• Journal of Microbiology and Biotechnology
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• v.33 no.6
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• pp.788-796
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• 2023

Canine parvovirus type 2 (CPV-2) has high morbidity and mortality rates in canines. Nonstructural protein 1 (NS1) of CPV-2 has endonuclease activity, initiates viral DNA replication, and is highly conserved. Thus, it is a promising target for antiviral inhibitor development. We overexpressed a 41.9 kDa active recombinant endonuclease in Escherichia coli and designed a nicking assay using carboxyfluorescein and quencher-linked ssDNA as substrates. The optimal temperature and pH of the endonuclease were 37℃ and pH 7, respectively. Curcumin, bisdemethoxycurcumin, demethoxycurcumin, linoleic acid, tannic acid, and α-tocopherol inhibited CPV-2 NS1 endonuclease with IC₅₀ values of 0.29 to 8.03 µM. The extracted turmeric, yerba mate, and sesame cake suppressed CPV-2 NS1 endonuclease with IC₅₀ values of 1.48, 7.09, and 52.67 ㎍/ml, respectively. The binding affinity between curcumin, the strongest inhibitor, and CPV-2 NS1 endonuclease by molecular docking was -6.4 kcal/mol. Curcumin inhibited CPV-2 NS1 endonuclease via numerous hydrophobic interactions and two hydrogen bonds with Lys97 and Pro111 in the allosteric site. These results suggest that adding curcuminoids, linoleic acid, tannic acid, α-tocopherol, extracted turmeric, sesame cake, and yerba to the diet could prevent CPV-2 infection.

• Molecules and Cells
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• v.47 no.1
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• pp.100001.1-100001.8
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• 2024

In eukaryotes, a primary protein quality control (PQC) process involves the destruction of conformationally misfolded proteins through the ubiquitin-proteasome system. Because approximately one-third of eukaryotic proteomes fold and assemble within the endoplasmic reticulum (ER) before being sent to their destinations, the ER plays a crucial role in PQC. The specific functions and biochemical roles of several E3 ubiquitin ligases involved in ER-associated degradation in mammals, on the other hand, are mainly unknown. We identified 2 E3 ligases, ubiquitin protein ligase E3 component N-recognin 1 (UBR1) and ubiquitin protein ligase E3 component N-recognin 2 (UBR2), which are the key N-recognins in the N-degron pathway and participate in the ER stress response in mammalian cells by modulating their stability. Cells lacking UBR1 and UBR2 are hypersensitive to ER stress-induced apoptosis. Under normal circumstances, these proteins are polyubiquitinated through Lys48-specific linkages and are then degraded by the 26S proteasome. In contrast, when cells are subjected to ER stress, UBR1 and UBR2 exhibit greater stability, potentially as a cellular adaptive response to stressful conditions. Although the precise mechanisms underlying these findings require further investigation, our findings show that cytoplasmic UBR1 and UBR2 have anti-ER stress activities and contribute to global PQC in mammals. These data also reveal an additional level of complexity within the mammalian ER-associated degradation system, implicating potential involvement of the N-degron pathway.

• Food Science and Biotechnology
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• v.17 no.1
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• pp.58-65
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• 2008

To see the possibility of irradiation as an alternative to ultra high temperature (UHT) sterilization, the quality characteristics of milk were analyzed. Milk treated by UHT ($135^{\circ}C$ for 4 sec) and irradiation at higher than 3 kGy showed no viable counts after 7 days of storage at $4^{\circ}C$. The contents of certain amino acids of milk, such as Arg, Asp, Glu, Ile, Leu, Lys, Pro, Ser, Thr, and Tyr, were lower in irradiated groups at 10 kGy than in UHT-treated one, but no difference was observed between irradiated milks at less than 5 kGy and UHT. The capillary electrophoresis (CE) patterns of the milk irradiated at 10 kGy showed a similar trend to the raw milk, low temperature long time (LTLT, $63^{\circ}C$ for 30 min), and high temperature short time (HTST, $72^{\circ}C$ for 15 sec) treated. However, the CE pattern of UHT-treated milk was different. Rennet coagulation test agreed with the CE results, showing that all milk samples were coagulated by rennet addition except for UHT-treated milk after 1 hr. These results suggest that irradiation of milk reduce the content of individual amino acids but it may not induce severe conformational change at a protein level when compared with UHT treatment.

• Asian-Australasian Journal of Animal Sciences
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• v.29 no.8
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• pp.1166-1172
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• 2016

This study was conducted to determine the effect of dietary supplementation of liquid DL-methionine hydroxy analog free acid (DL-MHA) on growth performance and gastrointestinal conditions of piglets. One hundred and eighty crossbred barrow piglets (Large White${\times}$Landrace, body weight: $12.48{\pm}0.33kg$) were divided into three groups with ten replications of six piglets each. Piglets received DL-MHA in diet at a concentration of 0 (control group), 0.15%, or 0.24%. The results indicated that increasing the standardized ileal digestible (SID) of sulfur amino acids (SAA) to lysine (SID SAA:Lys) ratio by supplementation of DL-MHA tended to increase (quadratic; p<0.10) weight gain and ADG, and showed slightly greater (linear; p<0.10) gain:feed ratio. The pH in the diet and cecum linearly decreased (p<0.01), whereas pH in colon had a quadratic response (p<0.01) with increasing supplementation of DL-MHA. By greater supplementation of DL-MHA, the population of Lactobacillus spp. in rectum was likely to increase (quadratic; p<0.10), but Escherichia coli population in the diet was reduced (quadratic; p<0.05). Acetic acid concentration and total short-chain fatty acids in cecum linearly increased (p<0.05), whereas valeric acid in cecum quadratically increased (p<0.05) with increasing DL-MHA levels. Moreover, the villous height of the jejunum quadratically increased (p<0.01) as the supplementation of DL-MHA was increased. It is concluded that the addition of DL-MHA in diet improved the growth performance and the morphology of gastrointestinal tract of piglets.

• Journal of Microbiology and Biotechnology
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• v.20 no.4
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• pp.700-707
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• 2010

A novel antioxidative peptide (APVPH I, antioxidative peptides from venison protein hydrolysates I) was purified from venison by enzymatic hydrolysis, column chromatography of DEAE-Sephacel, and high-performance liquid chromatography. The molecular mass of the purified peptide was found to be 9,853 Da and the amino acid sequences of the purified peptide was Met-Gln-Ile-Phe-Val-Lys-Thr-Leu-Thr-Gly. The purpose of this study was to evaluate the effects of APVPH I against $H_2O_2$-induced neuronal cells damage in PC-12 cells. Antioxidative enzyme levels in cultured neuronal cells were increased in the presence of the peptide. In addition, APVPH I inhibited productions of nitric oxide (NO), reactive oxygen species (ROS), malondialdehyde (MDA), and cell death against $H_2O_2$-induced neuronal cell damage in PC-12 cells. It was presumed to be APVPH I involved in regulating the apoptosis-related gene expression in the cell environment. The present results indicate that APVPH I substantially contributes to antioxidative properties in neuronal cells.

• Journal of Microbiology and Biotechnology
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• v.8 no.6
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• pp.639-644
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• 1998

Extracellular serine proteases were isolated from a soil bacterium, alkalophilic coryneform bacterium TU-19, which have been grown in a liquid medium optimized at 3$0^{\circ}C$ and pH 10.0. Three different sizes, 120 kDa (protease I), 80 kDa (protease II), and 45 kDa (protease III), of serine pro teases were purified using Sephadex G-150 and QAE-Sephadex chromatography (Kang et al. 1995. Agric. Chem Biotech. 38: 534-540). SDS-PAGE showed that the 120 kDa protease was degraded into the 80 kDa protease in 20 mM Tris-HCI (pH 8.0) buffer solution. This degradation was enhanced in the presence of 0.5 M NaCl and 5 mM EDTA, but was inhibited in the presence of 5 mM $CaCl_2$. These results indicated that the $Ca^{2＋}$ ion seems to stabilize the 120 kDa protease like other proteases derived from Bacillus species. The $NH_2$-terminal amino acid sequences of the 10 residues of both proteases were completely identical: Met-Asn-Thr-Gln-Asn-Ser-Phe-Leu-Ile-Lys. In contrast to this, the 80 kDa protease has 1.5 times higher specific activity than the 120 kDa protease does (Kang et al. 1995. Agric. Chern. Biotech. 38: 534-540). Therefore the C-terminal of the 120 kDa protease seems to be autolyzed to the 80 kDa protease but this autolysis did not decrease the protease activity. Optimum pH and temperature of both 80 kDa and 120 kDa proteases were pH 10.5 and $45^{\circ}C$, respectively, and pH and thermal stability were almost identical. Several divalent ions except the $Fe^{2＋}$ ion showed similar effects on activities of both proteases, which are similarly resistant to three different detergents.

• Journal of Microbiology and Biotechnology
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• v.12 no.2
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• pp.217-221
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• 2002

dTDP-D-glucose 4,6-dehydratase (TDPDH) catalyzes the conversion of dTDP-D-glucose to dTDP-4-keto-6-deoxy-D-glucose, and requires $NAD^＋$ as a coenzyme for its catalytic activity. The dTDP-D-glucose 4,6-dehydratase from Streptomyces antibioticus $Tu{\ddot}99$ tightly binds $NAD^＋$ [19]. In order to determine the role of lysine-148 in the $NAD^＋$ binding, the lysine of the dTDP-D-glucose 4,6-dehydratase from Streptomyces antibioticus $Tu{\ddot}99$ was mutated to various amino acids by site-directed mutagenesis. The catalytic activity of the four mutated enzymes of TDPDH did not recover after addition of $NAD^＋$ . However, the activity of K159A, the mutated enzyme of UDP-D-glucose 4-epimerase (UDPE), recovered after the addition of $NAD^＋$ [15]. Although dTDP-glucose 4,6-dehydratase, and UDP-galactose (glucose) 4-epimerase are members of the short-chain dehydrogenase/reductase SDR family and the lysine-148 of TDPDH was highly conserved as in UDPE (Lys-159), the function of the lysine-148 of TDPDH was different from that of UDPE. The mutated enzymes showed that the lysine-148 of the dTDP-D-glucose 4,6-dehydratase played no role in the $NAD^＋$ binding. Accordingly, it is suggested that the lysine-148 of the dTDP-D-glucose 4,6-dehydratase is involved in the folding of TDPDH.

• Journal of Microbiology and Biotechnology
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• v.20 no.1
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• pp.146-152
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• 2010

Streptomyces clavuligerus NRRL3585 produces a clinically important $\beta$-lactamase inhibitor, clavulanic acid (CA). In order to increase the production of CA, the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene (gap) was deleted in S. clavuligerus NRRL3585 to overcome the limited glyceraldehyde-3-phosphate pool; the replicative and integrative expressions of ccaR (specific regulator of the CA biosynthetic operon) and claR (Lys-type transcriptional activator) genes were transformed together into a deletion mutant to improve clavulanic acid production. We constructed two recombinant plasmids to enhance the production of CA in the gap1 deletion mutant of S. clavuligerus NRRL3585: pHN11 was constructed for overexpression of ccaR-claR, whereas pHN12 was constructed for their chromosomal integration. Both pHN11 and pHN12 transformants enhanced the production of CA by 2.59-fold and 5.85-fold, respectively, compared with the gap1 deletion mutant. For further enhancement of CA, we fed the pHN11 and pHN12 transformants ornithine and glycerol. Compared with the gap1 deletion mutant, ornithine increased CA production by 3.24- and 6.51-fold in the pHN11 and pHN12 transformants, respectively, glycerol increased CA by 2.96- and 6.21-fold, respectively, and ornithine and glycerol together increased CA by 3.72- and 7.02-fold, respectively.

• Reproductive and Developmental Biology
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• v.39 no.4
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• pp.105-115
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• 2015

Toll-like receptor 7 (TLR7) is critical for the triggering of innate immune response by recognizing the conserved molecular patterns of single-stranded RNA (ssRNA) viruses and mediated antigenic adaptive immunity. To understand how TLR7 distinguish pathogen-derived molecular patterns from the host self, it is essential to be able to identify TLR7 receptor interaction interfaces, such as active sites or R848-agonist binding sites. The functional interfaces of TLR7 can serve as targets for structure-based drug design in studying the TLR7 receptor's structure-function relationship. In contrast to mammalian TLR7, chicken TLR7 (chTLR7) is unknown for its important biological function. Therefore, it has been targeted to mediate contrasting evolutionary patterns of positive selection into non-synonymous SNPs across eleven species using TLR7 conservation patterns (evolutionary conserved and class-specific trace residues), where protein sequence differences to the TLR7 receptors of interest record mutation that have passed positive section across the species. In this study, we characterized the Lys609 residue on chTLR7-ECD homodimer interfaces to reflect the current tendency of evolving positive selection to be transfer into a stabilization direction of the R848-agonist/chTLR7-ECDs complex under the phylogenetically variable position across species and we suggest a potential indicator for contrasting evolutionary patterns of both the species TLR-ECDs.

• Asian-Australasian Journal of Animal Sciences
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• v.18 no.3
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• pp.384-389
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• 2005

These experiments were conducted to evaluate the feeding value of rice protein concentrate (RPC) in weaning pigs. In expt. I, a 5-week feeding trial was conducted with 126 pigs (L${\times}$Y${\times}$D; 21 d-old; 5.32${\pm}$0.34 kg). Treatments were spray-dried plasma protein (SDPP; control), soy protein concentrate (SPC) and RPC (phase 1), and dried porcine soluble (DPS; control), SPC and RPC (phase 2). An ileal digestibility trial was also conducted to compare digestibility of amino acids in the tested protein sources. In expt. II, 160 weaning pigs (L${\times}$Y${\times}$D; 21 d-old; 5.65${\pm}$0.35 kg) were used in a 5-week feeding trial to determine the optimal inclusion level of RPC in the diet. Treatments were control (9% SPC), and three levels of RPC instead of SPC in the diets (3, 6 and 9%). During phase 1, pigs fed SDPP showed better (p<0.05) ADG and FCR compared with those fed SPC or RPC, while there was no difference in ADFI among treatments. During phase 2, however, pigs fed DPS showed lower (p<0.05) ADG than those fed SPC or RPC. During the total period, there were no significant differences in ADG, ADFI and FCR among treatments. The apparent ileal digestibilities of his, lys, phe, thr and met were not different among the tested protein sources. The apparent ileal digestibilities of arg, ile, leu and val were lower (p<0.05) in RPC than SDPP. The true ileal digestibilities of arg and leu were lower (p<0.05) in RPC than SDPP and SPC. However, that of met was higher (p<0.05) in RPC than SDPP. In expt. II, there were no significant differences in ADG and FCR when SPC was substituted with RPC up to 9% during the total period. In conclusion, based on our experimental results, RPC would replace SPC in the complex prestarter diet, which is somewhat cheaper than SPC.