• Asian-Australasian Journal of Animal Sciences
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• 제23권4호
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• pp.530-536
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• 2010

Cdc25B is a mitotic regulator that might act as a starter phosphatase to initiate the positive feedback loop at the entry into mitotic (M) phase. In the present study, distribution of Cdc25B mRNA in duodenal mucosa of the chicken was demonstrated by means of in situ hybridization histochemistry (ISHH) using sense and antisense digoxigenin (DIG)-labeled RNA probes. The results showed that there were many labeled cells distributing in the duodenal mucosa of the adult chicken. Of these labeled cells, 81.60${\pm}$9.63% of Cdc25B mRNA positive cells was distributed in the basilar part and mid-portion of the intestinal gland and 36.21${\pm}$8.81% in the middle and basilar portion of villi of the small intestine of the chicken, respectively. Most of these labeled cells were positive in the regions of the stem cell and proliferation. The signals of ISHH decreased from basilar to upper part in the crypt of Lieberkuhn and weakened in the inferior villi of the duodenum. Moreover, the positive signals were both in the cytoplasm and cell nucleus. However, the labeled cells were negative in both the lamina muscularis mucosae and muscular layer. The results of ISHH suggested the existence of Cdc25B mRNA and vigorous proliferation activities in the duodenal mucosa of adult chicken, replenishing the cells which had sloughed off from the superior part of the villus. Our results provide some molecular evidence for a regular pattern of avian intestinal epitheliosis and functional partition and provide an approach to further study of the locations of Cdc25B in the chicken.

• Animal Bioscience
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• 제36권7호
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• pp.1022-1033
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• 2023

Objective: p66Shc, a 66 kDa protein isoform encoded by the proto-oncogene SHC, is an essential intracellular redox homeostasis regulatory enzyme that is involved in the regulation of cellular oxidative stress, apoptosis induction and the occurrence of multiple age-related diseases. This study investigated the expression profile and functional characteristics of p66Shc during preimplantation embryo development in sheep. Methods: The expression pattern of p66Shc during preimplantation embryo development in sheep at the mRNA and protein levels were studied by quantitative real-time polymerase chain reaction (RT-qPCR) and immunofluorescence staining. The effect of p66Shc knockdown on the developmental potential were evaluated by cleavage rate, morula rate and blastocyst rate. The effect of p66Shc deficiency on reactive oxygen species (ROS) production, DNA oxidative damage and the expression of antioxidant enzymes (e.g., catalase and manganese superoxide dismutase [MnSOD]) were also investigated by immunofluorescence staining. Results: Our results showed that p66Shc mRNA and protein were expressed in all stages of sheep early embryos and that p66Shc mRNA was significantly downregulated in the 4-to 8-cell stage (p<0.05) and significantly upregulated in the morula and blastocyst stages after embryonic genome activation (EGA) (p<0.05). Immunofluorescence staining showed that the p66Shc protein was mainly located in the peripheral region of the blastomere cytoplasm at different stages of preimplantation embryonic development. Notably, serine (Ser36)-phosphorylated p66Shc localized only in the cytoplasm during the 2- to 8-cell stage prior to EGA, while phosphorylated (Ser36) p66Shc localized not only in the cytoplasm but also predominantly in the nucleus after EGA. RNAi-mediated silencing of p66Shc via microinjection of p66Shc siRNA into sheep zygotes resulted in significant decreases in p66Shc mRNA and protein levels (p<0.05). Knockdown of p66Shc resulted in significant declines in the levels of intracellular ROS (p<0.05) and the DNA damage marker 8-hydroxy2'-deoxyguanosine (p<0.05), markedly increased MnSOD levels (p<0.05) and resulted in a tendency to develop to the morula stage. Conclusion: These results indicate that p66Shc is involved in the metabolic regulation of ROS production and DNA oxidative damage during sheep early embryonic development.

• Molecules and Cells
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• 제22권3호
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• pp.285-290
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• 2006

The light sensing system in the eye directly affects the circadian oscillator in the mammalian suprachiasmatic nucleus (SCN). To investigate this relationship in the rat, we examined the circadian expression of clock genes in the SCN and eye tissue during a 24 h day/night cycle. In the SCN, rPer1 and rPer2 mRNAs were expressed in a clear circadian rhythm like rCry1 and rCry2 mRNAs, whereas the level of BMAL1 and CLOCK mRNAs decreased during the day and increased during the night with a relatively low amplitude. It seems that the clock genes of the SCN may function in response to a master clock oscillation in the rat. In the eye, the rCry1 and rCry2 were expressed in a circadian rhythm with an increase during subjective day and a decrease during subjective night. However, the expression of Opn4 mRNA did not exhibit a clear circadian pattern, although its expression was higher in daytime than at night. This suggests that cryptochromes located in the eye, rather than melanopsin, are the major photoreceptive system for synchronizing the circadian rhythm of the SCN in the rat.

• 한국어병학회지
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• 제16권2호
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• pp.125-129
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• 2003

Conncxin (Cx) is an important and essential protein induction of oocyte maturation, which is present in almost all mammalian tissucs except cirulating blood cells and adult skeletal museles. In this study, goldfish Cx43 cDNA sequence is available in GenBank under the accession number AB078505. Homology analyses using the GenBank and EMBL general database searches indicated that goldfish Cx43 cDNA has a high homology with carp Cx43 (95.1% identity), zebrafish Cx43 (90.5% identity), and chicken Cx43 (81.9% identity). Goldfish Cx43 is similar to the Cx family in its general features, and all the typical Cx consensus sequences are also found. Moreover, significantly increased Cx43 transcrpts were observed in mature goldfish (GSI; 18.3-21.7) pituitary and ovary when compared with immature goldfish (GSI; 4.9-6.0). Cx43 transeripts were weakly detectrd in both liver and kidney of immature and mature goldfish. There is possible that Cx43 nctivity was relation to oocyte maturation in the goldfish.

• Asian Pacific Journal of Cancer Prevention
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• 제14권1호
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• pp.195-200
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• 2013

Secreted protein acidic and rich in cysteines-like protein 1 (SPARCL1), an extracellular matrix glycoprotein, has been implicated in the pathogenesis of several disorders including cancer. However, little is known about the expression and significance of SPARCL1 in human breast cancer. The aim of this study was to determine the expression pattern and clinicopathological significance of SPARCL1 in a Chinese breast cancer cohort. mRNA and protein expression of SPARCL1 in human breast cancer cell lines and breast cancer tissues was detected using the reverse transcription-polymerase chain reaction, real-time quantitative PCR, and Western blotting, respectively. Immunostaining of SPARCL1 in 282 Chinese breast cancer samples was examined and associations with clinicopathological parameters were analyzed. Compared to the positive expression in immortalized human breast epithelial cells, SPARCL1 was nearly absent in human breast cancer cell lines. Similarly, a significantly reduced expression of SPARCL1 was observed in human breast cancer tissues compared to that in normal breast epithelial tissues, for both mRNA and protein levels (P < 0.001). Immunohistochemical analysis showed that strong cytoplasmic immunostaining of SPARCL1 was observed in almost all normal breast samples (43/45) while moderate and strong immunostaining of SPARCL1 was only detected in 191 of 282 (67.7%) breast cancer cases. Moreover, down-regulation of SPARCL1 was significantly correlated with lymphatic metastasis (P = 0.020) and poor grade (P = 0.044). In conclusion, SPARCL1 may be involved in the breast tumorigenesis and serve as a promising target for therapy of breast cancer.

• Journal of Animal Science and Technology
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• 제44권6호
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• pp.677-684
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• 2002

한우 성장단계 특이발현 유전자 탐색에 관한 연구(장 등, 2002)에서 선정된 후보유전자 중, EPV 20, TCTP 및 aldolase A를 비롯하여 24개월령 cDNA probe에 대하여 강한 signal을 나타낸 ADRP 유전자의 발현양상을 분석하기 위하여 성장단계별 한우 등심조직 시료를 사용하여 semiquantitative RT-PCR 및 northern blot 분석을 실시하였다. EPV 20 유전자는 한우 등심조직에서 성장단계에 따른 발현량 차이가 거의 없는 결과를 근거로, 근육조직에서 항상 발현되는 유전자로 판단하였다. 또한 발달단계에 따라 발현량 차이가 있는 것으로 보고된 aldolase A 유전자 역시 뚜렷한 발현량 차이는 없었으며, aldolase A의 muscle specific promoter와 근육분화 관련 transcription factor에 관한 연구를 통하여 근육분화 및 근육수축에 있어 aldolase A의 역할 및 조절작용을 밝힐 수 있을 것으로 사료된다. 성장관련 단백질로 알려져 있는 TCTP의 발현양상을 분석한 결과, 한우 등심조직에서 성장함에 따라 발현량이 약간 증가하는 양상을 나타내었다. 이와 같은 발현양상 분석결과로 TCTP 유전자는 성장과 관련된 기능이 있지만 동물의 성장에 있어 직접적으로 관여하지는 않을 것으로 판단된다. 지방세포 분화의 초기단계에서 발현량이 급증하고 lipid droplet 형성에 관여하며 지방축적과도 관련이 있는 것으로 보고된 ADRP 유전자의 transcript의 크기는 약 1.82 kb 이었고, 24개월령 한우 등심조직에서 발현량이 급격히 증가하였으며, 30개월령 조직에서는 감소하는 양상을 나타내었다. 이와 같은 결과로부터 ADRP 유전자를 한우 성장단계 특이발현 유전자로 선정하였으며, ADRP 유전자의 발현양상은 근육내 지방축적과 관련이 있을 것으로 추정하였다. 이후에는 발현조절 기작의 해명 및 근육내 지방축적과의 관련성 분석을 위하여 ADRP 유전자의 전체적인 구조 및 전사조절 인자 분석을 비롯하여 다른 지방대사 및 지방축적 관련 유전자와의 상호작용 및 다형성 탐색분석에 관한 연구가 필요 할 것으로 판단하였다.

• Fisheries and Aquatic Sciences
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• 제18권1호
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• pp.73-80
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• 2015

To examine the interrelationship between transgenic insertion patterns and transgene expression profiles in established transgenic fish lines, four stable transgenic marine medaka Oryzias dancena germlines harboring ${\beta}$-actin regulator-driven RFP reporter constructs were selected. The established transgenic strains were characterized with regard to their transgenic genotypes (insertion pattern, concatemer formation, and transgene copy number based on genomic Southern blot hybridization and qPCR assay) and expression characteristics at the mRNA (qRT-PCR), protein (western blot), and phenotypic (fluorescent appearance) levels. From comparative examinations, it was found that transgenic expression at both the transcription and translation levels could be significantly downregulated in transgenic strains, potentially through methylation-mediated transgene silencing that was particularly associated with the formation of a long tail-to-head tandem concatemer in the chromosomal integration site(s). When this occurred, an inverse relationship between the transgene copy number and fluorescence intensity was observed in the resultant transgenic fish. However, with the other transgenic genotype, transgenic individuals with an identical Southern blot hybridization pattern, containing a tandem concatemer(s), had very different expression levels (highly robust vs. low expression strengths), which was possibly related to the differential epigenetic modifications and/or degrees of methylation. The concatemer-dependent downregulation of transgene activity could be induced in transgenic fish, but the overall pattern was strain-specific. Our data suggest that neither a low (or single) transgene copy number nor tandem transgene concatemerization is indicative of strong or silenced transgene expression in transgenic fish carrying a ubiquitous transgene. Hence, a sufficient number of transgenic lineages, with different genotypes, should be considered to ensure the establishment of the best-performance transgenic line(s) for practical applications.

• Fisheries and Aquatic Sciences
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• 제15권4호
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• pp.317-324
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• 2012

We characterized the cytoskeletal beta-actin (${\beta}$-ACT) gene (actb) and its 5'-upstream regulatory region in the Javanese ricefish Oryzias javanicus. The gene and protein structures were deduced from amino acid sequences of the actb gene and conserved in the teleost lineage. The O. javanicus actb gene has common transcription factor binding motifs in its regulatory region found in teleostean orthologues. Following quantitative reverse transcription-PCR, actb gene transcripts were detected in all tissues examined; however, the basal expression levels were different. During early development, O. javanicus actb mRNA levels showed a gradual increase and peaked between late somitogenesis and the heartbeat stage. Microinjection of O. javanicus embryos with the actb gene promoter-driven red fluorescent protein (RFP) gene reporter vector showed a ubiquitous distribution of RFP signals, although most exhibited a mosaic pattern of transgene expression. A small number of microinjected embryos displayed a wide distribution of RFP signals over their entire body, which resembled the expression pattern of endogenous actb. Data from this study provide a basis to develop a transgenic system with ubiquitous expression of foreign genes in O. javanicus.

• 자연과학논문집
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• 제11권1호
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• pp.95-98
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• 1999

$CO_2$ gas 처리가 배추 자가불화합성 타파에 미치는 영향을 조사하고자 배추 inbred line의 암술에서 mRNA를 분리하여 DD-PCR을 수행하였다. $CO_2$ gas를 처리한 암술에서 분리한 mRNA와 처리하지 않은 꽃에서 분리한 암술 mRNA 발현과의 특이적인 차이를 DD-PCR로 조사한 결과 세 가지 각기 다른 anchor primer에 대해 모두 18개의 특이적 증폭 DNA fragment를 얻을 수 있었다. 이 결과로 미루어 보아 $CO_2$ gas 처리에 의한 암술 mRNA발현의 변화가 배추 자가불화합성 타파에 영향을 미치는 것으로 생각된다.

• 한국어병학회지
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• 제36권2호
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• pp.337-348
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• 2023

본 연구에서는 넙치의 해독 과정에서 amprolium hydrochloride의 영향을 평가하기 위해 수행되었다. 이전 연구에서 보고된 amprolium의 LD₅₀ 값을 이용하여 두 가지 실험을 진행하였다. 첫 번째는 30마리의 넙치를 5개의 대조군 및 실험군으로 나누었고 4, 8, 16, 32 mg/kg 용량의 amprolium을 근육 내 주사 투여하였다. 주사 후 8, 24, 48 시간에 간과 신장을 적출하여 약물 대사 효소와 전염증성 사이토카인 유전자의 발현을 분석하였다. 32 mg/kg 용량의 실험군에서 IL-1β mRNA의 높은 발현을 확인하였고, CYP1A는 이와 반대의 결과를 보였으며, 간에서 UGT와 GST mRNA의 발현은 유의하게 감소하는 것을 확인하였다. 또한 신장에서 amprolium 주사 투여 후 약물 대사 효소와 사이토카인 유전자의 억제가 관찰되었다. 또 다른 실험에서는 4, 8, 16, 32 mg/kg과 60, 80, 100, 120 mg/kg의 용량을 설정하여 근육 내 주사 투여하였다. 주사를 완료하고 6일 후 간을 적출하여 유전자의 발현을 확인하였다. IL-1β의 발현은 4 mg/kg 용량 실험군에서 유의적으로 매우 높은 발현을 보였다. GST의 mRNA 발현 또한 4 mg/kg 용량 실험군에서 높은 발현을 보였다. 결론적으로 우리의 결과는 amprolium이 가축 산업의 가장 안전한 합성 항콕시듐 약물 중 하나로 간주되지만 넙치의 간접 또는 직접적인 물리적 또는 생물학적 독성을 유발하는 것으로 판단된다.