• Fisheries and Aquatic Sciences
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• v.25 no.4
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• pp.243-249
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• 2022

Bacillus SW1-1 is a probiotic isolated from shrimp intestines. We investigated the effects of Bacillus SW1-1 coated diets on the growth, feed utilization, innate immunity, hematological parameters and resistance to Edwardsiella tarda in olive flounder (Paralichthys olivaceus). A commercial diet was used as the control (AP0) and two other diets were prepared by coating 0.25% (AP25) or 0.50% (AP50) probiotic powder which contains 1.0 × 10⁷ CFU/g Bacillus SW1-1. Four replicate groups of olive flounder (153 ± 2 g) were fed one of the diets for 12 weeks. Growth performance and feed utilization of the fish were not significantly affected by the dietary Bacillus SW1-1. After the challenge with E. tarda, AP50 group showed significantly higher survival than AP0 and AP25 groups. Innate immunity and anti-oxidant capacity of the fish were not significantly affected after the feeding trial. However, after the E. tarda challenge, the innate immune parameters (immunoglobulin, lysozyme and anti-protease) were significantly improved in fish fed AP25 and AP50 diets compared to those in fish fed AP0 diet. After the challenge test, significantly lower glucose level was observed in AP50 group compared to AP0 group. These results indicate that dietary supplementation of Bacillus SW1-1 could increase the disease resistance of olive flounder against E. tarda infection. The optimum coating levels of Bacillus SW1-1 needs to be further elucidated.

• Journal of Microbiology and Biotechnology
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• v.33 no.10
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• pp.1390-1401
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• 2023

In this study, a 12-week feeding experiment was conducted to characterize the effects of exogenous α-amylase on the growth, feed utilization, digestibility, plasma α-amylase activity, feed degradation rate, and fecal particle size of olive flounder (Paralichthys olivaceus). Diet was supplemented with 0 (AA₀; control), 100 (AA₁₀₀), 200 (AA₂₀₀), or 400 (AA₄₀₀) mg/kg of α-amylase, respectively. Fish (273.1 ± 2.3 g) were stocked into 12 tanks (25 fish/1,000-L tank) and 3 tanks were randomly selected for each diet group. As a result, α-amylase was found to have no significant effects (p ≥ 0.05) on the growth, feed utilization parameters, and whole-body proximate compositions. α-Amylase-treated fish exhibited only a significant increase in the apparent digestibility coefficient of carbohydrates compared to the controls. In addition, in vitro analyses revealed that α-amylase dose-dependently increased (p < 0.05) the feed degradation rate, while photographs of the intestinal content after 2, 4, and 8 h of feeding demonstrated an improved degradation rate in the α-amylase-treated groups. Plasma α-amylase content was higher in the AA₂₀₀ and AA₄₀₀ groups, whereas the control group produced significantly larger-sized fecal particles (90% size class) than these two groups. In the intestine, no changes were observed in the expression levels of the immune-related TNF-α, IL-1β, IL-2, immunoglobulin-M, HSP-70, lysozyme, and amylase alpha-2A. However, growth-related genes IGF-1, IGF-2, TGF-β3, and growth hormone genes were upregulated in muscle tissues. Collectively, exogenous α-amylase has positive roles in the modulation of the digestibility coefficient, blood α-amylase concentration, growth-related gene expression, and diet degradation for improved digestion in olive flounder.

• Asian-Australasian Journal of Animal Sciences
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• v.31 no.1
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• pp.138-148
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• 2018

Objective: Fusarium mycotoxins deoxynivalenol (DON) and zearalenone (ZEN), common contaminants in the feed of farm animals, cause immune function impairment and organ inflammation. Consequently, the main objective of this study was to elucidate DON and ZEN effects on the mRNA expression of pro-inflammatory cytokines and other immune related genes in the kidneys of piglets. Methods: Fifteen 6-week-old piglets were randomly assigned to three dietary treatments for 4 weeks: control diet, and diets contaminated with either 8 mg DON/kg feed or 0.8 mg ZEN/kg feed. Kidney samples were collected after treatment, and RNA-seq was used to investigate the effects on immune-related genes and gene networks. Results: A total of 186 differentially expressed genes (DEGs) were screened (120 upregulated and 66 downregulated). Gene ontology analysis revealed that the immune response, and cellular and metabolic processes were significantly controlled by these DEGs. The inflammatory stimulation might be an effect of the following enriched Kyoto encyclopedia of genes and genomes pathway analysis found related to immune and disease responses: cytokine-cytokine receptor interaction, chemokine signaling pathway, toll-like receptor signaling pathway, systemic lupus erythematosus (SLE), tuberculosis, Epstein-Barr virus infection, and chemical carcinogenesis. The effects of DON and ZEN on genome-wide expression were assessed, and it was found that the DEGs associated with inflammatory cytokines (interleukin 10 receptor, beta, chemokine [C-X-C motif] ligand 9, CXCL10, chemokine [C-C motif] ligand 4), proliferation (insulin like growth factor binding protein 4, IgG heavy chain, receptor-type tyrosine-protein phosphatase C, cytochrome P450 1A1, ATP-binding cassette sub-family 8), and other immune response networks (lysozyme, complement component 4 binding protein alpha, oligoadenylate synthetase 2, signaling lymphocytic activation molecule-9, ${\alpha}$-aminoadipic semialdehyde dehydrogenase, Ig lambda chain c region, pyruvate dehydrogenase kinase, isozyme 4, carboxylesterase 1), were suppressed by DON and ZEN. Conclusion: In summary, our results indicate that high concentrations of DON and ZEN suppress the inflammatory response in kidneys, leading to potential effects on immune homeostasis.

• Microbiology and Biotechnology Letters
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• v.16 no.2
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• pp.150-155
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• 1988

In order to produce L-lysine from cellulosic substrates by the intergeneric protoplast fusion between cellulolytic bacteria, Cellulomonas flavigena KFCC31221 and amino acid producing bacteria, Brevibacterium flavum ATCC14067, Corynebacteriurn glutamicum ATCC13032, conditions for protoplast formation and regeneration of these strains were investigated. After the strains were mutated with 500$\mu\textrm{g}$/$m\ell$ N-methyl-N'-nitro N-nitrosoguanidine for 30 min and the mutants were enriched by treating 300$\mu\textrm{g}$/$m\ell$ penicillin-G for 2 hrs, B. flavum Hse- Str$^{r}$ , C. glutamicum Met$^{-}$Thr$^{-}$ Rif$^{r}$ and Cellulomonas flavigena Thr$^{-}$Val$^{-}$Kan$^{r}$ were isolated. The rate of protoplast formation ranged from 95 to 98% when strains were treated at the concentration of 500$\mu\textrm{g}$/$m\ell$ of lysozyme, pH 6.5, 33$^{\circ}C$, for 6 hrs. in Tris- malate buffer supplemented with 0.4M sucrose as osmotic stabilizer. Approximately 30-33% protoplast was regenerated on the regeneration complete medium(RCM) containing 1.5% agar and 0.5M sodium succinate overlaid with the same medium except 0.7% agar.

• The Korean Journal of Food And Nutrition
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• v.15 no.2
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• pp.104-111
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• 2002

The peptidyl prolyl sis-trans isomerase (PPIase, EC 5.2.1.8) from bacillus stearothermophilus was extracted from the cells treated with by lysozyme. PPIase was purified from the cell extracts by heat treatment, ammonium sulfate precipitation, ion exchange chromatography and finally gel filtration, sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE). The molecular weight of the purified PPIase was estimated as 18kDa by SDS-PAGE. The 39 amino acid residues from the N-terminus were determined by the protein sequencer. The enzyme showed the optimum pH at 8.0 and was stable at the range of pH 7.0∼8.0. The enzyme was considerably stable after heat treatment at 60$\^{C}$ for 30minutes, and the enzyme was quite stable up to 65$\^{C}$. The presence of the PPIase in the refolding solution accelerated the isomerization rate of the assay peptide. PPIase gene of Bacillus stearothermophilus was screened from a genomic library by plaque hybridization using the A-l primer as a probe. A PPIase positive plaque contained a 3.0kb insert of the chromosomal DNA. A 3.0kb fragment was subcloned into pUC18, resulting pPI-40. A DNA fragment encoding the N-terminal portion of the PPIase in pPI-40 was amplified by polymerase chain reaction(PCR) method using the A-1 and B-2 primers. The amplified fragment was cloned into the Sma I site of pUC18 and recombinant plasmid was designated as pSN-18. The nucleotide sequence of 167bp fragment was determined. The deduced amino acid sequence of PPIase was completely matched with the determined N-terminal amino acid sequence of PPIase B. stearothermophilus.

• Proceedings of the Korea Society of Poultry Science Conference
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• 2006.11a
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• pp.43-58
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• 2006

As a bioreactor, bird has proved to be most efficient system for producing useful therapeutic proteins. More than half of the egg white protein content derives from the ovalbumin gene with four other proteins(lysozyme, ovomucoid, ovomucin and conalbumin) present at levels of 50 milligrams or greater. And the naturally sterile egg also contains egg white protein at high concentration allowing for a long shelf life of recombinant protein without loss in activity. In spite of these advantages, transgenic procedures for the bird have lagged far behind because of its complex process of fertilized egg and developmental differences. Recently, a system to transplant mouse testis cells from a fertile donor male to the seminiferous tubules of an infertile recipient male has been developed. Spermatogenesis is generated from transplanted cells, and recipients are capable of transmitting the donor haplotype to progeny. After transplantation, primitive donor spermatogonia migrate to the basement membrane of recipient seminiferous tubules and begin proliferating. Eventually, these cells establish stable colonies with a characteristic appearance, which expands and produces differentiating germ cells, including mature spermatozoa. Thus, the transplanted cells self-renew and produce progeny that differentiate into fully functional spermatozoa. In this study, to develop an alternative system of germline chimera production that operates via the testes rather than through developing embryos, the spermatogonial stem cell techniques were applied. This system consisted of isolation and in vitro-culture of chicken testicular cells, transfer of in vitro-maintained cells into heterologous testes, production of germline chimeras and confirmation of germline transmission for evaluating production of heterologous, functional spermatozoa.

• Asian-Australasian Journal of Animal Sciences
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• v.23 no.9
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• pp.1137-1144
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• 2010

In this study, to examine genetic variability within a breed and genetic relationships between populations/breeds, we genotyped 606 birds from seven Japanese native chicken breeds at seven polymorphic loci of egg white proteins and compared those with Asian native chicken populations and commercial breeds. Genotyping of the Japanese native breeds showed that ovalbumin, two ovoglobulins and ovotransferrin were polymorphic, but ovomacroglobulin, ovoflavoprotein and lysozyme were monomorphic. The proportion of polymorphic loci ($P_{poly}$) and average heterozygosity ($\bar{H}$) within a population ranged from 0.286 to 0.429 and from 0.085 to 0.158, respectively. The coefficient of gene differentiation ($G_{ST}$) was 0.250 in the Japanese native chicken breeds. This estimate was higher than that of Asian native chicken populations ($G_{ST}$ = 0.083) and of commercial breeds ($G_{ST}$ = 0.169). Dendrogram and PCA plot showed that Satsuma-dori, Jitokko, Amakusa-daio and Hinai-dori were closely related to each other and grouped into Asian native chickens and that Tsushima-jidori, Nagoya and Chan (Utaichan) were ramified far from other Japanese native chicken breeds. The egg white protein polymorphisms demonstrated that the population differentiation of the seven Japanese native chicken breeds was relatively large.

• Journal of Microbiology and Biotechnology
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• v.19 no.9
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• pp.932-945
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• 2009

Several species of Gram-positive bacteria have cell wall peptidoglycan (syn. murein) in which not all of the sugar moieties are N-acetylated. This has recently been shown to be a secondary effect, caused by the action of a peptidoglycan N-acetylglucosamine deacetylase. We have found that the opportunistic pathogen Listeria monocytogenes is unusual in having three enzymes with such activity, two of which remain in the cytoplasm. Here, we examine the enzyme (PgdA) that crosses the cytoplasmic membrane and is localized in the cell wall. We purified a hexa-His-tagged form of PgdA to study its activity and constructed a mutant devoid of functional Lmo0415 (PgdA) protein. L. monocytogenes PgdA protein exhibited peptidoglycan N-acetylglucosamine deacetylase activity with natural substrates (peptidoglycan) from both L. monocytogenes and Escherichia coli as well as the peptidoglycan sugar chain component N-acetylglucosamine, but not with N-acetylmuramic acid. As was reported recently [6], inactivation of the structural gene was not lethal for L. monocytogenes nor did it affect growth rate or morphology of the cells. However, the pgdA mutant was more prone to autolysis induced by such agents as Triton X-100 and EDTA, and is more susceptible to the cationic antimicrobial peptides (CAMP) lysozyme and mutanolysin, using either peptidoglycan muramidases or autolysis-inducing agents. The pgdA mutant was also slightly more susceptible than the wild-type strain to the action of certain beta-lactam antibiotics. Our results indicate that protein PgdA plays a protective physiological role for listerial cells.

• Animal cells and systems
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• v.15 no.3
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• pp.219-225
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• 2011

We studied oxidative stress in cinnamon clownfish exposed to hypoosmotic (35 psu ${\rightarrow}$ 17.5 psu and 17.5 psu with prolactin (PRL)) and low temperature ($28^{\circ}C{\rightarrow}24^{\circ}C$ and $20^{\circ}C$) conditions by measuring the expression and activity of Cu/Zn-superoxide dismutase (Cu/Zn-SOD), catalase (CAT), and glutathione peroxidase (GPX). The expression and activity of the antioxidant enzymes were significantly higher after the fish were exposed to $24^{\circ}C$, $20^{\circ}C$, and 17.5 psu, and expression was repressed by PRL treatment. Furthermore, we measured $H_2O_2$ and lipid peroxidation levels and found that they were significantly higher after exposure to the hypoosmotic and low-temperature environments. Additionally, we investigated changes in plasma AST and ALT levels after exposure to low temperature and hypoosmotic stress. These levels increased upon exposure of the clownfish to $24^{\circ}C$, $20^{\circ}C$, and 17.5 psu, but the levels of these parameters decreased in the 17.5 psu with PRL treatment during a salinity change. The results indicate that hypoosmotic and low-temperature conditions induce oxidative stress in cinnamon clownfish and that the parameters tested in this study may be indices of oxidative stress in the cinnamon clownfish.

• Journal of Microbiology and Biotechnology
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• v.6 no.6
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• pp.414-419
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• 1996

The xylnX gene encoding a xylanase from Clostridium thermocellum ATCC27405 was cloned in the plasmid pJH27, an E. coli-Bacillus shuttle vector and the resultant recombinant plasmid, pJX18 was transformed into E. coli HB101. The overexpressed xylanase was found to be secreted into the periplasmic space of the recombinant E. coli cells. The crude enzyme was obtained by treating the E. coli cells with lysozyme, and purified by DEAE-Sepharose column chromatography. Molecular wieght of the xylanase was estimated to be 53 kDa by gel filtration. The pI value was determined to be pH 8.8. The N-terminal sequence of the enzyme protein was Asp-Asp-Asn-Asn-Ala-Asn-Leu-Val-Ser-Asn which was considered to be the sequence of that of the mature form protein. The Km value of the enzyme for oat spelt xylan was calculated to be 2.63 mg/ml and the Vmax value was $0.47 {\mu}mole/min$. The xylanase had a pH optimum for its activity at pH 5.4 and a temperature optimum at $60^{\circ}C$. The enzyme hydrolyzed xylan into xylooligosaccharides which were composed mainly of xylobiose (40%) and xyloltriose (12%) after 5 hour reaction. This result indicates that the xylanase from C. thermocellum ATCC27405 is an endo-acting enzyme.