Rapid Site-Directed Mutagenesis Using Two-PCR-Generated DNA Fragments Reproducing the Plasmid Template

We describe a new rapid and efficient polymerase chain reaction (PCR)-based site-directed mutagenesis method. This procedure is effective with any plasmid and it employs four oligonucleotide primers. One primer contains the desired mutation, the second is oriented in the opposite direction (one of these two primers should be phosphorylated), and the third and fourth should be coding in complementary fashion for a unique restriction site to be introduced in a nonessential region. The method consists of two simultaneous PCR reactions; the PCR products are digested with the enzyme that recognizes the newly introduced unique restriction site and then ligased and used to transform competent bacteria. Additionally, the use of Dpn I facilitates the elimination of template DNA. The newly introduced restriction site is essential for ligation in the correct orientation of the two-PCR products and is further used for mutant screening. Resulting plasmids carry both the new restriction site and the desired mutation. Using this method, more than 20 mutants have already been generated (using two different kinds of templates); all these mutants were sequenced for the desired mutation and transfected into AtT-20 cells and the expressed mutant proteins encoded by the vector were assayed

OR 47: Renal endothelin receptor type B upregulation in rats with low or high renin hypertension

Endothelin-1 (ET-1) is a potent renal and systemic vasoactive peptide. It acts through ETA and ETB receptors. We investigated density and subtype distribution of ET-1 receptors in hearts and kidneys of normotensive and hypertensive rats. Five groups of uninephrectomized Wistar rats were put on a low salt diet for six weeks. During this period, three groups of rats drank tap water and two groups received saline. One group of each regimen received DOCA subcutaneousely (1.6 mg/day). The fifth group of rats had the left renal artery clipped to induce 1K1C hypertension. At 6 weeks, mean arterial pressure (MAP) was recorded in conscious rats via a femoral artery catheter. Binding assays using 125I-ET-1 were carried out on membrane preparations in the presence and absence of the ETA receptor antagonist FR139317. On tap water, MAP was at 121.8±3.3 mmHg and DOCA or saline did not raise this MAP. On DOCA-salt and in 1K1C rats, MAP was increased to 154.5±5.8 mmHg (p<0.001) and 218.4±10.5 (p<0.001) mmHg, respectively. ET receptor subtypes were not equally expressed in the heart and the kidney: ETA was predominantly expressed in the heart, whereas ETB was predominant in the kidney. Both hypertensive models, the DOCA-salt and the 1K1C rats showed further significant changes: i) Cardiac weight index compared to controls of 2.49±0.06 mg/g was higher (p<0.001) at 3.89±0.10 and 4.86±0.18 mg/g in DOCA-salt and 1K1C hypertension, respectively, and kidney weight index compared to controls of 4.78±0.22 mg/g was higher at 10.10±0.54 mg/g in DOCA-salt (p<0.001) but tended to be below controls in 1K1C rats. ii) In the kidneys, the density of the ETB receptor subtype was upregulated in DOCA-salt and 1K1C rats from 160±8 to 217±12 and 190±2 fmol/mg (p<0.05), respectively, and ETA tended to be downregulated. iii) Plasma renin activity was decrased in DOCA-salt rats from 17±3 to 0.17±0.01 ng/ml/h and increased in 1K1C rats on low salt diet to 30±5 ng/ml/h (p<0.001). We conclude that upregulation of the ETB receptor mediating vasodilation and downregulation of the ETA receptor mediating vasoconstriction is compatible with a mainly renal counterregulatory effect of endothelin-1 to hypertension. This counterregulation may occur in both low and high renin models of hypertension. Am J Hypertens (2004) 17, 20A-21A; doi: 10.1016/j.amjhyper.2004.03.04

Measurement of plasma endothelin-1 in experimental hypertension and in healthy subjects

Background: Endothelin-1 is an endothelium-derived potent vasoconstrictor peptide of 21 amino acids. To establish reference values in different models of hypertension and in human subjects an assay for plasma immunoreactive endothelin-1 (ET-1) was optimized. Methods: ET-1 is extracted by acetone from 1 mL of plasma and subjected to a sensitive enzyme-linked immunosorbent assay. Results: The detection limit for plasma ET-1 is 0.05 fmol/mL. Mean recoveries of the 1, 2, 5, and 10 fmol of ET-1 added to 1 mL of plasma were 66%, 75%, 85%, and 92%, respectively. Within- and between-assay coefficients of variation were ≤12% and ≤10%, respectively. Assay accuracy was demonstrated by consistent recoveries of added ET-1 over the entire physiologic range of plasma concentrations and by the linearity of ET-1 concentrations measured in serially diluted plasma extracts (r = 0.99). No ET-1 was detected when albumin buffer was extracted instead of plasma. Using this method, we found increased ET-1 levels in plasma of three experimental rat models of hypertension: stroke prone spontaneously hypertensive rats (SP-SHR), deoxycorticosterone acetate-salt hypertensive rats, and one kidney-one clip hypertensive rats. In contrast, plasma ET-1 levels of SHR were half those of normotensive Wistar rats. In two kidney-one clip hypertensive rats, plasma ET-1 concentrations were not different from those found in sham-operated control rats. Plasma ET-1 concentrations of 37 healthy men were 0.85 ± 0.26 fmol/ml (mean ± SD). Conclusions: The present assay reliably measures ET-1 levels in rat and human plasma. It allows to discriminate between different forms of hypertension with high or low circulating levels of ET-1. Am J Hypertens 2003;16: 515-521 @ 2003 American Journal of Hypertension, Lt

Evidence for a role of sphingosine-1 phosphate in cardiovascular remodelling in Fabry disease

Aims A hallmark of Fabry disease is the concomitant development of left-ventricular hypertrophy and arterial intima-media thickening, the pathogenesis of which is thought to be related to the presence of a plasmatic circulating growth-promoting factor. We therefore characterized the plasma of patients with Fabry disease in order to identify this factor. Methods and results Using a classical biochemical strategy, we isolated and identified sphingosine-1 phosphate (S1P) as a proliferative factor present in the plasma of patients with Fabry disease. Plasma S1P levels were significantly higher in 17 patients with Fabry disease compared with 17 healthy controls (225 ± 40 vs. 164 ± 17 ng/mL; P = 0.005). There was a positive correlation between plasma S1P levels and both common carotid artery intima-media thickness and left-ventricular mass index (r2 = 0.47; P = 0.006 and r2 = 0.53; P = 0.0007, respectively). In an experimental model, mice treated with S1P developed cardiovascular remodelling similar to that observed in patients with Fabry disease. Conclusion Sphingosine-1 phosphate participates in cardiovascular remodelling in Fabry disease. Our findings have implications for the treatment of cardiovascular involvement in Fabry diseas

Identification of SLURP-1 as an epidermal neuromodulator explains the clinical phenotype of Mal de Meleda

Mal de Meleda is an autosomal recessive inflammatory and keratotic palmoplantar skin disorder due to mutations in the ARS B gene, encoding for SLURP-1 (secreted mammalian Ly-6/uPAR-related protein 1). SLURP-1 belongs to the Ly-6/uPAR superfamily of receptor and secreted proteins, which participate in signal transduction, immune cell activation or cellular adhesion. The high degree of structural similarity between SLURP-1 and the three fingers motif of snake neurotoxins and Lynx1 suggests that this protein interacts with the neuronal acetylcholine receptors. We found that SLURP-1 potentiates the human α7 nicotinic acetylcholine receptors that are present in keratinocytes. These results identify SLURP-1 as a secreted epidermal neuromodulator which is likely to be essential for both epidermal homeostasis and inhibition of TNF-alpha release by macrophages during wound healing. This explains both the hyperproliferative as well as the inflammatory clinical phenotype of Mal de Meled

Aortic remodelling in Fabry disease

Aims To evaluate thoracic aortic dilation in patients with Fabry disease (FD). Methods and results A cohort of 106 patients with FD (52 males; 54 females) from three European centres were studied. The diameter of the thoracic aorta was assessed at three levels (sinus of Valsalva, ascending aorta, and descending aorta) using echocardiograms and cardiovascular magnetic resonance imaging. Aortic dilation at the sinus of Valsalva was found in 32.7% of males and 5.6% of females; aneurysms were present in 9.6% of males and 1.9% of females. No aortic dilation was observed in the descending aorta. There was no correlation between aortic diameter at the sinus of Valsalva and cardiovascular risk factors. Conclusion Fabry disease should be considered as a cardiovascular disease that affects the heart and arterial vasculature, including the thoracic aorta. Thus, patients with FD should be closely monitored for the presence, and possible progression and complications of aortic dilation. Clinical Trial Registration: Protocol 101/01. Ethics committee, Faculty of Medicine, Lausann