• Journal of Life Science
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• v.28 no.3
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• pp.284-292
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• 2018

The melanin-concentrating hormone (MCH), a cyclic hypothalamic peptide composed of 17 amino acids, was initially identified in chum salmon (Oncorhynchus keta) as a regulator of pigmentation. Mammalian MCHs are cyclic hypothalamic peptides composed of 19 amino acids that regulate food intake and energy homeostasis. The present study examined not only MCH expression of different tissues but also the melanohore aggregation and intracellular $Ca^{2+}$ influx of fMCH and the other MCH. Real-time qPCR showed that MCH expressed specially in the brain, gonad, and ovary, and expression of MCH was observed during the developmental stages. In the application of synthetic fMCH and both types of synthetic fMCH, dN-fMCH and dC-fMCH, scale melanophore induced significant changes in aggregation activity with various concentrations of MCH. Also, compared to hMCH and sMCH, fMCH exhibited a 36~99.85% increase in relative potency (%), whereas aggregation of dN-fMCH and dC-fMCH remained in a high concentration. However, dispersion was induced rapidly according to be low concentration of dN-fMCH and dC-fMCH. We show that fMCH and its derivates were bound human MCHR1 and rat MCHR expressed in HEK293T cells with nano-molar affinity and are likely to be ligand-induced to mobilize intracellular $Ca^{2+}$. These results may provide new ligands for binding assay with MCHew ligands, as a structure similar to the mammalian MCH structure was discovered in fish. Once the fMCH receptor system is in place, it can be compared to the MCH system of mammals in terms of MCH function.

• BMB Reports
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• v.37 no.3
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• pp.282-291
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• 2004

Phytases catalyze the release of phosphate from phytic acid. Phytase-producing microorganisms were selected by culturing the soil extracts on agar plates containing phytic acid. Two hundred colonies that exhibited potential phytase activity were selected for further study. The colony showing the highest phytase activity was identified as Aspergillus niger and designated strain 113. The phytase gene from A. niger 113 (phyI1) was isolated, cloned, and characterized. The nucleotide and deduced amino acid sequence identity between phyI1 and phyA from NRRL3135 were 90% and 98%, respectively. The identity between phyI1 and phyA from SK-57 was 89% and 96%. A synthetic phytase gene, phyI1s, was synthesized by successive PCR and transformed into the yeast expression vector carrying a signal peptide that was designed and synthesized using P. pastoris biased codon. For the phytase expression and secretion, the construct was integrated into the genome of P. pastoris by homologous recombination. Over-expressing strains were selected and fermented. It was discovered that ~4.2 g phytase could be purified from one liter of culture fluid. The activity of the resulting phytase was 9.5 U/mg. Due to the heavy glycosylation, the expressed phytase varied in size (120, 95, 85, and 64 kDa), but could be deglycosylated to a homogeneous 64 kDa species. An enzymatic kinetics analysis showed that the phytase had two pH optima (pH 2.0 and pH 5.0) and an optimum temperature of $60^{\circ}C$.

• The Korean Journal of Zoology
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• v.33 no.4
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• pp.435-445
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• 1990

Gonadotropin releasing horrnone (GnRH) is known to be extrahypothalamically localized with a broad range including gonad. It remains, however, unknown whether GnRH is locally synthesized in the gonad. The present srudy aims to identity expression and cellular localization of GnRH-Iike mRNA and immunoreactive GnRH in the rat gonad. GnRH radioimmunoassay and chromatographic extracts on G-50 sephadex column showed that rat gonadal extracts contained a substantial amount of immunoreactive GnRH similar to the hypothalamic and synthetic GnRH. Although a wide distribution of immunostainable GnRH-like molecule with different cell types in the rat ovary was observed, the major cell population hybridized with GnRH probe appears to be granulosa. theca cells and corpus luteum. Immunoreactive GnRH-Iike peptides were distributed m various regions of testis, including spermatogenic cells, Sertoli cells and Leydig cells. In situ hybridization revealed that positive signals of GnRH-Iike mRNA were predominandy present in Sertoli cells within some seminiferous tubules, but absent in the outside of seminiferous tubules in the testis. This study clearly demonstrated that GnRH-Iike molecule present in the rat gonad may be resulted from the local synthetic machinery of GnRH supporting the notion that this peptide may act as autocrine and/or paracrine role in intra-gonadal communication.

• The Korean Journal of Pesticide Science
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• v.10 no.1
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• pp.56-65
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• 2006

An antagonistic bacterial isolate, that inhibits the growth of plant pathogens, was selected and identified from 5,000 isolates screened from the rhizosphere of various crop plants. An isolate Bacillus sp. N32, tested against Colletotrichum gloeosporioides causing anthracnose disease in hot pepper, produced both a heat resistant antifungal protein and a heat sensitive antifungal protein. The heat resistant protein was partially purified by Ammonium sulfate fractionation and gel filtration chromatography. The bioautography showed that the proteins possessed high antifungal activity. The biosynthetic gene cluster responsible for the heat resistant antifungal protein was cloned from cosmid library using DNA probe obtained from PCR product with the primers targeting the conserved nucleotide sequence of the synthetic genes reported earlier, Most of the clones obtained showed higher homology to fengycin antibiotic synthetic gene family reported earlier. On the other hand, the heat sensitive protein was isolated from SDS-PAGE and electroblotting to determine the N-terminal amino acid sequences. The heat sensitive antifungal protein gene was cloned from the ${\lambda}-ZAP$ libraries using a DNA probe based on the N-terminal amino acid sequences of the heat sensitive protein. We are contemplating to clone and sequence the whole gene cluster encoding the heat sensitive protein for further analysis.

• The Korean Journal of Physiology
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• v.23 no.1
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• pp.89-98
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• 1989

In order to produce antibody for use in radioimmunoassay of gastrin in physiological concentration, four rabbits of New Zealand white were immunized with synthetic human gastrin-17-I conjugated to hemocyanin with EDC. Among them, only one rabbit produced antibody that could bind 50% of $^{125}I-gastrin$ at a final dilution of 1:25,000. $^{125}I-gastrin$ was prepared with synthetic human gastrin-17-I and $NaI^{125}$ by lactoperoxidase technique. The product was then purified on a column of Sephadex Gl5/G5O (7:3, w/w) followed by a column of DEAE sephadex A-25. The specific radioactivity of the purified $^{125}I-gastrin$ was in the range of 347-1429 ${\mu}Ci/nmole$ when determined by the self-displacement method. The effective affinity constant $(K_{eff})$, total binding sites (N), heterogeneity index $({\alpha})$ and average affinity constant $(K_{0})$ of the anti-gastrin serum calculated from Scatchard plot as well as Sips plot were $1.77{\times}10^{11}/M$, 255 nM, 0.84 and $0.79{\times}10^{11}/M$, respectively. When radioimmunoassay was performed with the anti-gastrin serum, it was confirmed that the mean concentration of gastrin immunoreactivity in plasma was increased by feeding in humans and rats, and also increased by bombesin administration in rats. The results indicate that the anti-gastrin serum produced in the present investigation is suitable for radioimmunological determination of gastrin in physiological concentration.

• Journal of Microbiology and Biotechnology
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• v.17 no.12
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• pp.2005-2017
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• 2007

Diabetic nephropathy remains a major cause of morbidity and mortality in the diabetic population and is the leading cause of end-stage renal failure. Despite current therapeutics including intensified glycemic control and blood pressure lowering agents, renal disease continues to progress relentlessly in diabetic patients, albeit at a lower rate. Since synthetic drugs for diabetes are known to have side effects, fungal mushrooms as a natural product come into preventing the development of diabetes. Our previous report showed the hypoglycemic effect of extracellular fungal polysaccharides (EPS) in streptozotocin (STZ)-induced diabetic rats. In this study, we analyzed the differential expression patterns of rat kidney proteins from normal, STZ-induced diabetic, and EPS-treated diabetic rats, to discover diabetes-associated proteins in rat kidney. The results of proteomic analysis revealed that up to 500 protein spots were visualized, of which 291 spots were differentially expressed in the three experimental groups. Eventually, 51 spots were statistically significant and were identified by peptide mass fingerprinting. Among the differentially expressed renal proteins, 10 were increased and 16 were decreased significantly in diabetic rat kidney. The levels of different proteins, altered after diabetes induction, were returned to approximately those of the healthy rats by EPS treatment. A histopathological examination showed that EPS administration restored the impaired kidney to almost normal architecture. The study of protein expression in the normal and diabetic kidney tissues enabled us to find several diabetic nephropathy-specific proteins, such as phospholipids scramblase 3 and tropomyosin 3, which have not been mentioned yet in connection with diabetes.

• International Journal of Oral Biology
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• v.33 no.1
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• pp.25-31
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• 2008

Inhibition of proteasome activity may reduce many types of cancer, so it's pathway is effective in cancer as well as in clinical fields. Here the author has carried out experiment targeting on the elevation of apoptosis in oral cancer cells by combination of proteasome inhibitor, lactacystin, and DNA replication inhibitor, etoposide. The growth of KB cells was measured by MTT methods and apoptosis was analyzed by DNA fragmentation and Hochest nucleus staining. The proteasome activity was analyzed by fluorescent tagged peptide and cellular protein expression was detected by Western hybridization. Though lactacystin and etoposide inhibited KB cell growth alone, but low combined doses inhibited cell growth more strongly and induced apoptosis. The proteasome activity was also seriously inhibited by the combination of both chemicals. Tumor suppressor proteins and apoptosis inducing proteins were highly increased under the combination of both chemicals. From above studies we can conclude that proteasome inhibitors may be used for the treatment of oral cancer and proteasome inhibitors with DNA replication inhibitors may be effective in clinical trials of oral cancer.

• Bulletin of the Korean Chemical Society
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• v.26 no.5
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• pp.740-746
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• 2005

In this article, we report the tandem mass spectrometry investigations for the electron capture efficiencies of the protons belonging to the different locations (generations) in a poly(propylene imine) dendrimer with three layers of a repeat unit (named as the third generation dendrimer). The employed tandem mass spectrometry methods include SORI-CAD (sustained off-resonance irradiation collisional activation dissociation) and ECD(electron capture dissociation) mass spectrometry. We obtained SORI-CAD spectra for the dendrimer ions in the different charge states, ranging from 2+ to 4+. The analysis of fragmentation sites provides the information as to where the protons are distributed among various generations of the dendrimer. Based upon this, a new strategy to study the electron capture efficiencies of the protons is utilized to examine a new type of triplycharged ions by SORI-CAD, i.e., the 3+ ions generated from the charge reduction of the native 4+ ions by ECD: (M+4H)$^{4+}\;+\;e^-\;{\rightarrow}$ (M+4H)$^{3+\bullet}$ ${\rightarrow}\;({H^{\bullet}}_{ejected}$) + (M+3H)$^{3+}\;\rightarrow$ CAD. Interestingly, comparison of these four SORICAD spectra indicates that the proton distribution in the charge-reduced 3+ ions is very close to that in the native 4+ ions. It further suggests that in this synthetic polymer ($\sim$1.7 kDa) with an artificial architecture, the electron capture efficiencies of the protons are actually insensitive to where they are located in the molecule. This is somewhat contradictory to common expectations that the protons in the inner generations may not be well exposed to the incoming electron irradiation as much as the outer ones are, thus may be less efficient for electron capture. This finding may carry some implications for the case of medium sized peptide ions with similar masses, which are known to show no obvious site-specific fragmentations in ECD MS.

• Korean Journal of Medicinal Crop Science
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• v.13 no.5
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• pp.219-226
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• 2005

Sanguisorbae radix (SR) from Sanguisorba officinalis L. (Losaceae) is widely used in Korea and China due to its various pharmacological activity. The present study aims to investigate the effect of the methanol extract of SR on amyloid ${\beta}$ Protein(25-35) $(A{\beta}\;(25-35))$, a synthetic 25-35 amyloid peptide, -induced neurotoxicity using cultured rat cortical neurons. SR, over a concentration range of $10-50\;{\mu}g/ml$, inhibited the $A{\beta}$ (25-35) $(10\;{\mu}M)-induced$ neuronal cell death, as assessed by a 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) assay and the number of apoptotic nuclei, evidenced by Hoechst 33342 staining. Pretreatment of SR $(50\;{\mu}g/ml)$ inhibited $10\;{\mu}M\;A{\beta}$ (25-35)-induced} elevation of cytosolic calcium concentration $([Ca^{2+}]c)$, which was measured by a fluorescent dye, fluo-4 AM. SR $(10\;and\;50\;{\mu}g/ml)$ inhibited glutamate release into medium induced by $10\;{\mu}M\;A{\beta}(25-35)$, which was measured by HPLC, and generation of reactive oxygen species. These results suggest that SR prevents $A{\beta}$ (25-35)-induced neuronal cell damage in vitro.

• Natural Product Sciences
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• v.21 no.2
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• pp.134-140
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• 2015

The present study aims to investigate the effect of methanol extract of Korean mistletoe (KM; Viscum album var. coloratum), on amyloid $\beta$ protein ($A\beta$) (25-35), a synthetic 25-35 amyloid peptide, -induced neurotoxicity in cultured rat cerebral cortical neurons and memory impairment in mice. Exposure of cultured neurons to $10{\mu}M$ $A\beta$ (25-35) for 24 h induced a neuronal cell death, which was measured by a 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) assay and Hoechst 33342 staining. KM (10, 30 and $50{\mu}g/ml$) significantly inhibited the $A\beta$ (25-35)-induced apoptotic neuronal death. KM ($50{\mu}g/ml$) inhibited 10 μM Aβ (25-35)-induced elevation of intracellular calcium concentration ([Ca²⁺]_i), which was measured by a fluorescent dye, Fluo-4 AM. Glutamate release into medium and generation of reactive oxygen species (ROS) induced by $10{\mu}M$ $A\beta$ (25-35) were also inhibited by KM (10, 30 and $50{\mu}g/ml$). These results suggest that KM may mitigate the $A\beta$ (25-35)-induced neurotoxicity by interfering with the increase of [Ca²⁺]_i and then inhibiting glutamate release and generation of ROS in cultured neurons. In addition, orally administered KM (25 and 50 mg/kg, 7 days) significantly prevented memory impairment induced by intracerebroventricular injection of $A\beta$ (25-35) (8 nmol). Taken together, it is suggested that anti-dementia effect of KM is due to its neuroprotective effect against $A\beta$ (25-35)-induced neurotoxicity and that KM may have therapeutic role in prevention of the progression of Alzheimer's disease.