• ALGAE
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• v.36 no.4
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• pp.315-326
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• 2021

Ion channels are membrane protein complexes mediating passive ion flux across the cell membranes. Every organism has a certain set of ion channels that define its physiology. Dinoflagellates are ecologically important microorganisms characterized by effective physiological adaptability, which backs up their massive proliferations that often result in harmful blooms (red tides). In this study, we used a bioinformatics approach to identify homologs of known ion channels that belong to 36 ion channel families. We demonstrated that the versatility of the dinoflagellate physiology is underpinned by a high diversity of ion channels including homologs of animal and plant proteins, as well as channels unique to protists. The analysis of 27 transcriptomes allowed reconstructing a consensus ion channel repertoire (channelome) of dinoflagellates including the members of 31 ion channel families: inwardly-rectifying potassium channels, two-pore domain potassium channels, voltage-gated potassium channels (K_v), tandem K_v, cyclic nucleotide-binding domain-containing channels (CNBD), tandem CNBD, eukaryotic ionotropic glutamate receptors, large-conductance calcium-activated potassium channels, intermediate/small-conductance calcium-activated potassium channels, eukaryotic single-domain voltage-gated cation channels, transient receptor potential channels, two-pore domain calcium channels, four-domain voltage-gated cation channels, cation and anion Cys-loop receptors, small-conductivity mechanosensitive channels, large-conductivity mechanosensitive channels, voltage-gated proton channels, inositole-1,4,5-trisphosphate receptors, slow anion channels, aluminum-activated malate transporters and quick anion channels, mitochondrial calcium uniporters, voltage-dependent anion channels, vesicular chloride channels, ionotropic purinergic receptors, animal volage-insensitive cation channels, channelrhodopsins, bestrophins, voltage-gated chloride channels H⁺/Cl^- exchangers, plant calcium-permeable mechanosensitive channels, and trimeric intracellular cation channels. Overall, dinoflagellates represent cells able to respond to physical and chemical stimuli utilizing a wide range of G-protein coupled receptors- and Ca²⁺-dependent signaling pathways. The applied approach not only shed light on the ion channel set in dinoflagellates, but also provided the information on possible molecular mechanisms underlying vital cellular processes dependent on the ion transport.

• Journal of Animal Reproduction and Biotechnology
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• v.35 no.4
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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.

• The Plant Pathology Journal
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• v.37 no.6
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• pp.607-618
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• 2021

The pepper anthracnose fungus, Colletotrichum scovillei, causes severe losses of pepper fruit production in the tropical and temperate zones. RAC1 is a highly conserved small GTP-binding protein in the Rho GT-Pase family. This protein has been demonstrated to play a role in fungal development, and pathogenicity in several plant pathogenic fungi. However, the functional roles of RAC1 are not characterized in C. scovillei causing anthracnose on pepper fruits. Here, we generated a deletion mutant (𝜟Csrac1) via homologous recombination to investigate the functional roles of CsRAC1. The 𝜟Csrac1 showed pleiotropic defects in fungal growth and developments, including vegetative growth, conidiogenesis, conidial germination and appressorium formation, compared to wild-type. Although 𝜟Csrac1 was able to develop appressoria, it failed to differentiate appressorium pegs. However, 𝜟Csrac1 still caused anthracnose disease with significantly reduced rate on wounded pepper fruits. Further analyses revealed that 𝜟Csrac1 was defective in tolerance to oxidative stress and suppression of host-defense genes. Taken together, our results suggest that CsRAC1 plays essential roles in fungal development and pathogenicity in C. scovilleipepper fruit pathosystem.

• Food Science of Animal Resources
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• v.42 no.6
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• pp.1061-1073
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• 2022

Receptor activator of NF-kB ligand (RANKL) is known to play a major role in bone metabolism and the immune system, and its recombinant form has been expressed in bacterial systems for research since the last two decades. However, most of these recombinant forms are used after purification or directly using living cells. Here, there were cell extracts of recombinant Lactococcus lactis expressing mouse RANKL (mRANKL) used to evaluate its biological activity in mice. Mice were divided into three groups that were fed phosphate-buffered saline (PBS), wild-type L. lactis IL1403 (WT_CE), and recombinant L. lactis expressing mRANKL (mRANKL_CE). The small intestinal transcriptome and fecal microbiome were then profiled. The biological activity of mRANKL_CE was confirmed by studying RANK-RANKL signaling in vitro and in vivo. For small intestinal transcriptome, differentially expressed genes (DEGs) were identified in the mRANKL_CE group, and no DEGs were found in the WT_CE group. In the PBS vs. mRANKL_CE gene enrichment analysis, upregulated genes were enriched for heat shock protein binding, regulation of bone resorption, and calcium ion binding. In the gut microbiome analysis, there were no critical changes among the three groups. However, Lactobacillus and Sphingomonas were more abundant in the mRANKL_CE group than in the other two groups. Our results indicate that cell extracts of mRANKL_CE can play an effective role without a significant impact on the intestine. This strategy may be useful for the development of protein drugs.

• Journal of Nutrition and Health
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• v.32 no.6
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• pp.628-636
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• 1999

There is a marked increase in geriatric disease, especially liver disease, due to the continuous increase in alcohol and fat consumption. Since the fatty liver, induced by alcohol or fat, is basically from abnormalities in the lipid metabolism, it is possible that fatty acid binding protein(FABP) which is related to the fatty acid metabolism may also be abnormal in these livers. FABP is a small molecular weight protein family present in cytosol in high concentration. It has been proposed as a fatty acid transfer protein and as a binding protein responsible for controlling intracellular free fatty acid concentration. In this research, we have examined the relationship between liver FABP and fatty liver induced by alcohol or high cholesterol diet. Rats were fed one of either semipurified liquid diets; control diet containing 65% carbohydrate, 20% protein, and 15% fat or high cholesterol diet containing 1%(w/w) cholesterol or alcohol diet containing 37% of alcohol instead of carbohydrate. After 5 weeks of feeding period, all rats received commercial chow diet for 5 weeks to examine recovery effect. Liver and blood samples were collected at 0, 1, 3, 5 and 10 weeks to analyze lipid compositions. FABP was purified from liver cytosol and injected to rabbit to obtain antiserum. Liver FABP amount was determined by SDS-PAGE and western blotting methods. Fatty acid binding capacity was determined by binding of 14Cpalmitate with the delipidated liver cytosol. Consumption of alcohol increased serum cholesterol, triglyceride concentration and decreased HDL-cholesterol concentration after 5 weeks. Serum apolipoprotein B concentration increased after 3 weeks and LDL-cholesterol and apolipoprotein A concentration changed after 1 week. Liver cholesterol and triglyceride concentration increased after 3 weeks. Consumption of high cholesterol diet changed liver and serum lipid composition after 3 weeks. Swiching to normal diet for 5 weeks did not normalize most of lipid composition in serum and liver except serum and liver except serum cholesterol, triglyceride and liver cholesterol. Liver cytosol FABP content and the fatty acid binding capacity decreased dramatically after 1 week with alcohol consumption. This results indicate that FABP content changes before the changes before the changes of blood or liver lipid composition, suggesting changes of FABP may cause development of the fatty liver induced by alcohol and can be used as an index of detecting a early development of fatty liver.

• Asian-Australasian Journal of Animal Sciences
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• v.23 no.8
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• pp.1034-1042
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• 2010

This study aimed to determine the optimum dietary crude protein level in a typical diet for fattening pigs fed ad libitum under normal climate conditions in Central Vietnam. One hundred and ninety two gilts of Mong Cai local breed (MC), $F_1$ Large White${\times}$Mong Cai and $F_2$ crossbreds of (Landrace${\times}$Mong Cai)${\times}$Large White were used. At the start of the experiment, Mong Cai pigs weighed 12 kg at 11 weeks of age, $F_1$ pigs 12.1 kg at 8 weeks of age and $F_2$ pigs 12.2 kg at 8 weeks of age. Four diets differing in crude protein (CP) content (10.1, 13.1, 16.1 and 18.9% in DM) were formulated from rice bran, corn meal, cassava meal and fish meal. Calculated digestible energy content of the diets ranged from 13.5 to 13.8 MJ per kg DM. Pigs were housed individually in pens of $2.5\;m^2$ each and had ad libitum access to feed in a trough as well as water in bowls. The final weights after a growing period of 150 days were 66, 86 and 96 kg for MC, $F_1$ and $F_2$, respectively. Feed intake of MC pigs was highest at 13.1% CP while $F_1$ and $F_2$ had the highest feed intake at 16.1% CP. The results showed that for MC the maximum gain was obtained at levels between 13 to 16% CP. For the $F_1$ the maximum gain was at dietary protein levels of 16-17%. For $F_2$ the max gain was obtained at CP levels of 16 to 18%. Feed conversion was highest in MC pigs (~4.0) followed by $F_1$ (~3.3) and $F_2$ (~3.1), and within genotypes was lowest at the optimum CP level (p<0.05). Back fat thickness in MC (33.1 mm), $F_1$ (23.0 mm) and $F_2$ (20.5 mm) pigs was different and within genotypes was the lowest at intermediate CP levels. In conclusion, increasing the dietary crude protein contents in practical diets for pigs in Vietnam can increase production on small holder farms. Optimal performance for MC, $F_1$ and $F_2$ pigs is achieved at different dietary crude protein contents.

• Asian-Australasian Journal of Animal Sciences
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• v.10 no.6
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• pp.643-651
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• 1997

This experiment was conducted to determine apparent ileal and fecal digestibilities of protein sources for young pigs (15.6 kg BW) which were fitted with simple ileal T-cannulas. Experimental diets included one of the followings as a sole protein source: spray-dried plasma protein (SDPP), spray-dried blood meal (SDBM), soybean meal (SBM), isolated soy protein (ISP), dried skim milk (DSM), and fish meal (FM). The digestibilities of dry matter (DM), gross energy (GE) and crude protein (CP) in DSM-based diet were the highest (p < 0.05), and those of SDBM and SBM diets were the lowest at the terminal ileum. The average ileal digestibilities of essential amino acids (AAs) were 88.3, 84.7, 84.6, 83.4, 83.0 and 77.7% for pigs fed the DSM, ISP, SDBM, SBM, SDPP and FM diets, respectively. There were no significant differences in overall apparent ileal AAs digestibilities in DSM and ISP diets. Digestibilities of essential AAs in DSM, however, were significantly higher than those of ISP (p < 0.05). Among the essential AAs, the digestibility of arginine was higher except for SDBM or DSM, than other AAs. The ileal digestibilities of lysine, methionine and threonine were higher (p < 0.05) in pigs fed SDBM or DSM diet than in those fed SBM diet. Digestibilities for SDPP or ISP diet in these AAs were similar except for threonine which was higher (p < 0.05) for SDPP diet. The apparent ileal digestibilities of lysine and methionine ranged from 79.6 (FM) to 92.5 (DSM) and 75.4 (SDPP) to 88.7 (DSM), respectively. The apparent fecal digestibilities of average essential AAs were 90.4, 89.1, 86.0, 84.4, 84.2 and 80.8% in pigs fed the DSM, ISP, SDBM, SDPP, SBM and FM diets, respectively. The fecal digestibilities of essential AAs were higher (p < 0.05) in pigs fed the DSM and ISP. Lysine digestibility in pigs fed the DSM or ISP diet was higher (p < 0.05) than that of pigs fed SBM diet. Apparent essential AAs digestibility was the highest (p < 0.05) in pigs fed DSM diet, and was the lowest (p < 0.05) in pigs fed FM diet. Digestiibilities of essential AAs and nonessential AAs were higher when measured over the total digestive tract than at the end of the small intestine except for several AAs in SDPP, SDBM and SBM diets. In conclusion, whether it is fecal or ileal, the digestibilities of proximate nutrients and AAs in pigs fed DSM and ISP diet were the highest, and those of pigs fed FM was the lowest.

• Asian Pacific Journal of Cancer Prevention
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• v.14 no.9
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• pp.4999-5005
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• 2013

Objective: This study aimed to investigate expression of the proto-oncogene POK erythroid myeloid ontogenic factor (Pokemon) in colorectal cancer (CRC), and assess inhibitory effects of a small interference RNA (siRNA) expression vector in SW480 and SW620 cells. Methods: Semi-quantitative reverse transcription-polymerase chain reaction (PCR) and immunohistochemistry were performed to determine mRNA and protein expression levels of Pokemon in CRC tissues. Indirect immunofluorescence staining was applied to investigate the location of Pokemon in SW480 and SW620 cells. The siRNA expression vectors that were constructed to express a short hairpin RNA against Pokemon were transfected to the SW480 and SW620 cells with a liposome. Expression levels of Pokemon mRNA and protein were examined by real-time quantitative-fluorescent PCR and western blot analysis. The effects of Pokemon silencing on proliferation of SW480 and SW620 cells were evaluated with reference to growth curves with MTT assays. Results: The mRNA expression level of Pokemon in tumor tissues ($0.845{\pm}0.344$) was significantly higher than that in adjacent tumor specimens ($0.321{\pm}0.197$). The positive expression ratio of Pokemon protein in CRC (87.0%) was significantly higher than that in the adjacent tissues (19.6%). Strong fluorescence staining of Pokemon protein was observed in the cytoplasm of the SW480 and SW620 cells. The inhibition ratios of Pokemon mRNA and protein in the SW480 cells were 83.1% and 73.5% at 48 and 72 h, respectively, compared with those of the negative control cells with the siRNA. In the SW620 cells, the inhibition ratios of Pokemon mRNA and protein were 76.3% and 68.7% at 48 and 72 h, respectively. MTT showed that Pokemon gene silencing inhibited the proliferation of SW480 and SW620 cells. Conclusion: Overexpression of Pokemon in CRC may have a function in carcinogenesis and progression. siRNA expression vectors could effectively inhibit mRNA and protein expression of Pokemon in SW480 and SW620 cells, thereby reducing malignant cell proliferation.

• Korean Journal of Microbiology
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• v.43 no.3
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• pp.166-172
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• 2007

ERM proteins transfer the methyl group to $A_{2058}$ in 23S rRNA, which reduces the affinity of MLS (macrolide-lincosamide-streptogramin B) antibiotics to 23S rRNA, thereby confer the antibiotic resistance on micro-organisms ranging from antibiotic producers to pathogens and are classified into monomethyltransferase and dimethyltransferase. To investigate the differences between mono- and dimethyltransferase, tirD, a representative monomethylase gene was cloned in Escherichia coli from Streptomyces fradiae which contains ermSF, dimethylase gene as well to overexpress the TlrD for the first time. T7 promoter driven expression system successfully overexpress tlrD as a insoluble aggregate at $37^{\circ}C$ accumulating to around 55% of the total cell protein but unlike ErmSF, culturing at temperature as low as $18^{\circ}C$ did not make insoluble aggregate of protein into soluble protein. Coexpression of Thioredoxin and GroESL, chaperone was not helpful in turning into soluble protein either as in case of ErmSF. These results might suggest that differences between mono- and dimethylase could be investigated on the basis of the characteristics of protein structure. However, a very small amount of soluble protein which could not be detected by SDS-PAGE conferred antibiotic resistance on E. coli as in ErmSF which was expected from the activity exerted by monmethylase in a cell.

• Proceedings of the Korea Crystallographic Association Conference
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• 2003.05a
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• pp.18-18
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• 2003

Thioredoxin (Trx) is a small redox protein that is ubiquitously distributed from achaes to human. In diverse organisms, the protein is involved in various physiological roles by acting as electron donor and regulators of transcription and apoptosis as well as antioxidants. Sequences of Trx within various species are 27～69% identical to that of E. coli and all Trx proteins have the same overall fold, which consists of central five β strands surrounded by four α helices. The N-terminal cysteine in WCGPC motif of Trx is redox sensitive and the motif is highly conserved. Compared with general cysteine, the N-terminal cysteine has low pKa value. The result leads to increased reduction activity of protein. Recently, novel thio.edoxin-related protein (TRP14) was found from rat brain. TRP14 acts as disulfide reductase like Trx1, and its redox potential and pKa are similar to those of Trx1. However, TRP14 takes up electrons from cytosolic thioredoxin reductase (TrxR1), not from the mitochondrial thioredoxin reductase (TrxR2). Biological roles of TES14 were reported to be involved in regulating TNF-α induced signaling pathways in different manner with Trx1. In depletion experiments, depletion of TRP14 increased TNF-α induced phosphorylation and degradation of IκBα more than the depletion Trx1 did. It also facilitated activation of JNK and p38 MAP kinase induced by TNF-α. Unlike Trx1, TRP14 shows neither interaction nor interference with ASK1. Here, we determined three-dimensional crystal structure of TRP14 by MAD method at 1.8Å. The structure reveals that the conserved cis-Pro (Pro90) and active site-W-C-X-X-C motif, which may be involved in substrate recognition similar to Trx1 , are located at the beginning position of strand β4 and helix α2, respectively. The TRP14 structure also shows that surface of TRP14 in the vicinity of the active site, which is surrounded by an extended flexible loop and an additional short a helix, is different from that of Trx1. In addition, the structure exhibits that TRP14 interact with a distinct target proteins compared with Trx1 and the binding may depend mainly on hydrophobic and charge interactions. Consequently, the structure supports biological data that the TRP14 is involved in regulating TNF-α induced signaling pathways in different manner with Trx1.