• International Journal of Industrial Entomology and Biomaterials
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• v.46 no.1
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• pp.16-23
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• 2023

The silkworm's dormancy and embryonic development are accomplished through the interaction of various genes. Analysis of the expression of several interacting genes can predict the embryonic stage of silkworms. In this study, we analyzed the changes in the expression level of genes at each stage during the embryonic development of dormant silkworm eggs and selected genes that can predict the hatching time. Jam123 and Jam124 silkworms were collected after egg laying, and the silkworm eggs were preserved using a double refrigeration method and expression analysis was performed for 23 genes during embryogenesis. There were 5 genes showing significant changes during embryogenesis: UDP-glucuronosyltransferases (BmUGTs), heat shock protein hsp20.8 (BmHsp20.8), Cytochromes b5-like proteins (BmCytb5), Krüppel homolog 1 (BmKr-h1), and cuticular protein RR-1 motif 41 (BmCpr41). As a result of quantitative comparison of the expression levels of these 5 genes through real-time PCR, the BmUGTs gene showed a difference between Jam123 and Jam124, making it difficult to see it as an indicator for predicting hatching time. However, the BmHsp20.8 gene had a common expression decreased at the imminent hatching stage. In addition, it was confirmed that the expression level of the BmCytb5 gene decreased to the lowest level at the time of imminent hatching, and the expression of the BmKr-h gene was made only at the time of imminent hatching. The expression of the last BmCpr41 gene can be confirmed only at the time of imminent hatching, and it was confirmed that it shows a rapid increase right before hatching. Taken together, these results suggest that expression analysis of BmHsp20.8, BmCytb5, BmKr-h1, and BmCpr41 genes can determine the stage of embryogenesis, predict hatching time, which facilitate better management of silkworm eggs.

• Animal Bioscience
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• v.36 no.7
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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.

• Animal Bioscience
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• v.37 no.2
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• pp.193-202
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• 2024

Objective: Oxidative stress (OS) is a pathological process arising from the excessive production of free radicals in the body. It has the potential to alter animal gene expression and cause damage to the jejunum. However, there have been few reports of changes in the expression of long noncoding RNAs (lncRNAs) in the jejunum in piglets under OS. The purpose of this research was to examine how lncRNAs in piglet jejunum change under OS. Methods: The abdominal cavities of piglets were injected with diquat (DQ) to produce OS. Raw reads were downloaded from the SRA database. RNA-seq was utilized to study the expression of lncRNAs in piglets under OS. Additionally, six randomly selected lncRNAs were verified using quantitative real-time polymerase chain reaction (qRT-PCR) to examine the mechanism of oxidative damage. Results: A total of 79 lncRNAs were differentially expressed (DE) in the treatment group compared to the negative control group. The target genes of DE lncRNAs were enriched in gene ontology (GO) terms and Kyoto encyclopedia of genes and genomes (KEGG) signaling pathways. Chemical carcinogenesis-reactive oxygen species, the Foxo signaling pathway, colorectal cancer, and the AMPK signaling pathway were all linked to OS. Conclusion: Our results demonstrated that DQ-induced OS causes differential expression of lncRNAs, laying the groundwork for future research into the processes involved in the jejunum's response to OS.

• Biomolecules & Therapeutics
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• v.32 no.1
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• pp.154-161
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• 2024

Amyotrophic lateral sclerosis (ALS) is a fatal motor neuron disorder that causes progressive paralysis. L-Citrulline is a nonessential neutral amino acid produced by L-arginine via nitric oxide synthase (NOS). According to previous studies, the pathogenesis of ALS entails glutamate toxicity, oxidative stress, protein misfolding, and neurofilament disruption. In addition, L-citrulline prevents neuronal cell death in brain ischemia; therefore, we investigated the change in the transport of L-citrulline under various pathological conditions in a cell line model of ALS. We examined the uptake of [¹⁴C]L-citrulline in wild-type (hSOD1wt/WT) and mutant NSC-34/ SOD1^G93A (MT) cell lines. The cell viability was determined via MTT assay. A transport study was performed to determine the uptake of [¹⁴C]L-citrulline. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis was performed to determine the expression levels of rat large neutral amino acid transported 1 (rLAT1) in ALS cell lines. Nitric oxide (NO) assay was performed using Griess reagent. L-Citrulline had a restorative effect on glutamate induced cell death, and increased [¹⁴C]L-citrulline uptake and mRNA levels of the large neutral amino acid transporter (LAT1) in the glutamate-treated ALS disease model (MT). NO levels increased significantly when MT cells were pretreated with glutamate for 24 h and restored by co-treatment with L-citrulline. Co-treatment of MT cells with L-arginine, an NO donor, increased NO levels. NSC-34 cells exposed to high glucose conditions showed a significant increase in [¹⁴C]L-citrulline uptake and LAT1 mRNA expression levels, which were restored to normal levels upon co-treatment with unlabeled L-citrulline. In contrast, exposure of the MT cell line to tumor necrosis factor alpha, lipopolysaccharides, and hypertonic condition decreased the uptake significantly which was restored to the normal level by co-treating with unlabeled L-citrulline. L-Citrulline can restore NO levels and cellular uptake in ALS-affected cells with glutamate cytotoxicity, pro-inflammatory cytokines, or other pathological states, suggesting that L-citrulline supplementation in ALS may play a key role in providing neuroprotection.

• Animal Bioscience
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• v.37 no.3
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• pp.509-521
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• 2024

Objective: It was shown that microRNAs (miRNAs) play an important role in milk protein synthesis. However, the post-transcriptional regulation of casein expression by exogenous miRNA (xeno-miRNAs) in ruminants remains unclear. This study explores the regulatory roles of alfalfa xeno-miR162 on casein synthesis in bovine mammary epithelial cells (bMECs). Methods: The effects of alfalfa xenomiR-162 and G protein subunit gamma 11 (GNG11) on proliferation and milk protein metabolism of bMECs were detected by 5-Ethynyl-2'-Deoxyuridine (EdU) staining, flow cytometry, cell counting kit-8 (CCK-8), enzyme-linked immunosorbent assay, quantitative real-time polymerase chain reaction (qRT-PCR), and Western blot. Dual-luciferase reporter assay was used to verify the targeting relationship between GNG11 and xenomiR-162. Results: Results showed that over-expression of xenomiR-162 inhibited cell proliferation but promoted apoptosis, which also up-regulated the expression of several casein coding genes, including CSN1S1, CSN1S2, and CSN3, while decreasing the expression of CSN2. Furthermore, the targeting relationship between GNG11 and xenomiR-162 was determined, and it was confirmed that GNG11 silencing also inhibited cell proliferation but promoted apoptosis and reduced the expression of casein coding genes and genes related to the mammalian target of rapamycin (mTOR) pathway. Conclusion: Alfalfa xenomiR-162 appears to regulate bMECs proliferation and milk protein synthesis via GNG11 in the mTOR pathway, suggesting that this xeno-miRNA could be harnessed to modulate CSN3 expression in dairy cows, and increase κ-casein contents in milk.

• IMMUNE NETWORK
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• v.21 no.6
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• pp.43.1-43.14
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• 2021

Group 3 innate lymphoid cells (ILC3), which express IL-22 and IL-17A, has been introduced as one of pathologic cells in axial spondyloarthritis (axSpA). Dyslipidaemia should be managed in axSpA patients to reduce cardiovascular disease, and dyslipidaemia promotes inflammation. This study aimed to reveal the role of circulating ILC3 in axSpA and the impact of dyslipidaemia on axSpA pathogenesis. AxSpA patients with or without dyslipidaemia and healthy control were recruited. Peripheral blood samples were collected, and flow cytometry analysis of circulating ILC3 and CD4⁺ T cells was performed. The correlation between Ankylosing Spondylitis Disease Activity Score (ASDAS)-C-reactive protein (CRP) and circulating immune cells was evaluated. The effect of oxidized low-density lipoprotein cholesterol (oxLDL-C) on immune cell differentiation was confirmed. AxSpA human monocytes were cultured with with oxLDL-C, IL-22, or oxLDL-C plus IL-22 to evaluate osteoclastogenesis using tartrate-resistant acid phosphatase (TRAP) staining and real-time quantitative PCR of osteoclast-related gene expression. Total of 34 axSpA patients (13 with dyslipidaemia and 21 without) were included in the analysis. Circulating IL-22⁺ ILC3 and Th17 were significantly elevated in axSpA patients with dyslipidaemia (p=0.001 and p=0.034, respectively), and circulating IL-22⁺ ILC3 significantly correlated with ASDAS-CRP (Rho=0.4198 and p=0.0367). Stimulation with oxLDL-C significantly increased IL-22⁺ ILC3, NKp44^- ILC3, and Th17 cells, and these were reversed by CD36 blocking agent. IL-22 and oxLDL-C increased TRAP⁺ cells and osteoclast-related gene expression. This study suggested potential role of circulating IL-22⁺ ILC3 as biomarker in axSpA. Furthermore, dyslipidaemia augmented IL-22⁺ ILC3 differentiation, and oxLDL-C and IL-22 markedly increased osteoclastogenesis of axSpA.

• IMMUNE NETWORK
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• v.20 no.5
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• pp.40.1-40.15
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• 2020

The protein encoded by the Gene Associated with Retinoid-Interferon-Induced Mortality-19 (GRIM-19) is located in the mitochondrial inner membrane and is homologous to the NADH dehydrogenase 1-alpha subcomplex subunit 13 of the electron transport chain. Multiple sclerosis (MS) is a demyelinating disease that damages the brain and spinal cord. Although both the cause and mechanism of MS progression remain unclear, it is accepted that an immune disorder is involved. We explored whether GRIM-19 ameliorated MS by increasing the levels of inflammatory cytokines and immune cells; we used a mouse model of experimental autoimmune encephalomyelitis (EAE) to this end. Six-to-eight-week-old male C57BL/6, IFNγ-knockout (KO), and GRIM-19 transgenic mice were used; EAE was induced in all strains. A GRIM-19 overexpression vector (GRIM19 OVN) was electrophoretically injected intravenously. The levels of Th1 and Th17 cells were measured via flow cytometry, immunofluorescence, and immunohistochemical analysis. IL-17A and IFNγ expression levels were assessed via ELISA and quantitative PCR. IL-17A expression decreased and IFNγ expression increased in EAE mice that received injections of the GRIM-19 OVN. GRIM19 transgenic mice expressed more IFNγ than did wild-type mice; this inhibited EAE development. However, the effect of GRIM-19 overexpression on the EAE of IFNγ-KO mice did not differ from that of the empty vector. GRIM-19 expression was therapeutic for EAE mice, elevating the IFNγ level. GRIM-19 regulated the Th17/Treg cell balance.

• Korean Journal of Microbiology
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• v.49 no.2
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• pp.137-145
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• 2013

This study was performed at 2 sites of Nak-Dong River to investigate the changes of nitrifiers depending on the presence and absence of organic pollutants (due to the effluents of domestic wastewater treatment plant, WWTP). Conventional chemical parameters such as T-N, $NH_4$-N, $NO_2$-N, $NO_3$-N were measured and the quantitative nitrifiers at the 2 sites were analyzed comparatively by fluorescent in situ hybridization (FISH) with NSO190 and NIT3, after checking the presence of gene amoA of ammonia oxidizing bacteria (AOB) and 16S rDNA signature sequence for Nitrobacter sp. that belongs to nitrite oxidizing bacteria (NOB). Also ${\alpha}{\cdot}{\beta}{\cdot}{\gamma}$-Proteobacteria were detected using FISH to get a glimpse of the general bacterial community structure of the sites. Based on the distribution structure of the ${\alpha}{\cdot}{\beta}{\cdot}{\gamma}$-Proteobacteria and the measurement of nitrogen in different phases, it could be said that the site 2 was more polluted with organics than site 1. Corresponding to the above conclusion, the average numbers of AOB and NOB detected by NSO160 and NIT3, respectively, at site 2 [AOB, $9.3{\times}10^5$; NOB, $1.6{\times}10^6$ (cells/ml)] was more than those at site 1 [AOB, $7.8{\times}10^5$; NOB, $0.8{\times}10^6$ (cells/ml)] and also their ratios to total counts were higher at site 2 (AOB, 27%; NOB, 34%) than those at site 1 (AOB, 18%; NOB, 23%). Thus, it could be concluded that the nitrification at site 2 was more active due to continuous loading of organics from the effluents of domestic WWTP, compared to site 1 located closed to raw drinking water supply and subsequently less polluted with organics.

• Journal of Life Science
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• v.30 no.9
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• pp.804-812
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• 2020

The giant mealworm beetle, Zophobas atratus (Coleoptera: Tenebrionidae) has been used as a protein source for small pets and mammals. Recently, it was temporarily registered in the list of the Food Code. We previously performed an in silico analysis of the Zophobas atratus transcriptome to identify putative antimicrobial peptides and identified several antimicrobial peptide candidates. Among them, we assessed the antimicrobial and anti-inflammatory activities of zophobacin 1 that was selected bio-informatically based on its physicochemical properties against microorganisms and mouse macrophage Raw264.7 cells. Zophobacin 1 showed antimicrobial activities against microorganisms without inducing hemolysis and decreased the nitric oxide production of the lipopolysaccharide-induced Raw264.7 cells. Moreover, ELISA and Western blot analysis revealed that zophobacin 1 reduced expression levels of pro-inflammatory enzymes such as inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). We also investigated expression of pro-inflammatory cytokines (interleukin-6 and interleukin-1β) production through quantitative real time-PCR and ELISA. Zophobacin 1 markedly reduced the expression level of cytokines through the regulation of mitogen-activated protein kinases (MAPKs) and nuclear factor kappa B (NF-κB) signaling. We confirmed that zophobacin 1 bound to bacterial cell membranes via a specific interaction with lipopolysaccharides. These data suggest that zophobacin 1 could be promising molecules for development as antimicrobial and anti-inflammatory therapeutic agents.

• Journal of Life Science
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• v.26 no.4
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• pp.419-430
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• 2016

Powdery mildew disease caused by Podosphaera xanthii is a major concern for Cucumis melo production worldwide. Knowledge on genetic behavior of the related genes and their modulating phytohormones often offer the most efficient approach to develop resistance against different diseases. Mildew Resistance Locus O (MLO) genes encode proteins with seven transmembrane domains that have significant function in plant resistance to powdery mildew fungus. We collected 14 MLO genes from ‘Melonomics’ database. Multiple sequence analysis of MLO proteins revealed the existence of both evolutionary conserved cysteine and proline residues. Moreover, natural genetic variation in conserved amino acids and their replacement by other amino acids are also observed. Real-time quantitative PCR expression analysis was conducted for the leaf samples of P. xanthii infected and phyto-hormones (methyl jasmonate and salicylic acid) treated plants in melon ‘SCNU1154’ line. Upon P. xanthii infection using 7 different races, the melon line showed variable disease reactions with respect to spread of infection symptoms and disease severity. Three out of 14 CmMLO genes were up-regulated and 7 were down-regulated in leaf samples in response to all races. The up- or down-regulation of the other 4 CmMLO genes was race-specific. The expression of 14 CmMLO genes under methyl jasmonate and salicylic acid application was also variable. Eleven CmMLO genes were up-regulated under salicylic acid treatment, and 7 were up-regulated under methyl jasmonate treatments in C. melo L. Taken together, these stress-responsive CmMLO genes might be useful resources for the development of powdery mildew disease resistant C. melo L.