Comparison of conventional plasmid vector and Semliki forest virus-derived vectors in expressing growth hormone releasing hormone (GHRH)

2008 ◽  
Vol 5 (3) ◽  
pp. 211-216
Author(s):  
Ren Xiao-Hui ◽  
Luo Hu-Ying ◽  
Liu Song-Cai ◽  
Zhang Ming-Jun ◽  
Ouyang Song-Ying ◽  
...  
Keyword(s):  
Gene Expression ◽  
Growth Hormone ◽  
Semliki Forest Virus ◽  
Expression Vectors ◽  
Growth Hormone Releasing Hormone ◽  
Plasmid Vectors ◽  
Releasing Hormone ◽  
Replicon Vector ◽  
Foreign Genes ◽  
Rna Replicon

AbstractThe elements for the Semliki forest virus (SFV) RNA replicon were obtained from the Alphavirus genome. It was designed to overcome the poor efficacy of some current plasmid vectors. Genes coding for viral replicases are preserved while genes coding for structural proteins are replaced by foreign genes in the RNA replicon. High levels of RNA replication and expression of foreign genes in the cytoplasm are regulated by the replicases. To evaluate the effects of the SFV RNA replicon on the improvement of gene expression, a LacZ gene was inserted into pIRES-neo digested by BamHI and dephosphorylated by alkaline phosphatase to construct pIRES-neo-LacZ. The RNA replicon vector pCMV-rep-LacZ and two conventional cytomegalovirus (CMV) promoter-based vectors (pLNCX-LacZ and pIRES-neo-LacZ) were transfected, using Lipofectin, to prepared 293 cells. Growth hormone releasing hormone (GHRH) expression vectors (pCMV-Rep-GHRH, pCDNA3.1(+)-GHRH and pIRES-neo-GHRH) were also tested using the same procedure. Target gene expression was detected with radioimmunoassay (RIA) and reverse transcription-polymerase chain reaction (RT-PCR). Results showed that the expression level of the RNA replicon vector was 2–3 times higher than with normal plasmid vectors. This result will help to improve the efficiency of gene expression in eukaryotic cells.

scholarly journals SAT-603 Growth Hormone-Releasing Hormone (GHRH) Antagonists Stimulate Feeding in Mice

2020 ◽  
Vol 4 (Supplement_1) ◽  
Author(s):  
Sheila Leone ◽  
Lucia Recinella, PharmD ◽  
Annalisa Chiavaroli, PharmD ◽  
Giustino Orlando, PharmD ◽  
Claudio Ferrante, PharmD ◽  
...  
Keyword(s):  
Gene Expression ◽  
Growth Hormone ◽  
Body Weight ◽  
Locomotor Activity ◽  
Food Intake ◽  
Brown Fat ◽  
Growth Hormone Releasing Hormone ◽  
Antitumor Effects ◽  
Releasing Hormone ◽  
Fat Depots

Abstract Growth hormone-releasing hormone (GHRH) is a hypothalamic neuropeptide which stimulates the synthesis and secretion of growth hormone (GH) in pituitary gland. GHRH was also found to modulate food intake in mammals. MIA-690 is a synthetic GHRH antagonist of the Miami (MIA) series with potent antitumor effects. To date, its role in hypothalamic feeding modulation has not been evaluated. In the present study, we aimed to investigate the effects of chronic MIA-690 administration on feeding behavior, locomotor activity and hypothalamic dopamine (DA), norepinephrine (NE), serotonin (5-hydroxytriptamine, 5-HT), orexigenic peptides [agouti-related peptide (AgRP) and neuropeptide Y (NPY)] and anorexigenic peptides [cocaine and amphetamine-regulated transcript (CART) and proopiomelanocortin (POMC)] activity. Adult C57/BL6 mice were treated daily for 4 weeks by subcutaneous administration of (5 µg) MIA-690 or vehicle solution. Food intake and body weight were recorded every 4 days throughout the study. Immediately after the last injection, locomotor activity in the home cage was recorded, and thereafter animals were sacrificed. Visceral, subcutaneous and brown fat depots were quickly excised and weighed. Hypothalamus was also dissected for evaluating gene expression of AgRP, NPY, CART and POMC by real-time reverse transcription polymerase chain reaction. In addition, hypothalamic DA, NE and 5-HT levels were measured by high performance liquid chromatography (HPLC) coupled to electrochemical detection. Our findings show that administration of MIA-690 increased food intake and body weight, without affecting locomotor activity. No difference was observed in visceral, subcutaneous and brown fat mass in animals treated with MIA-690 or vehicle. As for neuromodulatory effects, a significant increase of AgRP gene expression and NE levels, along with a reduction of 5-HT levels were found after MIA-690 treatment. On the other hand, we did not observe any alteration in NPY, POMC and CART gene expression, as well as DA levels, following MIA-690 administration. In conclusion, chronic peripheral administration of MIA-690 could play an orexigenic role paralleled by increased body weight. The stimulation of feeding could be mediated, at least in part, by increased AgRP gene expression and NE levels and decreased 5-HT levels, in the hypothalamus.

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