• The Korean Journal of Physiology and Pharmacology
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• v.7 no.2
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• pp.79-84
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• 2003

We have previously reported that expression of the somatostatin receptor subtypes, sst1-5, is differentially regulated by growth hormone (GH)-releasing hormone (GHRH) and forskolin (FSK), in vitro. GHRH binds to membrane receptors selectively located on pituitary somatotropes, activates adenylyl cyclase (AC) and increases sst1 and sst2 and decreases sst5 mRNA levels, without significantly altering the expression of sst3 and sst4. In contrast FSK directly activates AC in all pituitary cell types and increases sst1 and sst2 mRNA levels and decreases sst3, sst4 and sst5 expression. Two explanations could account for these differential effects: 1) GHRH inhibits sst3 and sst4 expression in somatotropes, but this inhibitory effect is masked by expression of these receptors in unresponsive pituitary cell types, and 2) FSK inhibits sst3 and sst4 expression levels in pituitary cell types other than somatotropes. To differentiate between these two possibilities, somatotropes were sequentially labeled with monkey anti-rat GH antiserum, biotinylated goat anti-human IgG, and streptavidin-PE and subsequently purified by fluorescent-activated cell sorting (FACS). The resultant cell population consisted of 95% somatotropes, as determined by GH immunohistochemistry using a primary GH antiserum different from that used for FACS sorting. Purified somatotropes were cultured for 3 days and treated for 4 h with vehicle, GHRH (10 nM) or FSK ($10{\mu}M$). Total RNA was isolated by column extraction and specific receptor mRNA levels were determined by semi-quantitative multiplex RT-PCR. Under basal conditions, the relative expression levels of the various somatostatin receptor subtypes were sst2＞sst5＞sst3=sst1＞ sst4. GHRH treatment increased sst1 and sst2 mRNA levels and decreased sst3, sst4 and sst5 mRNA levels in purified somatotropes, comparable to the effects of FSK on purified somatotropes and mixed pituitary cell cultures. Taken together, these results demonstrate that GHRH acutely modulates the expression of all somatostatin receptor subtypes within GH-producing cells and its actions are likely mediated by activation of AC.

• Clinical and Experimental Reproductive Medicine
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• v.21 no.3
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• pp.241-245
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• 1994

Despite increasing success rate of IVF, poor response to ovarian stimulation remains a problem. So, attempts to improve ovarian responses, for example, by using combined gonadotropin-releasing hormone analogue(GnRH-a) and human menopausal gonadotropin(hMG) have shown limited success. It is reported that response of granulosa cells in vitro to FSH is stimulated by co-incubation with IGF-l, and IGF-l production can be increased by growth hormone. This suggest that combination regimen of G.H. and hMG may augment follicle recruitment. In fifteen patients who had previous history of poor ovarian response to gonadotropin stimulation after pituitary suppression with mid -luteal GnRH-a, the effectiveness of cotreatment with G.H. in IVF program was evaluated using a combination regimen of G.R. and hMG at Korea University Hospital IVF Clinic. Ovarian responses to gonadotropin stimulation in control and GH-treated cycles assessed by total dose and duration of hMG treatment, follicular development and peak $E_2$ level, number of eggs retrieved, and fertilization rates were also assessed. In each group, serum and follicular fluid IGF-1 concentrations on day of egg collection were measured by RIA after acidification and extraction by reveresed phase chromatography. Patients receiving G.H. required fewer days and ampules of gonadotropins, developed more oocytes, and more embryos transferred. But, the differences were not statistically significant, except the duration of hMG treatment. Our data showed a significantly higher concentration of IGF-l in the serum, not in the follicular fluid, of patients treated with G.H. compared with control group. These data suggest that growth hormone treatment does not improve the ovarian response in women with limited ovarian reserve to gonadotropin stimulation for IVF.

• KSBB Journal
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• v.14 no.5
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• pp.593-599
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• 1999

In this study, optimal conditions to infect CD34 positive cells containing hematopoietic stem cells obtained from cord blood and bone marrow were found using two different retroviral vectors expressing human growth hormone (hGH) and $\beta$-galactosidase. CD34 positive cells were successfully infected with recombinant retroviruses only when the CD34 positive cells were co-cultured with packaging cells secreting recombinant retroviruses. To find the highest infection efficiency for the gene transfer, CD34 positive cells from cord blood were co-cultured with packaging cells secreting recombinant retroviruses encoding E. coli lacZ gene. The highest infection efficiency was obtained when CD34 positive cells were cultured for 3 days, and then co-culturing was done for another 2 days. When CD34 positive cells from bone marrow were co-cultured with packaging cells secreting recombinant retroviruses encoding hGH gene, the maximum amount of hGH was also secreted at the same conditions found above, i.e. 3 days of culture and 2 days of co-culture. These results show that there are optimal conditions for the gene transfer into hematopoietic stem cells regardless of sources of target cells or retroviral vectors used to infect.

• Clinical and Experimental Pediatrics
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• v.52 no.12
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• pp.1370-1376
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• 2009

Purpose:Treatment of precocity with gonadotropin releasing hormone analogue (GnRHa) might theoretically exert a detrimental effect on the bone mass during pubertal development. We investigated the short-term changes in bone mineral density (BMD) during GnRHa treatment and the enhancement in the changes with the co-administration of GnRHa and human growth hormone (hGH). Methods:Forty girls with precocious or early puberty who were using GnRHa for more than 1 year were enrolled. Of them, 14 concurrently received hGH. Lumbar bone mineral density was measured before and after the treatment, and bone mineral density-standard deviation scores (BMD-SDSs) were compared according to chronologic age (CA) and bone age (BA), as well as according to the administration of GnRHa alone (Group I) or the co-administration of hGH and GnRHa (Group II). Results:BMDs before and after treatment were in the normal range according to CA but were significantly lower according to BA (P<0.05). During treatment, BMD-SDSs did not change according to CA but significantly increased according to BA (P<0.05). BMD-SDSs in group I did not change during treatment according to CA or BA, while those in group II increased significantly according to BA (P<0.05), but not according to CA. Conclusion:Lumbar BMD was adequate according to CA at initial manifestation of precocity but was lower if compared to BA, that is, BMD did not increase with BA. Because co-treatment with hGH significantly increased BMD-SDSs according to BA, hGH co-treatment could be considered during GnRHa therapy.

• Korean Journal of Animal Reproduction
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• v.22 no.4
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• pp.419-424
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• 1998

The present study was carried out to examine the efficiency of cloning of transgenic embryos by nuclear transfer(NT) using gene-injected rabbit embryos. The rabbit embryos at pronuclear stage were microinjected with methallothionein-human growth hormone(MT-hGH) gene and cultured to 8- and 16-cell in TCM-199 containing 10% FCS with a monolayer of rabbit oviductal epithelial cells in a 5% $CO_2$incubator. The recipient oocytes were collected from the oviducts 14~16 h after hCG injection. The oocytes were enucleated and activated with 5$\mu$M ionomycin and 2mM 6-dimethylaminopurine. Blastomeres form gene-injected embryos were transferred into the enucleated oocytes by micromanipulation. The nuclear transplant oocytes were electrofused and co-cultured with rabbit oviductal cells. Following 120 h of culture, blastocysts were prepared for gene analysis by polymerase chain reaction(PCR). In previous experiment, the rate of gene-positive embryos detected by the nested PCR analysis was significantly decreased while developing to blastocyst(25%)(Kang et al., 1998). The fusion rate of gene-injected blastomeres was significantly(P＜0.05) lower than non-injected blastomeres(66% vs 80%). However, the NT embryos that were derived from gene-injected donor embryos did not differ from control embryos in development to the blastocyst stage(39% vs 31%). Of the 43 NT blastocysts developed from the gene-injected donor embryos, twelve(28%) were positive for the injected DNA. The results indicate that NT with gene-injected embryos can be successfully used for cloning and multiplication of transgenic embryos, furthermore applicable to improvement of transgenic animal production.

• YAKHAK HOEJI
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• v.57 no.2
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• pp.125-131
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• 2013

Biologically active peptides, including growth factors and cytokines, participate in various biological processes in human skin. They could provide a great advantage of maintaining healthy skin. Many peptide growth factors like epidermal growth factor (EGF) and human growth hormone (hGH) have been used in cosmetic formulations. The delivery of peptide growth factors across the Stratum corneum, however, seems not sufficient because of their physical properties such as high molecular weight and hydrophilicity. So increasing the penetration of growth factors of interest into skin would be a major concern for ensuring their maximum biological efficacy. In this study, we have identified several skin penetration-enhancing peptides which facilitate delivery of growth factors, when fused at N-terminus of the target protein, into skin. For efficient and rapid screening, we constructed a skin-penetrating assay system using Franz cell and porcine skin. Next, we carried out phage display screening using M-13 bacteriophage with random 12 -amino acid library on its coat protein P3 on that system. After several selection rounds, peptide sequences facilitate the penetration of phages through the porcine skin were identified from a large population of phages. We found that phages with the most potent peptide (S3-2, NGSLNTHLAPIL) could penetrate the porcine skin eight times more than those with control peptide (12 mino acids scrambled peptide). Furthermore, growth factors conjugated with S3-2 peptide penetrate porcine skin three to five times efficiently than non-conjugated growth factors. In conclusion, our data shows that the skin penetration-enhancing peptide we have characterized could increase the delivery of growth factors and is useful for cosmeceutical application.

• BMB Reports
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• v.31 no.2
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• pp.123-129
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• 1998

Foreign proteins, $endo-{\beta}-1,4-glucanase$ of Bacillus subtilis, preS1+S2 region of hepatitis B virus large surface antigen, human ${\beta}_2-adrenergic$ receptor ($h{\beta}_{2}AR$), and bovine growth hormone (bGH) were expressed in Saccharomyces cerevisiae and secreted into the medium. These proteins were expressed using the alcohol dehydrogenase I (ADH1) promoter of Saccharomyces cerevisiae and secreted by signal sequence of the 97 K killer toxin gene of doublestranded linear DNA plasmid (pGKL1) of S. cerevisiae. All these proteins underwent severe modifications; in particular, N-glycosylation in the case of $endo-{\beta}-1,4-glucanase$, $h{\beta}_2AR$, and preS1+S2. Seventy four percent of the expressed $endo-{\beta}-1,4-glucanase$ was secreted into the culture medium. Highly modified proteins were detected in the culture medium and in the cell. Expressed $h{\beta}_2AR$, which has seven transmembrane domains, remained in the cell. The degrees of secretion and modification and the states of proteins in the culture medium and in the cell were quite different. These results indicated that the nature of the protein has a critical role in its secretion and modifications.

• 한국생물공학회:학술대회논문집
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• 2001.11a
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• pp.85-88
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• 2001

We scaled up a covalent immobilization system of urokinase to the activated Sepharose and used it repeatedly to cleave a fusion protein consisting of human growth hormone and GST fragment. After scale up from 6 ml to 250 ml, the column system still demonstrated basically the same performance in terms of urokinase immobilization and fusion protein cleavage. When the column was washed with 6M guanidine HCl after the cleavage reaction. the immobilized urokinase showed no activity probably because it was fully unfolded. However. as the denaturant was gradually removed from the column the immobilized urokinase fully regained its bioactivity. which indicated it was properly refolded into its native conformation as covalently attached to the solid matrix. After 20 cycles of this 'solid-phase unfolding/refolding', the immobilized urokinase maintained approx. 80% of the initial bioactivity. This method provides an efficient protocol to apply the solid-phase refolding technique to improve the longevity of immobilized enzyme columns.

• KSBB Journal
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• v.16 no.5
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• pp.500-504
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• 2001

We scaled up a covalent immobilization system of urokinase to the activated Sepharose and used it repeatedly to cleava a fusion protein consisting of human growth hormone and GST fragment. After scale up from 6 ml to 250 ml. the column system still demonstrated basically the same performance in terms of urokinase immobilization and fusion protein cleavage. When the column was washed with 6 M guanidine HCI after the cleavage reaction, the immobilized urokinase showed no activity probably becasue it was fully unfoled. However, as the denaturant was gradually removed from the column the immobilized urokinase fully regained its bioactivity, which indicated it was properly refolded into is natie conformation as covalently attached to the solid matrix. After 20 cycles of this solid-phase unfolding/refolding. the immobilized urokinase maintained approx. 80% of the initial bioactivity. This method provides and efficient protocol to apply the solid-phase refolding technique to improve the longevity of immobilized enzyme columns.

• KSBB Journal
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• v.18 no.4
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• pp.306-311
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• 2003

Solid-phase refolding of immobilized proteins can be an effective way to reuse an immobilized enzyme column. Oriented immobilization methods are known to provide higher activity of the immobilized enzymes. In this study, using recombinant EK (enterokinase) as a model enzyme and a fusion protein, that consisted of recombinant human growth hormone and six His tag that was linked by the peptide of EK-specific recognition sequence, as a model substrate, we evaluated two oriented immobilization methods, i. e., reductive alkylation of N-terminus ${\alpha}$-amine and affinity interaction between poly-histidine tag and Ni-NTA (nickel-nitrilotriacetic acid). The immobilization yield, activity and cleavage of the immobilized enzymes, and the yield of solid-phase refolding were compared. The Ni affinity immobilization and the covalent immobilization yields were about 100％ and 65％, respectively. But the specific activities were the same, about 50％ of that of the soluble enzyme. The cleavage rate by the covalently immobilized EK was higher than the soluble enzyme and the side reaction of cryptic cleavage was significantly decreased. Covalently immobilized EK showed almost 100％ refolding yield but the affinity immobilized EK showed only 70％ yield, which suggested the covalent conjugation provided more rigid ‘reference structure’ for the solid-phase refolding. The monomeric hGH could be easily obtained by capturing the cleaved poly Histidine tag by the Ni affinity column.