming unit (CFU) and the types C. perfringens with PCR assay. According to the gross lesions on the mucose of small intestine with 10-days-old broilers, the non-antibiotic group showed a higher value (0.6) than the antibiotic group (0.0). Whereas 20-days-old broilers with, the antibiotic treatment had a slightly lower value (1.0) than the non-antibiotic group (1.3). In the histological examination on the villi of the small intestine, there was no damage of the villi of the small intestine with 1-day-old broilers in both groups; however, the non-antibiotic group showed a higher value (0.4) than the antibiotic group (0.0) with 10-days-old broilers. In the non-antibiotic group, the CFU of C. perfringens of the fecal samples from the small intestine increased from 10 days of raising broilers and rapidly increase after 20 and 30 days of raising broilers. There was no detection of C. perfringens types with PCR assy in 1-day-old broilers, but we found C. perfringens type A in 10-, 20- and 30-days-old broilers. Although it is possible to raise healthy broilers by using antibiotics, the addition of antibiotics to concentrate feed is prohibited for public health. The results of this study would contribute to proper feeding management through the careful use of antibiotics.(Key words : necrotic enteritis, Clostridium perfringens, broiler, antibiotics)

Acetylcholine controls mouse oocyte maturation via downregulation of cAMP

1. In mice, acetylcholine (ACh) plays an important role in oocyte activation and embryonic development. However, the role of ACh in mouse oocyte maturation has not been investigated.

Major medical causes by breed and life stage for dogs presented at veterinary clinics in the Republic of Korea: a survey of electronic medical records

Background Age and breed are considered the greatest risk factors for disease prevalence and mortality in companion dogs. Understanding the prevalence of diseases, in relation to age and breed, would support appropriate guidance for future health care strategies and provide useful information for the early diagnosis of diseases. The purpose of this study was to investigate the major medical causes for dogs visiting primary-care veterinary clinics in the Republic of Korea, stratified by age and breed. Methods A total of 15,531 medical records of canine patients were analyzed from 11 veterinary clinics who shared data from January 1, 2016 to December 31, 2016. An electronic medical record (EMR) system was used for data collection, which included the animal identification number, age, breed, gender, neuter status, clinical information, and diagnosis. EMR data were classified using the International Classification of Disease system from the World Health Organization; presenting signs or diagnoses were identified according to breed and life stage. Results Within the age groups, preventive medicine (16.7% confidence intervals (CI) [15.9–17.5]) was the most common cause for clinic visits for the <1 year and 1–3 year groups. Additionally, neutering surgery (6.6% CI [6.0–7.1]) and patella luxation (1.4% CI [1.8–2.7]) were frequently performed in these age groups. In the 4–6 year group, otitis externa (8.8% CI [7.8–10.0]) and dermatitis or eczema (8.5% CI [7.5–9.6]) were common medical problems. In older dogs (>10 year), the prevalences of heart disease, kidney disease, Cushing’s disease, and mammary tumors were higher than in the other age groups. Small and toy breed dogs comprised 67.7% of all dogs in this analysis. For all breeds, otitis externa, dermatitis or eczema, vomiting, and diarrhea were common medical problems. Discussion This study identified the most common medical disorders and differences in prevalences of diseases, according to age and breeds. The information from EMRs for dogs visiting primary-care veterinary clinics can provide background knowledge that is required to enable a better understanding of disease patterns and occurrence by age and breeds. The information from this study could enable the creation of strategies for preventing diseases and enable the identification of health problems for more effective disease management in companion dogs.

Relationship between Sloan-Kettering virus expression and mammalian follicular development-RETRACTION

Zygote wishes to inform its readers that its Editor-in-Chief has decided to retract the above article after an investigation carried out in compliance with the Committee on Publication Ethics guidelines found that the authors duplicated substantial parts of the following two articles: 1. Kim H, Yamanouchi K, Nishihara M. (2006) Expression of ski in the granulosa cells of atretic follicles in the rat ovary. J. Reprod. Dev . 52 , 715–721 2. Kim H, Yamanouchi K, Matsuwaki T, Nishihara M. (2012) Induction of Ski protein expression upon luteinization in rat granulosa cells without a change in its mRNA expression. J. Reprod. Dev . 58 , 254–259