Xie, Q.F.
(College of Animal Science and Technology, China Agricultural University)
,
Wu, C.X.
(College of Animal Science and Technology, China Agricultural University)
,
Meng, Q.Y.
(State Key Laboratory for Agrobiotechnology, China Agricultural University)
,
Li, N.
(State Key Laboratory for Agrobiotechnology, China Agricultural University)
Ghrelin is an acylated peptide recently identified as the endogenous ligand for the growth hormone (GH) secretagogues receptor 1a (GHS-R1a) and is involved in a novel system for regulating GH release. To understand the long-term effects of ghrelin, here we constructed six myogenic expression vectors...
Ghrelin is an acylated peptide recently identified as the endogenous ligand for the growth hormone (GH) secretagogues receptor 1a (GHS-R1a) and is involved in a novel system for regulating GH release. To understand the long-term effects of ghrelin, here we constructed six myogenic expression vectors containing the cDNA of swine mature ghrelin (pGEM-wt-sGhln, pGEM-wt-hGhln), ghrelin mutant of $Ser^3$ with $Trp^3$ (pGEM-mt-sGhln, pGEM-mt-hGhln) and truncated ghrelin derivative (pGEM-tmtsGhln, pGEM-tmt-hGhln) encompassing the first 7 residues of ghrelin (including $Ser^3$ substituted with $Trp^3$) and adding a basic amino acid, Lys (K) in the C-terminus. The constructs, pGEM-wt-sGhln, pGEM-mt-sGhln and pGEM-tmt-sGhln were linked with the ghrelin leader sequence, while the pGEM-wt-hGhln, pGEM-mt-hGhln and pGEM-tmt-hGhln were linked with a leader sequence from the human growth hormone releasing hormone (hGHRH). Intramuscular injection of 200 ${\mu}g$ pGEM-wt-sGhln or pGEM-tmt-sGhln augmented growth over 3 weeks in normal rats and peaked at day 21 or 14 post-injection respectively, whose body weight gains were on average approximately 6% or 19% heavier over controls. However, other injectable vectors had no such enhanced growth effects. Our results suggested that the efficacy of the ghrelin leader sequence was more effective than that of hGHRH in our system. Moreover, the results indicated that skeletal muscle might have the ability to posttranslationally modify the in vivo expressed ghrelin. And the most strikingly, the short ghrelin analog seems to mimic the biological effects more efficiently when compared with the full-length ghrelin.
Ghrelin is an acylated peptide recently identified as the endogenous ligand for the growth hormone (GH) secretagogues receptor 1a (GHS-R1a) and is involved in a novel system for regulating GH release. To understand the long-term effects of ghrelin, here we constructed six myogenic expression vectors containing the cDNA of swine mature ghrelin (pGEM-wt-sGhln, pGEM-wt-hGhln), ghrelin mutant of $Ser^3$ with $Trp^3$ (pGEM-mt-sGhln, pGEM-mt-hGhln) and truncated ghrelin derivative (pGEM-tmtsGhln, pGEM-tmt-hGhln) encompassing the first 7 residues of ghrelin (including $Ser^3$ substituted with $Trp^3$) and adding a basic amino acid, Lys (K) in the C-terminus. The constructs, pGEM-wt-sGhln, pGEM-mt-sGhln and pGEM-tmt-sGhln were linked with the ghrelin leader sequence, while the pGEM-wt-hGhln, pGEM-mt-hGhln and pGEM-tmt-hGhln were linked with a leader sequence from the human growth hormone releasing hormone (hGHRH). Intramuscular injection of 200 ${\mu}g$ pGEM-wt-sGhln or pGEM-tmt-sGhln augmented growth over 3 weeks in normal rats and peaked at day 21 or 14 post-injection respectively, whose body weight gains were on average approximately 6% or 19% heavier over controls. However, other injectable vectors had no such enhanced growth effects. Our results suggested that the efficacy of the ghrelin leader sequence was more effective than that of hGHRH in our system. Moreover, the results indicated that skeletal muscle might have the ability to posttranslationally modify the in vivo expressed ghrelin. And the most strikingly, the short ghrelin analog seems to mimic the biological effects more efficiently when compared with the full-length ghrelin.
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가설 설정
We then asked if a single injection of pGEM-wt-sGhln, pGEM-wt-hGhln in adolescent rats would be sufficient to elicit augmented GH systemic levels and enhance growth.
, 2001). We then asked if a sin이e injection of pGEM-wt-sGhln, pGEM-wt-hGhln in adolescent rats would be sufficient to elicit augmented GH systemic levels and enhance growth. Since N-octanoyl posttranslational modification at serine residue 3 of ghrelin is required for biological activity, and at present, there do not provide any information whether skeletal muscle can perform the cellular machinery for this acylation, so the pGEM-mt-sGhln and pGEM-mt-hGhln replacing the octanoylated serine with an aromatic amino acid, Trp(W) were constructed.
제안 방법
, 1997, 1999). In this study, six expression vectors containing natural mature ghrelin and its analogs cDNA were constructed. The effects of signal peptides of either ghrelin itself or human GHRH were also tested.
, 1996) and the 3’ untranslated region of hGH cDNA in a 613 bp Sac I/Nsi I fragment. To construct expression vectors, pGEM-A5f3f was first cut by Mlu I, then blunted using Klenow Fragment (Takara) and then cut by Spe I; The wild-type ghrelin and its variants cDNA were obtained by PCR amplification from pMD18-T-Ghln constructs respectively and were inserted into blunt-ended Mlu I/Spe I to create pGEM-wt-sGhln, pGEM-mt-sGhln, pGEM-tmt-sGhln, pGEM-wt-hGhln, pGEM-mt-hGhln and pGEM-tmt-hGhln. The amino acid sequence of ghrelin and its variants encoded by the vectors above were shown as Table 2.
, 1996) and the 3’ untranslated region of hGH cDNA in a 613 bp Sac I/Nsi I fragment. To construct expression vectors, pGEM-A5f3f was first cut by Mlu I, then blunted using Klenow Fragment (Takara) and then cut by Spe I; The wild-type ghrelin and its variants cDNA were obtained by PCR amplification from pMD18-T-Ghln constructs respectively and were inserted into blunt-ended Mlu I/Spe I to create pGEM-wt-sGhln, pGEM-mt-sGhln, pGEM-tmt-sGhln, pGEM-wt-hGhln, pGEM-mt-hGhln and pGEM-tmt-hGhln. The amino acid sequence of ghrelin and its variants encoded by the vectors above were shown as Table 2.
대상 데이터
Wistar male SPF rats (National Institute for Control of Pharmaceuticals and Biological Products, Beijing, China) were housed and cared for in the animal facility of Institute for Space Medical Engineering (Beijing, China). All animal experimentations were performed in accordance with guidelines for the care and use of laboratory animals, under environmental condition of 10 h light/14 h darkness.
데이터처리
The statistical significance of differences between plasmid DNA injection groups and control group was evaluated independently with Ducan’s multiple range test, preceded by the analysis of variance (ANOVA).
Values are expressed as mean±standard error of the mean (SEM). The statistical significance of differences between plasmid DNA injection groups and control group was evaluated independently with Ducan’s multiple range test, preceded by the analysis of variance (ANOVA). A p value of less than 0.
후속연구
Injected pGEM-wt-sGhln encoding natural mature ghrlein augmented growth up to 5 weeks. This may indicate that skeletal muscle may have the ability to perform posttranslational modification of ghrelin, which is necessary for its subsequent biological activities, but this hypothesis needs further confirmation. Injected pGEM-tmt-sGhln encoding truncated serine residue replacement ghrelin variant was the most potent stimulus to increase growth in normal rats.
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