• Korean Journal of Microbiology
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• v.47 no.4
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• pp.281-288
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• 2011

To screen downstream genes of Aspergillus nidulans MsnA showing amino acid sequence similarity to the zinc finger region of Msn2/4 stress response transcription factors in Saccharomyces cerevisiae, differentially expressed genes (DEG) in MsnA overexpressed or msnA null mutant strains compared to wild type have been isolated. The cognate gene IDs were identified by DNA sequencing of the selected DEGs. Among those, DEG6 was known as mstB encoding a putative monosaccharide transporter. Expression level of mstB mRNA was increased in MsnA overproducing strains and MsnA bound directly to the promoter region of mstB in vitro. MstB containing twelve transmembrane domains exhibited 80% of amino acid sequence identities to A. niger MstA a high-affinity monosaccharide transporter. A null mutant of mstB was phenotypically undistinguishable to wild type. On the other hand, forced overexpression of MstB caused the increased formation of sexual structure cleistothecia in 0.1% glucose condition where wild type showed almost no cleistothecia. This result implies that mstB is involved in transport of monosaccharide required for sexual differentiation.

• Journal of Animal Science and Technology
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• v.65 no.3
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• pp.652-663
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• 2023

The rumen fluids contain a wide range of bacteria, protozoa, fungi, and viruses. The various ruminal microorganisms in the rumen provide nutrients by fermenting the forage they eat. During metabolic processes, microorganisms present in the rumen release diverse vesicles during the fermentation process. Therefore, in this study, we confirmed the function of rumen extracellular vesicles (EVs) and their interaction with the host. We confirmed the structure of the rumen EVs by transmission electron microscope (TEM) and the size of the particles using nanoparticle tracking analysis (NTA). Rumen EVs range in size from 100 nm to 400 nm and are composed of microvesicles, microparticles, and ectosomes. Using the Caenorhabditis elegans smart animal model, we verified the interaction between the host and rumen EVs. Exposure of C. elegans to rumen EVs did not significantly enhance longevity, whereas exposure to the pathogenic bacteria Escherichia coli O157:H7 and Staphylococcus aureus significantly increased lifespan. Furthermore, transcriptome analysis showed gene expression alterations in C. elegans exposed to rumen EVs, with significant changes in the metabolic pathway, fatty acid degradation, and biosynthesis of cofactors. Our study describes the effect of rumen EV interactions with the host and provides novel insights for discovering biotherapeutic agents in the animal industry.

• Asian-Australasian Journal of Animal Sciences
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• v.28 no.6
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• pp.876-887
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• 2015

Dicer, an ribonuclease type III type endonuclease, is the key enzyme involved in biogenesis of microRNAs (miRNAs) and small interfering RNAs (siRNAs), and thus plays a critical role in RNA interference through post transcriptional regulation of gene expression. This enzyme has not been well studied in the Indian water buffalo, an important species known for disease resistance and high milk production. In this study, the primary coding sequence (5,778 bp) of bubaline dicer (GenBank: AB969677.1) was determined and the bubaline Dicer1 biocomputationally characterized to determine the phylogenetic signature among higher eukaryotes. The evolutionary tree revealed that all the transcript variants of Dicer1 belonging to a specific species were within the same node and the sequences belonging to primates, rodents and lagomorphs, avians and reptiles formed independent clusters. The bubaline dicer1 is closely related to that of cattle and other ruminants and significantly divergent from dicer of lower species such as tapeworm, sea urchin and fruit fly. Evolutionary divergence analysis conducted using MEGA6 software indicated that dicer has undergone purifying selection over the time. Seventeen divergent sequences, representing each of the families/taxa were selected to study the specific regions of positive vis-$\grave{a}$-vis negative selection using different models like single likelihood ancestor counting, fixed effects likelihood, and random effects likelihood of Datamonkey server. Comparative analysis of the domain structure revealed that Dicer1 is conserved across mammalian species while variation both in terms of length of Dicer enzyme and presence or absence of domain is evident in the lower organisms.

• Asian-Australasian Journal of Animal Sciences
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• v.12 no.1
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• pp.129-138
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• 1999

If rumen bacteria can be manipulated to utilize nutrients (i.e., ammonia and plant cell wall carbohydrates) more completely and efficiently, the need for protein supplementation can be reduced or eliminated and the digestion of fiber in forage or agricultural residue-based diets could be enhanced. However, these approaches require a complete and accurate description of the rumen community, as well as methods for the rapid and accurate detection of microbial density, diversity, phylogeny, and gene expression. Molecular ecology techniques based on small subunit (SSU) rRNA sequences, nucleic acid probes and the polymerase chain reaction (PCR) can potentially provide a complete description of the microbial ecology of the rumen of ruminant animals. The development of these molecular tools will result in greater insights into community structure and activity of gut microbial ecosystems in relation to functional interactions between different bacteria, spatial and temporal relationships between different microorganisms and between microorganisms and reed panicles. Molecular approaches based on SSU rRNA serve to evaluate the presence of specific sequences in the community and provide a link between knowledge obtained from pure cultures and the microbial populations they represent in the rumen. The successful development and application of these methods promises to provide opportunities to link distribution and identity of gastrointestinal microbes in their natural environment with their genetic potential and in situ activities. The use of approaches for assessing pupulation dynamics as well as for assessing community functionality will result in an increased understanding and a complete description of the gastrointestinal communities of production animals fed under different dietary regimes, and lead to new strategies for improving animal growth.

• Journal of Microbiology and Biotechnology
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• v.33 no.9
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• pp.1213-1227
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• 2023

Fetal growth restriction (FGR) is a prevalent obstetric condition. This study aimed to investigate the role of Toll-like receptor 9 (TLR9) in regulating the inflammatory response and gut microbiota structure in FGR. An FGR animal model was established in rats, and ODN1668 and hydroxychloroquine (HCQ) were administered. Changes in gut microbiota structure were assessed using 16S rRNA sequencing, and fecal microbiota transplantation (FMT) was conducted. HTR-8/Svneo cells were treated with ODN1668 and HCQ to evaluate cell growth. Histopathological analysis was performed, and relative factor levels were measured. The results showed that FGR rats exhibited elevated levels of TLR9 and myeloid differentiating primary response gene 88 (MyD88). In vitro experiments demonstrated that TLR9 inhibited trophoblast cell proliferation and invasion. TLR9 upregulated lipopolysaccharide (LPS), LPS-binding protein (LBP), interleukin (IL)-1β and tumor necrosis factor (TNF)-α while downregulating IL-10. TLR9 activated the TARF3-TBK1-IRF3 signaling pathway. In vivo experiments showed HCQ reduced inflammation in FGR rats, and the relative cytokine expression followed a similar trend to that observed in vitro. TLR9 stimulated neutrophil activation. HCQ in FGR rats resulted in changes in the abundance of Eubacterium_coprostanoligenes_group at the family level and the abundance of Eubacterium_coprostanoligenes_group and Bacteroides at the genus level. TLR9 and associated inflammatory factors were correlated with Bacteroides, Prevotella, Streptococcus, and Prevotellaceae_Ga6A1_group. FMT from FGR rats interfered with the therapeutic effects of HCQ. In conclusion, our findings suggest that TLR9 regulates the inflammatory response and gut microbiota structure in FGR, providing new insights into the pathogenesis of FGR and suggesting potential therapeutic interventions.

• Fisheries and Aquatic Sciences
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• v.17 no.4
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• pp.471-478
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• 2014

Genomic organization, including the structural characteristics of 5'-flanking regions of two 65-kDa protein (WAP65) isoform genes associated with warm temperature acclimation, were characterized and their transcriptional responses to immune challenges were examined in the intestine, kidney and spleen of the mud loach (Misgurnus mizolepis; Cypriniformes). Both mud loach Wap65 isoform genes displayed a 10-exon structure that is common to most teleostean Wap65 genes. The two mud loach Wap65 isoforms were predicted to possess various stress- and immune-related transcription factor binding sites in their regulatory regions; however, the predicted motif profiles differed between the two isoforms, and the inflammation-related transcription factor binding motifs, such as NF-${\kappa}B$ and CREBP sites, were more highlighted in the Wap65-2 isoform than the Wap65-1 isoform. The results of qRT-PCR indicated that experimental immune challenges using Edwardsiella tarda, lipopolysaccharide or polyI:C induced the Wap65-2 isoform more than Wap65-1 isoform, although modulation patterns in response to these challenges were tissue- and stimulant-dependent. This study confirms that functional diversification between the two mud loach Wap65 isoforms (i.e., closer involvement of Wap65-2 in the acute phase of inflammation and innate immunity) occurs at the mRNA level in multiple tissues, and suggests that such differential modulation patterns between the two isoforms are related to the different transcription factor binding profiles in their regulatory regions.

• Journal of Microbiology and Biotechnology
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• v.26 no.2
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• pp.347-355
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• 2016

An exo-β-D-glucosaminidase (AorCsxA) from Aspergillus oryzae FL402 was heterologously expressed and purified. The deduced amino acid sequence indicated that AorCsxA belonged to glycoside hydrolase family 2. AorCsxA digested colloid chitosan into glucosamine but not into chitosan oligosaccharides, demonstrating exo-β-D-glucosaminidase (CsxA) activity. AorCsxA exhibited optimal activity at pH 5.5 and 50℃; however, the enzyme expressed in Pichia pastoris (PpAorCsxA) showed much stronger thermostability at 50℃ than that expressed in Escherichia coli (EcAorCsxA), which may be related to glycosylation. AorCsxA activity was inhibited by EDTA and most of the tested metal ions. A single amino acid mutation (F769W) in AorCsxA significantly enhanced the specific activity and hydrolysis velocity as revealed by comparison of V_max and k_cat values with those of the wild-type enzyme. The three-dimensional structure suggested the tightened pocket at the active site of F769W enabled efficient substrate binding. The AorCsxA gene was heterologously expressed in P. pastoris, and one transformant was found to produce 222 U/ml activity during the high-cell-density fermentation. This AorCsxA-overexpressing P. pastoris strain is feasible for large-scale production of AorCsxA.

• Animal cells and systems
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• v.8 no.1
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• pp.27-32
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• 2004

Endothellial cells are subjected to hemodynamic shear stress, the dragging force generated by blood flow. Shear stress regulates endothelial cell shape, structure, and function, including gene expression. Since endothelial cells must be anchored to their extracellular matrices(ECM) for their survival and growth, we hypothesized that ECMs are crucial for shear-dependent activation of extracellular signalactivated regulated kinase(ERK) that is important for cell proliferation. Shear stress-dependent activation of ERK was observed in cells plated on two different matrices, fibronectin and vitronectin(the two most physiologically relevant ECM in endothelial cells). We then treated bovine aortic endothelial cells(BAECs) with Arg-Gly-Asp(RGD) peptides that block the functional activation of integrin binding to fibronectin and vitronectin, and a nonfunctional peptide as a control. Treatment of cells with the RGD peptides, but not the control peptide, significantly inhibited ERK activity in a concentration-dependent manner. This supports the idea that integrin adhesion to the ligands, fibronectin and vitronectin, mediates shear stress-dependent activation of ERK. Subsequently, whereas antagonists of vitronectin(LM 609, an antibody for integrin ${\alpha}_{\gamma}$/${\beta}_3$ and XT 199, an antagonist specific for integrin ${\alpha}_{\gamma}$/${\beta}_3$) did not have any effect on shear-dependent activation of ERK, antagonists of fibronectin(a neutralizing antibody for integrin ${\alpha}_5$/${\beta}_1$or ${\alpha}_4$${\beta}_1$ and SM256) had an inhibitory effect. These results clearly demonstrate that mechanoactivation of ERK requires anchoring of endothelial cells to fibronectin through integrins.

• Korean Journal of Environmental Agriculture
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• v.38 no.3
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• pp.185-196
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• 2019

BACKGROUND: This study investigated the effects of rice genetically modified to be resistant against rice blast and rice bacterial blight on the soil microbial community. A comparative analysis of the effects of rice genetically modified rice choline kinase (OsCK1) gene for disease resistance (GM rice) and the Nakdong parental cultivar (non-GM rice) on the soil microbial community at each stage was conducted using rhizosphere soil of the OsCK1 and Nakdong rice. METHODS AND RESULTS: The soil chemistry at each growth stage and the bacterial and fungal population densities were analyzed. Soil DNA was extracted from the samples, and the microbial community structures of the two soils were analyzed by pyrosequencing. No significant differences were observed in the soil chemistry and microbial population density between the two soils. The taxonomic analysis showed that Chloroflexi, Proteobacteria, Firmicutes, Actinobacteria, and Acidobacteria were present in all soils as the major phyla. Although the source tracking analysis per phylogenetic rank revealed that there were differences in the bacteria between the GM and non-GM soil as well as among the cultivation stages, the GM and non-GM soil were grouped according to the growth stages in the UPGMA dendrogram analysis. CONCLUSION: The difference in bacterial distributions between Nakdong and OsCK1 rice soils at each phylogenetic level detected in microbial community analysis by pyrosequencing may be due to the genetic modification done on GM rice or due to heterogeneity of the soil environment. In order to clarify this, it is necessary to analyze changes in root exudates along with the expression of transgene. A more detailed study involving additional multilateral soil analyses is required.

• Proceedings of the Korean Aquaculture Society Conference
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• 2003.10a
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• pp.44-44
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• 2003

Gene expression in five tissues of the abalone (Haliotis discus hannai) was investigated using an expressed sequence tag (EST) analysis. Randomly selected clones were obtained from cDNA libraries constructed with gill (GI), digestive diverticula(DD), hepatopancreas (HP), foot/mucus (FM) and rectangular muscle (RM). Of 1,235 clonesanalyzed (288 clones for GI, DD, HP each,166 for FM, and 205 for RM), 741 (60.0%) clones in total turned out to share significant similarity with the sequences from NCBI GenBank (less than 10/sup -3/ of e-values), 423 sequences showed poor similarity (> 10/sup -3/), and 71 sequences didn't match with any sequences in GenBank. The percent unique sequence (singleton) was ranged from 56.1% (RM) to 74.7% (FM) among libraries. On the other hand, overall percent singleton was 55.3% when all the ESTs from five libraries were assembled into contigs. Analysis of the organisms represented by the best hit for each EST (e-values < 10/sup -3/) showed that 23.8% matched with mammalian entries, 24.0% with mollusks, 14.4% with insects, 11.6% with fish and 26.2% with others. The expressed patterns differed among the tissues when judged by the categorization of the sequences from each library into 10 broad functional classes. In all the libraries, the class I (no hit o. poor similarity) was the largest category with an average of 40.1%. This largest class was followed by class V (general metabolisms) in DD (21.9%), GI (14.6%) and HP (16.7%), while the 'cell structure and motility'(class VI) was the second largest class in remaining two libraries (31.2% for RM and 9.6% for FM). The class IX (cell division and proliferation) was the smallest class in all the libraries (less than 3%). This report provides the first tissue-specific lists of expressed abalone genes, which could be a fundamental basis for genomics program of abalone species.