• Applied Biological Chemistry
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• v.40 no.6
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• pp.495-500
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• 1997

To isolate a gene for cyclodextrin glycosyltransferase (CGTase) from alkalophilic Bacillus sp. E1, polymerase chain reaction (PCR) amplification was carried out. Direct molecular cloning of 1.2 kbp fragment and partial nucleotide sequence analysis of the PCR amplified clone, pH12, showed close homology with CGTases from Bacillus species. To investigate the genomic structure of the gene, Southern blot analysis of genomic DNA was carried out with the clone pH12 as a molecular probe. It showed that 5.3 kbp XbaI fragment was hybridized with the probe pH12. To isolate a genomic clone, genomic DNA library was constructed and a genomic clone for CGTase, pCGTE1, was isolated. Nucleotide sequence analysis of the clone pCGTE1 revealed that BCGTE1 contained 2,109 bp open reading frame encoding a polypeptide of 703 amino acids and showed over 94.3% amino acid sequence homology with CGTase of ${\beta}-cyclodextrin$ producer, Bacillus sp. KC201.(Received October 7, 1997; accepted October 20, 1997)

• Applied Biological Chemistry
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• v.39 no.2
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• pp.112-117
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• 1996

Arginine decarboxylase (ADC) is the first enzyme in one of the two pathways of diamine putrescine biosynthesis in plants. The genes encoding ADC have previously been cloned from Escherichia coli, oat and tomato genome. Two degenerate oligonucleotides (17-mer) corresponding to two conserved regions of ADC were used as primers in polymerase chain reaction of rice (Oryza sativa L.) genomic DNA, and an approximately 1.0 kbp fragment was obtained. This amplified PCR product showed an open reading frame which contains 1,022 bp of nucleotide sequences. This PCR product was cloned into pGEM-originated T vector and the short 500 bp PstI digested fragment was subcloned into pGEM-3zf(+/-) vectors to facilitate sequencing. The nucleotide sequence of this PCR product showed about 74% and 70% identity with the same regions of the oat and tomato ADC cDNA sequences, respectively. The predicted amino acid sequence exhibited 45% and 62% identity with oat and tomato ADC polypeptide fragments, respectively. The sequence similarities of 34%, 47% and 38% were previously reported in oat and E. coli, tomato and oat, and tomato and E. coli ADC amino acids, respectively. Therefore, similarities and identities between rice and oat or tomato are remarkably higher than those others of the previous reports. In the highly conserved regions in both the amino acid sequence and spacing regions among the sequences of these three, rice ADC open reading frame also has the exactly same regions with the striking similarity. RNA blot analysis showed that hnc is expressed as a transcript of approximately 2.5 kbP in the rice seedling leaf tissues.

• Nutrition Research and Practice
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• v.11 no.2
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• pp.121-129
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• 2017

BACKGROUND/OBJECTIVES: The study was conducted to evaluate the effects of dietary leucine supplementation on mitochondrial biogenesis and energy metabolism in the liver of normal birth weight (NBW) and intrauterine growth-retarded (IUGR) weanling piglets. MATERIALS/METHODS: A total of sixteen pairs of NBW and IUGR piglets from sixteen sows were selected according to their birth weight. At postnatal day 14, all piglets were weaned and fed either a control diet or a leucine-supplemented diet for 21 d. Thereafter, a $2{\times}2$ factorial experimental design was used. Each treatment consisted of eight replications with one piglet per replication. RESULTS: Compared with NBW piglets, IUGR piglets had a decreased (P < 0.05) hepatic adenosine triphosphate (ATP) content. Also, IUGR piglets exhibited reductions (P < 0.05) in the activities of hepatic mitochondrial pyruvate dehydrogenase (PDH), citrate synthase (CS), ${\alpha}$-ketoglutarate dehydrogenase (${\alpha}$-KGDH), malate dehydrogenase (MDH), and complexes I and V, along with decreases (P < 0.05) in the concentration of mitochondrial DNA (mtDNA) and the protein expression of hepatic peroxisome proliferator-activated receptor-${\gamma}$ coactivator $1{\alpha}$ (PGC-$1{\alpha}$). Dietary leucine supplementation increased (P < 0.05) the content of ATP, and the activities of CS, ${\alpha}$-KGDH, MDH, and complex V in the liver of piglets. Furthermore, compared to those fed a control diet, piglets given a leucine-supplemented diet exhibited increases (P < 0.05) in the mtDNA content and in the mRNA expressions of sirtuin 1, PGC-$1{\alpha}$, nuclear respiratory factor 1, mitochondrial transcription factor A, and ATP synthase, $H^+$ transporting, mitochondrial F1 complex, ${\beta}$ polypeptide in liver. CONCLUSIONS: Dietary leucine supplementation may exert beneficial effects on mitochondrial biogenesis and energy metabolism in NBW and IUGR weanling piglets.

• Journal of The Korean Society of Inherited Metabolic disease
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• v.12 no.2
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• pp.108-112
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• 2012

Glycogen storage disease type III (GSD type III, OMIM #232400) is a rare autosomal recessive disease caused by a deficiency of the glycogen-debranching enzyme (GDE) with a mutation in the AGL gene (OMIM *610860). It is known to be bifunctional enzyme, that is, having two independent catalytic activities; 1,4-${\alpha}$-D-glucan 4-${\alpha}$-D-glycosyltransferase (EC 2.4.1.25) and amylo-1,6-glucosidase (EC 3.2.1.33) that occur at separate active sites on a single polypeptide chain. Most patients with GSD type III usually have symptoms related to decreased glycogenolysis in liver and muscles, such as hepatomegaly, hypoglycemia, failure to thrive, hyperlipidemia, muscle weakness and cardiomyopathy (type IIIa), however some patients show symptoms restricted to liver (type IIIb). GSD type III is diagnosed by enzyme test through liver or muscle biopsy or mutation analysis of the AGL gene. We report the case of GSD type III proven by gene study after liver biopsy, which revealed c.476delA, c.3444_3445insA in exon 6, 27 of AGL gene in Korean patient.

• Clinical and Experimental Reproductive Medicine
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• v.25 no.2
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• pp.199-205
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• 1998

The present study was performed to analyze the expression of LH genes in the rat ovary. Expression of LH subunit genes in the rat ovary was demonstrated by amplification of ovarian RNA by RT-PCR. The ovarian $LH_\beta$ transcripts contained at least two parts of the published cDNA structure, the pituitary exons 1, 2 and 3 and the part of testicular ex on 1 in the major trancripts form in rat testis. Using RIA, significant amount of LH-like molecules were detected in crude ovarian extracts, and the competition curves with increasing amount of tissue extracts were parallel with those of standard peptide, indicating that the ovarian immunoreactive LH-like material is similar to authentic pituitary LH molecule. The administration of PMSG to immature rats resulted in a sharp decrease of the ovarian LH contents after 24 h post-injection. In conclusion, these findings demonstrate that genes for LH subunits are expressed in the rat ovary, and suggest that LH can playa central role in regulation of female reproduction with both endocrine (by pituitary LH) and auto- and/or para-crine (by ovarian LH) manner.

• Clinical and Experimental Reproductive Medicine
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• v.23 no.3
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• pp.269-275
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• 1996

Biosynthesis and secretion of anterior pituitary hormones are under the control of specific hypothalamic stimulatory and inhibitory factors. Among them, Growth Hormone Releasing Hormone (GHRH) is the major stimulator of pituitary somatotrophs activating GH gene expression and secretion. Human GHRH is a polypeptide of 44 amino acids initially isolated from pancreatic tumors, and the gene for the hypothalamic form of GHRH is organized into 5 exons spanning over 10 kilobases (kb) on genomic DNA and encodes a messenger RNA of 700-750 nucleotides. Several neuropeptides classically associated with the hypothalamus have been found in the extrahypothalamic regions, suggesting the existence of novel sources, targets and functions. GHRH-like immunoreactivity has been found in several peripheral sites, including placenta, testis, and ovary, indicating that GHRH may also have regulatory roles in peripheral reproductive organs. Furthermore, higher molecular weight forms of the GHRH transcripts were identified from these organs (1.75 kb in testis; 1.75 and >3 kb in ovary). These tissue-specific expression of GHRH gene suggest the existence of unique regulatory mechanism of GHRH expression and function in these organs. In fact, placenta-specific and testis-specific promoters for GHRH transcripts which are located in about 10 kb upstream region of hypothalamic promoter were reported. The use of unique promoters in extrahypothalamic sites could be refered in a different control of GHRH gene and different functions of the translated products in these tissues. Somatotrophs and lactotrophs have been thought to be derived from a common bipotential progenitor, the somatolactotrophs, which give origins to either phenotypes. Although the precise mechanism responsible for the lactotroph differentiation in the anterior pituitary gland has not been yet clalified, there are several candidators for the generation of lactotrophs. In human, the presence of GHRH peptides with different size from authentic hypothalamic form in the normal anterior pituitary and several types of adenoma were demonstrated. Recently our group found the existence of immunoreactive GHRH and its transcript from the normal rat anterior pituitary (gonadotroph> somatotroph> lactotroph), and the GHRH treatment evoked the increased proliferation rate of anterior pituitary cells in vitro. The transgenic mouse models clearly shown that GHRH or NGF overexpression by anterior pituitary cells induced development of pituitary hyperplasia and adenomas particularly GH-oma and prolactinoma. Taken together, we hypothesize that the pituitary GHRH could serve not only as a modulator of hormone secretion but as a paracrine or autocrine regulator of anterior pituitary cell proliferation and differentiation. Interestingly enough, the expression of Pit-1 homeobox gene (the POU class transcription factor) was confined to somatotrophs, lactotrophs and somatolactotrophs in which GHRH receptors are expressed commonly. Concerning the mechanism of somatolactotroph and lactotroph differentiation in the anterior pituitary, we have focused following two possibilities; (1) changes in the relative levels or interactions of both hypothalamic and intrapituitary factors such as dopamine, VIP, somatostatin, NGF and GHRH; (2) alterations of GHRH-GHRH receptor signaling and Pit-1 activity may be the cause of lactotroph differentiation or pituitary hyperplasia and adenoma formation. Extensive further studies will be necessary to solve these complicated questions.

• Polymer(Korea)
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• v.30 no.5
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• pp.445-452
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• 2006

In this study, we investigated interaction of Schwann cells (SCs) with various cell-adhesive coated polymer surface. We used cell-adhesives that like a fibronectin (FN), fibrinogen(FG), laminin(LM), vitronectin (VN), poly-D-Iysine (PDL), and poly-L-Iysine (PLL) to coat PLGA film surface and evaluated the surface property of coated or not PLGA films by measurement of water contact angle and ESCA. SCs were cultured on coated or non-coated PLGA film surface, and then examined the cell adhesion and proliferation by cell count and SEM observation. Cell count results revealed initial cell adhesion related to protein adsorption on PLGA surface. In addition, serum content in media related to cell proliferation rate. In this result, we recognized that adhesion and proliferation of SCs were affected by specific cell-adhesives. In these results, we recognized that is important to provide the suitable surface environment according to cell types and culture condition for improvement of cell adhesion and proliferation.

• Animal cells and systems
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• v.4 no.2
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• pp.157-163
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• 2000

Yeast pheromone a-factor is a 13-amino acid peptide hormone that is synthesized as a part of a larger precursor, prepro-$\alpha$-factor, consisting of a signal peptide and a proregion of 64 amino acids. The carboxy-terminal half of the precursor contains four tandem copies of mature $\alpha$-factor. To investigate the molecular basis of intracellular sorting, proteolytic processing, and storage of the peptide hormone, yeast prepro-$\alpha$-factor precursors were heterologously expressed in rat pituitary $GH_3 cells. When cells harboring the precursor were metabolically labeled, a species of approximately 27 kD appeared inside the cells. Digestion with peptide: N-glycosidase F (PNG-F) shifted the molecular mass to a 19 kD, suggesting that the 27 kD protein was the glycosylated form as in yeast cells. The nascent polypeptide is efficiently targeted to the ER in the $GH_3 cells, where it undergoes cleavage of its signal peptide and core glycosylation to generate glycosylated pro-a-factor. To look at the post ER intracellular processing, the pulse-labelled cells were chased up to 2 hrs. The nascent propeptides disappeared from the cells at a half life of 30 min and only 10-25% of the newly synthesized, unprocessed precursors were stored intracellularly after the 2 h chase. However, about 20% of the pulse-labeled pro-$\alpha$-factor precursors were secreted into the medium in the pro-hormone form. With increasing chase time, the intracellular level of propeptide decreased, but the amount of secreted propeptide could not account for the disappearance of intracellular propeptide completely. This disappearance was insensitive to lysosomotropic agents, but was inhibited at $16^{circ}C or 20^{\circ}C$, suggesting that the turnover of the precursors was not occurring in the secretory pathway to trans Golgi network (TGN) or dependent on acidic compartments. From these results, it is concluded that a pan of these heterologous precursors may be processed at its paired dibasic sites by prohormone processing enzymes located in TGN/secretpry vesicles producing small peptides, and that the residual unprocessed precursors may be secreted into the medium rather than degraded intracellularly.

• Korean Journal of Veterinary Research
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• v.40 no.2
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• pp.197-211
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• 2000

The pineal body have been known to be affected by superior cervical ganglia, and most of its nerve fibers containing peptidergic neurotransmitters have been considered to be originated from this ganglia. To confirm this relationships, some peptidergic neurotransmitters were identified in both of pineal body and superior cervical ganglia of the Korean native goat, which were divided into two group; breeding season and non-breeding season. The localizations of two catecholamine-synthesizing enzymes; tyrosine hydroxylase (TH) and dopamine beta-hydroxylase (DBH), were investigated by immunohistochemistry in the superior cervical ganglia and the pineal body of adult Korean native goats. Substance P (SP), calcitonin gene-related peptide (CGRP), vasoactive intestinal polypeptide (VIP), neuropeptide Y (NPY) and galanin (GAL) were also identified in these organs by immunohistochemical and double immunofluorescent methods. In superior cervical ganglia, immunoreactivities for TH and DBH were confirmed in the same ganglion cells. The immunoreactivites for SP, VIP(only in male), NPY and GAL were identified in both of ganglion cell bodies and nerve fibers in the ganglia. CGRP immunoreactivity, however, was observed only in nerve fibers. Most NPY- and VIP-immunoreactive(IR) ganglion cells also contained TH. SP and TH were colocalized in the cell bodies, but not in the nerve fibers. TH immunoreactivity was shown in almost all of ganglion cells in the superior cervical ganglia. The immunoreactivity for NPY had some seasonal variation and was stronger in breeding season than in non-breeding season. In pineal body, lots of TH-IR fibers were observed throughout the parenchyma including the pineal stalk and most of them also contained DBH. SP- and NPY-IR fibers were also immunostained with TH or DBH. But a few SP- and NPY-IR fibers were not colocalized with TH or DBH. Exceptionally, a bipolar neuron-like cell was observed to be immunostained with NPY in the pineal body. A few CGRP and GAL-IR fibers were observed, while VIP-IR fibers were not present. It is concluded that most TH- and DBH-IR fibers as well as the peptidergic immunoreactive fibers of the pineal body might be originated from the superior cervical ganglia. Some peptidergic immunoreactive fibers, however, might be come from other regions of brain. We also suggest that NPY in pineal body plays a important role for pineal function. The seasonal variation of NPY immunoreactivity indicates that the synthesis and use of NPY may be different between in breeding and non-breeding seasons.

• Journal of Marine Bioscience and Biotechnology
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• v.2 no.4
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• pp.234-237
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• 2007

Calmodulin (CaM) is a $Ca^{2+}$-binding protein essential for biological functions mediated through $Ca^{2+}$-dependent mechanism. A cDNA clone for CaM was isolated from a cDNA library of olive flounder Paralichthys olivaceus. The CaM cDNA concists of 782 bp and encodes a polypeptide of 149 amino acids with four $Ca^{2+}$-binding motifs EF-hands (EF-I, EF-II, EF-III, and EF-IV). The deduced amino acid sequence of CaM shows 97-100% amino acid sequence identity to other CaM sequences. Semi-quantitative PCR analysis revealed that the CaM transcription was began during early development and the CaM mRNA is expressed highly in brain and intestine, and moderately in kidney, gill, and eye of healthy olive flounder. Taken together, CaM may be necessary for early olive flounder development and that it may have a part in homeostasis.