• BMB Reports
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• 제40권6호
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• pp.1016-1020
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• 2007

In this study, dioscin was isolated from Dioscoreae Rhizoma (DR), which is the rhizome of Dioscorea batatas DECNE. that inhabits broad areas of Korea and Japan. To determine whether dioscin induced growth hormone (GH) release, we evaluated its induction effects on GH release both in vitro and in vivo. The 70% methanol extract of DR, and its n-hexane and n-BuOH fractions, induced rat GH (rGH) release in rat pituitary cells 10-fold, 8-fold, and 5-fold higher than the control ($0.36{\pm}0.02 nM$), respectively (p < 0.05 each). The dioscin-induced rGH release of the cells was concentration-dependent and its $ED_{50}$ was $1.14{\times}10^{-5} M$. Within 90 minutes after intravenous administration of $10{\mu}g$/kg (p < 0.05 at $t_{max}$), dioscin caused the greatest increase in rGH concentration ($C_{max}$) in the rat plasma ($34.16{\pm}14.10 ng/ml$) (n = 4), which was twice as high as the control group ($12.88{\pm}3.29 ng/ml$) (n = 27).

• 한국발생생물학회지:발생과생식
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• 제2권2호
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• pp.189-196
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• 1998

Growth Hormone Releasing Hormone (GHRH)은 척추동물의 시상하부로부터 합성, 분비되어 시상하부-뇌하수체간의 문맥계를 통해 뇌하수체 전엽에 작용하여 Growth Hormone (GH)의 분비를 촉진한다. 시상하부에서 발현되는 일부 Releasing Hormone 들이 여러 시상하부외 조직에서도 검출되고 조직특이적인 기능을 수행한다는 사실이 여러 연구자들에 의해 밝혀졌다. 이러한 사실들을 배경으로 본 연구자는 GHRH가 흰쥐의 뇌하수체 전엽과 뇌하수체로부터 유래된 종양세포주들에서 발현될 가능성을 조사하였다. GHRH 펩타이드와 mRNA의 존재와 구조를 규명하기 위하여 뇌하수체와 배양 세포를 사용하여 GHRH immunocytochemistry, 방사면역측정법, GHRH PCR과 RNase protection assay를 시행하였다. Immunocytochemistry의 결과 gonadotrope (대형)와 somat-olactotrope (중간형)로 추정되는 세포들에서 GHRH 염색이 나타났고, Somatolactotrope성 종양세포인 GH3 cell 추출물에서 immunoreactive GHRH가 방사면역측정법으로 검출되었다. 3'rapid amplification of cDNA end (3'-RACE)를 시행한 결과, 흰쥐 뇌하수체에 GHRH transcript가 존재하고, 그 3'end 부분이 다른 조직내의 GHRH와 동일함을 확인하였다. GHRH RT-PCR에서도 뇌하수체와 종양세포주들인 $\alpha$T3 cell (gonadotrope성)과 GH3 cell에서 예상 산물들이 증폭되었다. RNase protection assay를 시행한 결과 난소절제에 의해 뇌하수체내 GHRH 유전자 발현이 증가됨을 확인하였다. 이상의 결과는 GHRH가 뇌하수체 전엽의 gonadotrope와 somatotrope에서 발현되고, paracrine 또는 autocrine조절물질로 작용하여 GH 분비 외에도 뇌하수체 전엽 세포들의 분화와 분열등에 관여함을 시사한다.

• Journal of Microbiology and Biotechnology
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• 제1권3호
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• pp.157-162
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• 1991

Conditions for glutathione production in E. coli cells which possess pGH501 (2 gshI+gshII) were studied. In terms of ATP supply for the glutathione synthesis, two different systems have been constructed and compared. When the acetate kinase reaction of E. coli was used for ATP generation, 20 mM of L-cysteine was completely converted to glutathione by toluene-treated E. coli cells (100 mg/ml) harboring pGH501 within 2 h at $37^{\circ}C$. However, considering the economical aspects, the glycolytic pathway of yeast was chosen as a better system for ATP generation. The optimal concentrations of reactants for glutathione production were determined to be as follows; 80 mM L-glutamate, 20 mM L-cysteine, 20 mM glycine, 20 mM $MgCl_2$, 50 mM potassium phosphate buffer (pH 7.5), 400 mM glucose, polyoxyethylene stearylamine ($5\;\mul/ml$), toluene-treated E. coli HB101/pGH501 (100 mg/ml), and dried yeast cells (400 mg/ml). The conversion ratio of L-cysteine to glutathione was 80% (about 5 mg/ml) under optimal condition within 6 h at $37^{\circ}C$.

• The Korean Journal of Physiology and Pharmacology
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• 제5권2호
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• pp.139-146
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• 2001

L-type $Ca^{2+}$ channels play an important role in regulating cytosolic $Ca^{2+}$ and thereby regulating hormone secretions in neuroendocrine cells. Since hormone secretions are also regulated by various kinds of protein kinases, we investigated the role of some kinase activators and inhibitors in the regulation of the L-type $Ca^{2+}$ channel currents in rat pituitary $GH_3$ cells using the patch-clamp technique. Phorbol 12,13-dibutyrate (PDBu), a protein kinase C (PKC) activator, and vanadate, a protein tyrosine phosphatase (PTP) inhibitor, increased the $Ba^{2+}$ current through the L-type $Ca^{2+}$ channels. In contrast, bisindolylmaleimide I (BIM I), a PKC inhibitor, and genistein, a protein tyrosine kinase (PTK) inhibitor, suppressed the $Ba^{2+}$ currents. Forskolin, an adenylate cyclase activator, and isobutyl methylxanthine (IBMX), a non-specific phosphodiesterase inhibitor, reduced $Ba^{2+}$ currents. The above results show that the L-type $Ca^{2+}$ channels are activated by PKC and PTK, and inhibited by elevation of cyclic nucleotides such as cAMP. From these results, it is suggested that the regulation of hormone secretion by various kinase activity in $GH_3$ cells may be attributable, at least in part, to their effect on L-type $Ca^{2+}$ channels.

• Animal cells and systems
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• 제4권2호
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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.

• 생명과학회지
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• 제10권4호
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• pp.422-430
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• 2000

In order to search for the effects of Shine-Dalgarno (SD) sequence and nucleotide sequence of spacer region (SD-ATG) on bGH expression, oligonucleotides containing random SD sequences and a spacer region were chemically synthesized. The distance between SD region and initiation codon (ATG) was fixed to 9 nucleotides in length. The expression vectors have been constructed using pT7-1 vector containing a T7 promoter. Positive clones were screened with colony hybridization and named pT7A or pT7B plasmid series. The selected clones were confirmed by DNA sequencing and finally, 19 clones having various SD combinations were obtained. When bovine growth hormone was induced by IPTG in E. coli BL21(DE3), all cells harboring these plasmids produced a detectable level of bGH in western blot analysis. However, various SD sequences did not affect on bGH expression, indicating that the sequences of SD and the spacer region did not sufficiently destabilize mRNA secondary structure of bGH gene. Therefore, these results indicate that the disruption of mRNA secondary structure might be a major factor for regulating bGH expression in the translational initiation process.

• Animal cells and systems
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• 제3권2호
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• pp.103-113
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• 1999

Many in-depth reviews related to regulations of prolactin secretion are available. We will, therefore, focus on controversial aspects using personal opinion in this review. The neuroendocrine control of prolactin secretion from the anterior pituitary gland involves multiple factors including prolactin-release inhibiting factor (PIF) and prolactin releasing factor (PRF). The PIF exerts a tonic inhibitory control in the physiological conditions. The PIF should be able to effectively inhibit prolactin release or a lifetime, but the inhibitory action of dopamine cannot be sustained for a long period of time. Perifusion of a high concentration of dopamine (l ,000 nM) could not sustain inhibitory action on prolactin release but when a small amount of ascorbic acid (0.1 mM) is added in a low concentration of dopamine (3 nM) solution, prolactin release was inhibited for a long period. Ascorbate is essential for dopamine action to inhibit prolactin release. We have, therefore, concluded that the PIF is dopamine plus ascorbate. The major transduction system for dopamine to inhibit prolactin release is the adenylyl cyclase system. Dopamine decreases cyclic AMP concentration by inhibiting adenylyl cyclase, and cyclic AMP stimulates prolactin release. However, the inhibitory mechanism of dopamine on prolactin release is much more complex than simple inhibition of CAMP production. The dopamine not only inhibits cyclic AMP synthesis but also inhibits prolactin release by acting on a link(s) after the CAMP event in a chain reaction for inhibiting prolactin release. Low concentrations of dopamine stimulate prolactin release. Lactotropes are made of several different subtypes of cells and several different dopamine receptors are found in pituitary. The inhibitory and stimulatory actions induced by dopamine can be generated by different subtype of receptors. The GH$_4$ZR$_7$ cells express only the short isoform (D$_{2s}$) of the dopamine receptor, as a result of transfecting the D$_{2s}$ receptors into GH$_4$C$_1$ cells which do not express any dopamine receptors. When dopamine stimulates or inhibits prolactin release in GH$_4$ZR$_7$ cells, it is clear that the dopamine should act on dopamine D$_{2s}$ receptors since there is no other dopamine receptor in the GH$_4$ZR$_7$. Dopamine is able to stimulate prolactin release in a relatively low concentration while it inhibits in a high concentration in GH$_4$ZR$_7$. These observations indicate that the dopamine D$_2$ receptor can activate stimulatory and/or inhibitory transduction system depending upon dopamine concentrations.

• 대한수의학회지
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• 제38권3호
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• pp.497-507
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• 1998

The localizations and morphological characteristics of immunoreactive cells for prolactin(PRL) and growth hormone(GH) antisera were studied with double immunohistochemistry in the adenohypophysis of the Korean native goat. PRL immunoreactive cells(mammotropes) and GH immunoreactive cells(somatotropes) were present in the pars distalis and pars tuberalis, but not in the pars intermedia. A few cells were stained with both PRL and GH antisera (somatomammotropes). The possessional percentages of mammotropes, somatotropes and somatomammotropes were 37.0%, 32.6%, 1.0% in females and 35.6%, 32.6%, 1.1% in males, respectively. Mammotropes were oval or round in shape, and $10{\sim}20{\mu}m{\times}17{\sim}25{\mu}m$ in size. These cells were distributed throughout the pars distalis, but were more abundant on the dorsal part adjacent to the hypophyseal cavity and along the lateral and ventral peripheral regions. Somatotropes were elliptical, triangular or polygonal in shape, and $8{\sim}17{\mu}m{\times}17{\sim}18{\mu}m$ in size. The distribution pattern of somatotropes was similar to that of mammotropes. Some somatomammotropes were intercalated between clusters of mammotropes while the others were dispersed independently among the secretory cells.

• 멤브레인
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• 제11권1호
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• pp.29-37
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• 2001

본 논문은 인공 진피와 조직공학용 scaffold로 이용하기 위해 다공성 membrane로서 gelatin-based sponge의 효율성을 연구하였다. 불용성의 다공성 membrane은 1-ethyl-(3-3dimethylaminopropyl)carbodiimide(EDC)로 가교하여 제조하였다. Fourier-transformed infrared (FT-IR) spectroscopy, scanning electron microscopy(SEM) 그리고 Instron analysis로 다공성 membrane의 특성을 조사하였다. 다공성 membrane은 용적당 큰 표면적을 제공하는 micro porous한 구조를 가지고 있다. Gelatin/hyaluronic acid (HA) membrane의 공경크기는 40~200$\mu\textrm{m}$이다. HA의 첨가는 다공성 membrane의 기계적 강도와 세포부착능력에 영향을 미쳤다. Gelatin/HA 다공성 membrane의 압축강도는 collagen과 비슷하며, 세포배양과 인공진피 transplantation에 있어서의 충분한 기계적 강도를 가지고 있다. Fibroblasts를 함유한 진피기질을 제조하기 위해 직경 8mm의 다공성 membran에 4$\times$10(sup)5cells/membrane의 세포밀도로 fibroblast를 배양하였다. GH91 porous membrane에서의 fibroblast 부착성은 GH55 porous membrane에서보다 우수하였다. 삼차원 구조의 gelatin/HA membrane matrix에서의 fibroblast의 배양은 생체내 조건과 유사한 생리적 환경을 제공하였다.

• 한국동물학회:뉴스레터
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• 제12권2호
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• pp.116.1-116
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• 1995

No Abstract, See Full Text