• Journal of Plant Biotechnology
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• 제36권4호
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• pp.407-414
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• 2009

A cDNA clone encoding phytocystatin was isolated from Brassica rapa seedlings, through rapid amplification of cDNA ends (RACE). This gene (name as Brcpi1; GenBank accession no.: EF079953) had a total length of 881 bp with an open reading frame of 609 bp, and encoded predicted polypeptide of 203 amino acid (aa) residues including a putative N-terminal signal peptide. Other relevant regions found its sequence included the G and PW conserved aa motifs, and the consensus LARFAV sequence for phytocystatins and the reactive site QVVAG. The BrCPI1 protein shared 95, 94, 81, 80 and 78% identity with other CPI proterins isolated from Brassica oleracea (BoCPI-1), Arabidopsis thaliana (AtCY SB), Glycine max (GmCPI), Oryza sativa (OsCYS-2) and Zea may (ZmCPI) at amino acid level, respectively. Southern blot analysis showed that Brcpi1 was a low copy gene. Expression pattern analysis revealed that Brcpi1 was a tissue-specific expressing gene during reproductive growth and strongly expressed at mature seedling stages. Furthermore, overexpression of Brcpi1 in transgenic Arabidopsis was enhanced tolerance to salt and cold stresses. Meanwhile the juvenile seedling of Brcpi1 transgenic plants was not affected by various concentrations ABA in MS medium. Taken together, the results showed that Brcpi1 functioned as a cysteine protease inhibitor and it exhibited a protective agent against diverse types of abiotic stress, which induced this gene in a tissue- and stress-specific manner.

• Reproductive and Developmental Biology
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• 제33권4호
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• pp.217-222
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• 2009

The purpose of this study is to evaluate the effects of alcohol or cigarette smoking on seminal parameters in a large group of mice model. Nine groups (n=20/group) of mice were treated intensive noxious materials that abdominal injection of 21% (v/v) of ethanol, cigarette smoke (10, 20, 30 minutes/day), and combination of ethanol and 30 minutes of smoking. In addition, vitamin C and selenium were also treated to mice exposed to combination of alcohol and smoking to identify the recovering effect. Sperm viability and motility were significantly decreased in either alcohol consumption or smoking exposed group, and combination of both materials have additive detrimental effects on seminal parameters. Mice groups that exposed to alcohol and smoking showed statistically significant decrease in motility and increase of static spermatozoa. Moreover, combination of both treatments showed cumulative effect in increase of static spermatozoa. Treatments of either vitamin C or selenium dramatically recovered detrimental effects of alcohol and smoking on seminal quality, although combination of both antioxidant molecules did not show any additive effect. In conclusion, detrimental effects of alcohol and cigarette consumption on sperm quality and motility were identified in mice model, and these detrimental effects can be compensated to uptake of anti-oxidant molecules.

• Reproductive and Developmental Biology
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• 제34권4호
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• pp.289-297
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• 2010

Erythropoietin (EPO) is a glycoprotein hormone secreted from primarily cells of the peritubular capillary endothelium of the kidney, and is responsible for the regulation of red blood cell production. We constructed and expressed dimeric cDNAs in Chinease hamster ovary (CHO) cells encoding a fusion protein consisting of 2 complete human EPO domains linked by a 2-amino acid linker (Ile-Asp). We described the activity of dimeric hyperglycosylated EPO (dHGEPO) mutants containing additional oligosaccharide chains and characterized the function of glycosylation. No dimeric proteins with mutation at the $105^{th}$ amino acid were found in the cell medium. Growth and differentiation of the human EPO-dependent leukemiae cell line (F36E) were used to measure cytokine dependency and in vitro bioactivity of dHGEPO proteins. MIT assay at 24 h increased due to the survival of F36E cells. The dHGEPO protein migrated as a broad band with an average molecular mass of 75 kDa. The mutant, dHGEPO, was slightly higher than the wild-type (WT) dimeri-EPO band. Enzymatic N-deglycosylation resulted in the formation of a narrow band with a molecular mass twice of that of of monomeric EPO digested with an N-glycosylation enzyme. Hematocrit values were remarkably increased in all treatment groups. Pharmacokinetic analysis was also affected when 2.5 IU of dHGEPO were intravenously injected into the tails of the mice. The biological activity and half-life of dHGEPO mutants were enhanced as compared to the corresponding items associated the WT dimeric EPO. These results suggest that recombinant dHGEPO may be attractive biological and therapeutic targets.

• Reproductive and Developmental Biology
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• 제36권1호
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• pp.71-77
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• 2012

The objective of this study was to examine the effect of thymidine treatment during $in$ $vitro$ maturation (IVM) of porcine follicular oocytes on blastocyst development. Porcine oocytes were treated with thymidine (10 mM, 20 mM and 30 mM) for 2 or 6 hr in the preiods of IVM I and/or II. The survival rates of the blastocysts in the 6 hr treatment groups of 10 mM and 20 mM during IVM I period were significantly higher than those of control group ($p$<0.05). However, the survival rate of the blastocysts in the 2 hr treatment group of 20 mM during IVM II period was significantly higher than control group ($p$<0.05). Furthermore, the survival rate of the blastocysts in the 6 hr treatment group of 30 mM during IVM II period was significantly lower than control group ($p$<0.05). Consistent with the previous result, blastocyst development of both IVM I and II treatment group was also showed as similar pattern. Total and apoptotic cell numbers of blastocysts derived from thymidine treated porcine oocytes were examined by using Tunel assay. The results showed that there was no significant differences in total cell number of blastocysts between thymidine treated and untreated groups. However, apoptosis-positive cells in the thymidine treated group (6 hr IVM I) were significantly lower than those of other groups ($p$<0.05). Taken together, these results indicate that high quality oocytes were selected by DNA synthesis mechanism according to high concentration thymidine treatment during porcine oocyte maturation. Therefore, we concluded that presumptive selected oocytes by thymidine treatment during maturation periods improved the further embryo development and embryonic quality of IVF embryos by decreasing the incidence of apoptosis in preimplantation porcine embryos.

• 한국균학회소식:학술대회논문집
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• 한국균학회 2016년도 춘계학술대회 및 임시총회
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• pp.27-27
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• 2016

The ascomycete fungus Fusarium graminearum is the most common pathogen of Fusarium head blight (FHB), a devastating disease for major cereal crops worldwide. FHB causes significant crop losses by reducing grain yield and quality as well as contaminating cereals with trichothecenes and zearalenone (ZEA) that pose a serious threat to animal health and food safety. ZEA is a causative agent of hyperestrogenic syndrome in mammals and can result in reproductive disorders in farm animals. In F. graminearum, the ZEA biosynthetic cluster is composed of four genes, PKS4, PKS13, ZEB1, and ZEB2, which encode a reducing polyketide synthase, a nonreducing polyketide synthase, an isoamyl alcohol oxidase, and a transcription factor, respectively. Although it is known that ZEB2 primarily acts as a regulator of ZEA biosynthetic cluster genes, the mechanism underlying this regulation remains undetermined. In this study, two isoforms (ZEB2L and ZEB2S) from the ZEB2 gene in F. graminearum were characterized. It was revealed that ZEB2L contains a basic leucine zipper (bZIP) DNA-binding domain at the N-terminus, whereas ZEB2S is an N-terminally truncated form of ZEB2L that lacks the bZIP domain. Interestingly, ZEA triggered the induction of both ZEB2L and ZEB2S transcription. In ZEA producing condition, the expression of ZEB2S transcripts via alternative promoter usage was directly or indirectly initiated by ZEA. Physical interaction between ZEB2L and ZEB2L as well as between ZEB2L and ZEB2S was observed in the nucleus. The ZEB2S-ZEB2S interaction was detected in both the cytosol and the nucleus. ZEB2L-ZEB2L oligomers activated ZEA biosynthetic cluster genes, including ZEB2L. ZEB2S inhibited ZEB2L transcription by forming ZEB2L-ZEB2S heterodimers, which reduced the DNA-binding activity of ZEB2L. This study provides insight into the autoregulation of ZEB2 expression by alternative promoter usage and a feedback loop during ZEA production.

• 한국수정란이식학회지
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• 제31권1호
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• pp.39-46
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• 2016

Cryopreservation has been applied successfully in many mammalian species. Nevertheless, pig embryos, because of their greater susceptibility to cryoinjuries, have shown a reduced developmental competence. The aim of this study was to evaluate the survival status of vitrified-warmed porcine embryos. Forced blastocoele collapse (FBC) and non-FBC blastocysts are vitrified and concomitantly cultured in culture media which were supplemented with/without fetal bovine serum (FBS). Porcine vitrified-warmed embryos were examined in four different methods: group A, non-FBC without FBS; group B, non-FBC with FBS; group C, FBC without FBS; group D, FBC with FBS. After culture, differences in survival rates of blastocysts derived from vitrified-warmed porcine embryos were found in group A~D (39.5 (A) vs 52.5 (B) and 54.8 (C) vs 66.7% (D), respectively, p<0.05). Reactive oxygen species (ROS) level of survived blastocysts was lower in group D than that of another groups (p<0.05). Moreover, total cell number of survived blastocysts was higher in group D than that of other groups (p<0.05). Otherwise, group D showed significantly lower number of apoptotic cells than other groups ($2.0{\pm}1.5$ vs $3.2{\pm}2.1$, $2.8{\pm}1.9$, and $2.7{\pm}1.6$, respectively, p<0.05). Taken together, these results showed that FBS/FBC improves the developmental competence of vitrified porcine embryos by modulating intracellular levels of ROS and the apoptotic index during the vitrification/warming procedure. Therefore, we suggest that FBS and FBC are effective treatment techniques during the vitrification/warming procedures of porcine blastocysts.

• Reproductive and Developmental Biology
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• 제37권4호
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• pp.247-253
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• 2013

Here, we present an approach of blood plasma proteome profiling and their comparisons between the young and the adult pigs as prerequisite for the identification of bio-markers related to the health conditions, growth performance and meat quality. To profile the proteome in porcine plasma, blood samples were collected from 19 young piglets and 20 adult male barrows and the plasma was retrieved. Then, protein profiling was initiated using one and two-dimensional electrophoresis. Proteins were spotted and then identified by MALDI-TOF-TOF and LC-MS-MS. In the results, more than thirty-six and twenty eight protein spots were selected in young piglets and adult pigs, respectively and twenty three proteins were identified. The proteome profile images were compared between those ones using Image Master Version 7.0. The image of expressed proteome showed that most of proteins from plasma of young piglet separated clearly and concentrated in 2DE display compared to ones from adult. Image analysis in detail was carried out to look for the specific proteins related to age progression. It demonstrated that the characteristics of proteome expression could be distinct to their age stages. Further investigations needed to proceed to understand the age dependent change of protein conformation and biological meaning of those differences in proteome expression between young and mature adult pigs.

• 한국패류학회지
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• 제26권3호
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• pp.245-254
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• 2010

For the studies of germ cell development and maturation in the ovary, the gametogenic cycle and the number of spawning seasons per year in female Ruditapes philippinarum were investigated by quantitative statistical analysis using an Image Analyzer System. Compared with the results by qualitative and quantitative analyses, monthly variations in female gonad indice by qualitative histological analysis showed a pattern similar to that of the female gametogenic cycle calculated by quantitative statistical analysis. The number of spawning seasons occurred once per year, from June to October. In quantitative statistical analysis using an image analyzer system, monthly changes in the portions (%) of the ovary area to total tissue areas in females increased in March and reached a maximum in May, and then showed a rapid decrease from June to October when spawning occurred. And also monthly changes in portions (%) of follicle areas to the ovary area and in portions of oocyte areas to ovarian tissue areas in females began to increase in March and reached a maximum in May, and then. rapidly dropped from June to October when spawnig occurred. From these data, it is apparent that the number of spawning seasons occurred once per year, from June to October. Monthly changes in the number of the oocyte per $mm^2$ and in mean diameter of the oocyte in captured image which were calculated for each female slide showed a maximum in May and reached the minimum from December to February. Therefore, female R. philippinarum showed a unimodal gametogenic cycle during the year.

• 한국동물생명공학회지
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• 제35권4호
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• pp.315-322
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• 2020

Aquaporin channels (AQPs) are known to play an important role in the development of ovarian follicles through their function in water transport pathways. Compared to other AQPs, research on the role of AQP4 in female reproductive physiology, particularly in cattle, remains limited. In our previous study, gene chip microarray data showed a downregulation of AQP4 in bovine cystic follicles. This study was performed to validate the AQP4 expression level at the protein level in bovine follicles using immunohistochemistry, Western blotting, and immunoprecipitation assays. Immunostaining data showed that AQP4 was expressed in granulosa and theca cells of bovine ovarian follicles. The ovarian follicles were classified according to size as small (< 10 mm) or large (> 25 mm) in diameter. Consistent with earlier microarray data, semi-quantitative PCR data showed a decrease in AQP4 mRNA expression in large follicles. Western blot analysis showed a downregulation of the AQP4 protein in large follicles. In addition, AQP4 was immunoprecipitated and blotted with anti-AQP4 antibody in small and large follicles. Accordingly, AQP4 exhibited a low expression in large follicles. These results show that AQP4 is downregulated in bovine ovarian large follicles, suggesting that the downregulation of AQP4 expression may interfere with follicular water transport, leading to bovine follicular cysts.

• Journal of Microbiology and Biotechnology
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• 제32권6호
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• pp.792-799
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• 2022

As a vital problem in reproductive health, recurrent spontaneous abortion (RSA) affects about 1% of women. We performed this study with an aim to explore the molecular mechanism of interleukin-23 (IL-23) and find optimal or effective methods to improve RSA. First, ELISA was applied to evaluate the expressions of IL-23 and its receptor in HTR-8/SVneo cells after IL-23 treatment. CCK-8, TUNEL, wound healing and transwell assays were employed to assess the proliferation, apoptosis, migration and invasion of HTR-8/SVneo cells, respectively. Additionally, the expressions of apoptosis-, migration-, epithelial-mesenchymal transition- (EMT-) and p38 MAPK signaling pathway-related proteins were measured by western blotting. To further investigate the relationship between IL-23 and p38 MAPK signaling pathway, HTR-8/SVneo cells were treated for 1 h with p38 MAPK inhibitor SB239063, followed by a series of cellular experiments on proliferation, apoptosis, migration and invasion, as aforementioned. The results showed that IL-23 and its receptors were greatly elevated in IL-23-treated HTR-8/SVneo cells. Additionally, IL-23 demonstrated suppressive effects on the proliferation, apoptosis, migration, invasion and EMT of IL-23-treated HTR-8/SVneo cells. More importantly, the molecular mechanism of IL-23 was revealed in this study; that is to say, IL-23 inhibited the proliferation, apoptosis, migration, invasion and EMT of IL-23-treated HTR-8/SVneo cells via activating p38 MAPK signaling pathway. In conclusion, IL-23 inhibits trophoblast proliferation, migration, and EMT via activating p38 MAPK signaling pathway, suggesting that IL-23 might be a novel target for the improvement of RSA.