In situ synthesis of oligonucleotide arrays by using surface tension.

This work describes the in situ synthesis of oligonucleotide arrays on glass surfaces. These arrays are composed of features defined and separated by differential surface tension (surface tension arrays). Specifically, photolithographic methods were used to create a series of spatially addressable, circular features containing an amino-terminated organosilane coupled to the glass through a siloxane linkage. Each feature is bounded by a perfluorosilanated surface. The differences in surface energies between the features and surrounding zones allow for chemical reactions to be readily localized within a defined site. The aminosilanation process was analyzed using contact angle, X-ray photoelectron spectroscopy (XPS), and time-of-flight/secondary ion mass spectroscopy (TOF-SIMS). The efficiency of phosphoramidite-based oligonucleotide synthesis on these surface tension arrays was measured by two methods. One method, termed step-yields-by-hybridization, indicates an average synthesis efficiency for all four (A,G,C,T) bases of 99.9 +/- 1.1%. Step yields measured for the individual amidite bases showed efficiencies of 98.8% (dT), 98.0% (dA), 97.0% (dC), and 97.6% (dG). The second method for determining the amidite coupling efficiencies was by capillary electrophoresis (CE) analysis. Homopolymers of dT (40- and 60mer), dA (40mer), and dC (40mer) were synthesized on an NH(4)OH labile linkage. After cleavage, the products were analyzed by CE. Synthesis efficiencies were calculated by comparison of the full-length product peak with the failure peaks. The calculated coupling efficiencies were 98.8% (dT), 96.8% (dA), and 96.7% (dC).

Renal hemodynamics in young and old spontaneously hypertensive rats during intrarenal infusion of arginine vasopressin.

BACKGROUND/AIMS: To gain insight into the effect of arginine vasopressin (AVP) on renal hemodynamics in hypertensive rats, we investigated the vasoconstrictive response to AVP on total renal blood flow (RBF) and total and zonal glomerular filtration rate (GFR) in young and old spontaneously hypertensive rats (SHR). A hypothesis of increased AVP sensitivity in the juxtamedullary cortex of SHR was tested. METHODS: Total RBF and total and zonal GFR were studied in 10- and 40-week-old SHR and normotensive Wistar-Kyoto rats (WKY). RBF was recorded by a flowmeter before infusion of AVP and immediately after injection of a bolus dose of 10 ng AVP. Whole kidney GFR and its intracortical distribution was measured by the tubular uptake of 125I- and 131I-labelled aprotinin before and during a continuous infusion of AVP 5 ng/min. Ligand binding measurements of preglomerular V1a receptors were performed in young and old rats. RESULTS: RBF decreased by 43 +/- 3% in 10-week SHR (9.2 +/- 0.5 vs. 5.2 +/- 0.3 ml x min(-1) x g(-1)), significantly more than 10-week WKY where RBF decreased by 35 +/- 3% (9.6 +/- 0.7 vs. 6.5 +/- 0.5 ml x min(-1) x g(-1)) (p < 0.05). The effect of AVP on RBF was attenuated in 40-week-old rats where the decline in RBF was 29 +/- 5% in SHR and 23 +/- 4% in WKY (p > 0.05). GFR decreased by 6 +/- 3% (1.03 +/- 0.04 vs. 0.96 +/- 0.04 ml x min(-1) x g(-1), p < 0.05) in 10-week SHR and was unchanged in 10-week WKY (1.10 +/- 0.07 vs. 1.08 +/- 0.04 ml x min(-1) x g(-1), p > 0.10). GFR decreased by 11 +/- 10% in 40-week SHR and by 4 +/- 4% in 40-week WKY (p > 0.05). AVP infusion significantly increased filtration fraction in all groups except 40-week SHR, indicating that AVP has the strongest vasoconstrictive effect on postglomerular vessels. The intrarenal distribution of GFR was unchanged in the normotensive and hypertensive groups. V1a receptor density was upregulated in young SHR compared to young WKY (p < 0.05), but downregulated in old compared to young SHR (p = 0.05). CONCLUSION: The results indicate that AVP sensitivity is not increased in the juxtamedullary cortex in SHR and the reduced vasoconstrictive effect in old SHR is due to a reduced density of V1a receptors.