ABSTRACT
The yeast Saccharomyces cerevisiae is known as an available host for human G-protein-coupled receptor (GPCR) ligand screening. Although several types of yeast signal sequences (SS) attached with the GPCRs could improve their productivities and facilitate transportation of the GPCRs to the yeast plasma membrane, the effects of additional SS on ligand-specific signalling functions of GPCRs are not reported. Here, we demonstrated the controlling signalling properties by addition of SS using engineered yeast as a host. Prepro and pre regions of alpha-factor and amino-terminal sequence of Ste2 (Ste2N) were used as SS, and somatostatin (SST) receptor subtype-5 (SSTR5) was used as a model GPCR. We also constructed a yeast-based fluorescent assay system for monitoring the activation levels of SSTR5 signalling by a green fluorescent protein (GFP) reporter gene. The production levels and localisation patterns of the SS-attached SSTR5 were more significantly improved than those of wild-type SSTR5. In addition, we successfully controlled the pharmacological efficacy and potency by introducing SS. Among four types of SSTR5 receptors, Ste2N-SSTR5 responded at the lowest ligand concentration. This finding will be informative for constructing optimal yeast-based ligand screening systems to discriminate the cells on the basis of signalling levels.
Subject(s)
Protein Sorting Signals/physiology , Receptors, G-Protein-Coupled/chemistry , Receptors, G-Protein-Coupled/metabolism , Receptors, Somatostatin , Flow Cytometry , Green Fluorescent Proteins , Humans , Microscopy, Fluorescence , Receptors, Somatostatin/chemistry , Receptors, Somatostatin/metabolism , Recombinant Fusion Proteins/metabolism , Saccharomyces cerevisiae , Signal Transduction , Somatostatin/metabolism , TransfectionABSTRACT
To allow the comprehensive assessments of yeast expression systems, a simple and immediate method for simultaneously evaluating the expression level and plasmid maintenance in yeast was demonstrated. This method uses green fluorescent protein (GFP) and flow cytometry (FCM) and is characterized by a dual analysis of the average intensity of GFP fluorescence and the population of GFP-expressing cells. The FCM analysis of GFP fluorescence intensity rapidly quantifies the expression level without complex manipulations, such as the enzymatic reaction of a lacZ reporter assay. Moreover, the single-cell analysis revealed that the proportion of cells expressing GFP in the cell cluster reflects the plasmid retention rate; therefore, the FCM analysis of the GFP-expressing population allows the immediate estimation of the plasmid retention rate without the 2- or 3-day incubation required for colony counting. We show that the FCM analysis with GFP reporter is a suitable method to explore the hopeful expression vector and host strain or establish the several expression systems exhibiting the characteristic properties in yeast.
Subject(s)
Flow Cytometry/methods , Gene Expression/genetics , Genetic Vectors/genetics , Plasmids/genetics , Saccharomyces cerevisiae/genetics , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Models, Genetic , Saccharomyces cerevisiae/cytology , Saccharomyces cerevisiae/metabolismABSTRACT
Agents to inhibit the renin-angiotensin system have been reported to suppress the progression of abdominal aortic aneurysm (AAA). However, the effects of calcium channel blockers (CCBs) are still unclear in terms of the inhibition of the progression of AAA. Recently, several effects of CCBs beyond those associated with blood pressure lowering have attracted much interest. In this study, we examined the effects of nifedipine on AAA progression. AAA was induced in rats by transient aortic perfusion with elastase. Then, nifedipine (10 mg/kg/day) and saline (control) were administered to rats by osmotic mini-pump. At 2 and 4 weeks, the size of the AAA, blood pressure and heart rate were measured. Then, to further explore the mechanisms of the progression of AAA, we used human vascular smooth muscle cells (VSMCs). Especially, we focused on NF-kappaB and matrix metalloproteinase-9 (MMP-9). Treatment with nifedipine resulted in a significant inhibition of the progression of AAA such as aneurismal dilation at 14 and 28 days compared to the control (week 2: control, 2.98+/-0.71 mm; nifedipine, 2.37+/-0.64 mm; p<0.05 and week 4: control, 3.28+/-0.98 mm; nifedipine, 2.41+/-0.17 mm; p<0.05). Neither nifedipine nor saline changed blood pressure and heart rate, significantly. Nifedipine (1 microM) significantly suppressed angiotensin II-induced (10(-6) M) NF-kappaB activity in VSMCs by reporter assay (p<0.01). Furthermore, nifedipine (1 microM) inhibited MMP-9 protein expression and activity. Saline did not show such inhibitory effects. Taken together, these results indicated that nifedipine inhibits the progression of experimental AAA possibly through suppression of NF-kappaB and MMP-9 activity, leading to protective effects against AAA beyond those associated with blood pressure lowering.
Subject(s)
Aortic Aneurysm, Abdominal/drug therapy , Aortic Aneurysm, Abdominal/pathology , Calcium Channel Blockers/therapeutic use , Nifedipine/therapeutic use , Animals , Aortic Aneurysm, Abdominal/diagnostic imaging , Aortic Aneurysm, Abdominal/physiopathology , Blood Pressure/drug effects , Blotting, Western , Cells, Cultured , Disease Models, Animal , Disease Progression , Electrophoretic Mobility Shift Assay , Heart Rate/drug effects , Humans , Male , NF-kappa B/genetics , NF-kappa B/metabolism , Nifedipine/pharmacology , Rats , Rats, Wistar , Sodium Chloride/pharmacology , Time Factors , Transcription, Genetic/drug effects , UltrasonographyABSTRACT
Many clinical trials have demonstrated that angiotensin converting enzyme inhibitors have protective effects on organ damage, suggesting the importance of inhibition of the renin-angiotensin system. In this study, we investigated the effects of a non-depressor dose of imidapril on organ damage induced by diabetes and hypertension. Diabetes was induced by an intravenous injection of streptozotocin (STZ, 40 mg/kg) in 15-week-old male spontaneously hypertensive rats (SHR). Imidapril (2 mg/kg/day) or vehicle was given orally for 28 days, and then the heart weight, left ventricle mass (LVM), urinary albumin excretion (UAE) and endothelial function were examined, as well as the urinary NOx level and local hepatocyte growth factor (HGF) expression. There were no significant differences between the treated groups in systolic blood pressure and plasma parameters. On the other hand, UAE was significantly suppressed in the imidapril-treated group (450+/-44 mg/day) compared to the vehicle-treated group (963+/-182 mg/day) (p<0.01). Moreover, endothelial function assessed by dilative reaction to acetylcholine as well as cardiac hypertrophy assessed by both heart/body weight ratio and LVM were significantly improved in the imidapril-treated group (p<0.05 and p<0.01, respectively). The urinary NOx concentration and local HGF expression in vessel walls were also significantly increased in the imidapril-treated group (p<0.01). A non-depressor dose of imidapril showed protective effects against organ damage in diabetic SHR, which may be partially due to the increase of HGF and NO.
