Effects of pulsed ultrasound on sphenoid bone temperature and the heart rate in guinea-pig foetuses.

Temperature increase induced by exposure to unscanned pulsed ultrasound at an intensity (I(SPTA)) 2.82 W/cm2 was measured in the brain adjacent to the sphenoid bone of foetal guinea-pigs in late gestation under in vitro and in vivo (in utero) conditions. After 120 s exposure a mean temperature increase of 2.6 degrees C was measured in vitro. Removal of the overlying parietal bones increased this value to 5.2 degrees C. Mean temperature increases at the sphenoid bone recorded in utero were 1.5 degrees C live and 2.0 degrees C post mortem. Measurement of foetal ECG showed that ultrasound-induced heating of the hypothalamic region did not significantly alter foetal heart rate.

Multidimensional chromatography coupled with mass spectrometry for target-based screening.

The synthesis of structural analogs and the process of drug discovery have evolved dramatically through recent advances in solid-phase synthesis reagents and automated screening systems. As molecular diversity strategies emerge, the need for automated target-based selection of lead candidates becomes equally important. Multidimensional automated chromatographic techniques coupled to electrospray ionization mass spectrometry facilitate the selection process and provide maximum characterization information in a single screening run. The capture of tightly bound affinity leads by target biomolecules, followed by subsequent release and high-resolution separation with sensitive detection, significantly reduces the time required to identify and characterize lead compounds. This automated multidimensional chromatographic approach coupled with mass spectrometry, Selectronics, was used with several organic and natural libraries to demonstrate an automated target-based screening technique to select for high-affinity binders as potential lead compounds.

Stimulation of bovine milk galactosyltransferase activity by bovine colostrum N-acetylglucosaminyltransferase I.

Purified bovine milk galactosyltransferase was stimulated by purified bovine colostrum N-acetylglucosaminyltransferase I by more than 10-fold. Only slight stimulation of the N-acetylglucosaminyltransferase I by galactosyltransferase was observed. Heat inactivation destroyed the ability of the N-acetylglucosaminyltransferase I to stimulate the galactosyltransferase. The stimulation of galactosyltransferase was accompanied by a decrease in Km of this enzyme from 9.7 to 3.3. mM and an increase in Vmax from 1.87 to 3.71 nmol galactose transferred/min per mg galactosyltransferase when GlcNAc was the substrate. When the Km for UDPgalactose was determined, it increased from 0.19 to 0.42 mM in the presence of N-acetylglucosaminyltransferase I and the Vmax increased from 0.66 to 2.76 nmol galactose transferred/min per mg galactosyltransferase. In phosphatidylcholine vesicles, no effect on Km values with GlcNAc as substrate was noted, while an increase in the Km of UDPgalactose was observed. The Vmax values were generally higher in the lipid vesicles. Complex formation between galactosyltransferase and N-acetylglucosaminyltransferase I was demonstrated both by glycerol density gradient centrifugation and Bio-Gel P-100 column chromatography. An approximate molecular weight for the complex was obtained on a calibrated Sephadex G-200 column and found to be about 75 000, consistent with a 1:1 complex. The stimulation of galactosyltransferase involved the N-acetyllactosamine synthetase activity of this enzyme and not the lactose synthetase activity, since the latter activity was only slightly affected. Since N-acetylglucosaminyltransferase I is not involved in the lactose synthetase reaction, the stimulation is consistent with the known biosynthetic role of N-acetylglucosaminyltransferase I in the biosynthesis of asparagine-linked oligosaccharides.

Control of glycoprotein synthesis. IX. A terminal Man alpha l-3Man beta 1- sequence in the substrate is the minimum requirement for UDP-N-acetyl-D-glucosamine: alpha-D-mannoside (GlcNAc to Man alpha 1-3) beta 2-N-acetylglucosaminyltransferase I.

Twenty low molecular weight compounds were tested as substrates for UDP-GlcNAc:alpha-D-mannoside (GlcNAc to Man alpha 1-3) beta 2-N-acetylglucosaminyltransferase I (GlcNAc-transferase I) purified from bovine colostrum. This enzyme is at a key control point in the biosynthetic path leading to complex Asn-linked oligosaccharides. The highest activity was obtained with the substrate Man alpha 1-3(R1 alpha 1-6)Man beta 1-R2 where R1 was Man alpha 1-3(Man alpha 1-6)Man- (Km = 0.20 mM) and R2 was -4GlcNAc beta 1-4GlcNAc-Asn. Somewhat less effective were substrates in which R1 was Man- (Km = 0.4-0.6 mM) and R2 was either-4GlcNAc or -4GlcNAc beta 1-4(Fuc alpha 1-6)GlcNAc-Asn. Removal of the Man alpha 1-6 arm (R1 = H-) or replacing R2 with an isopropyl group had no effect on Vmax but increased the Km about 10-fold, thereby leading to an 85% reduction in enzyme activity as measured under standard conditions. An 85% reduction in activity was also observed if R2 was replaced with N-acetylglucosaminitol. Enzyme activity was reduced 33% if R1 was Gal beta 1-4GlcNAc beta 1-2Man-. Any compounds lacking a Man alpha 1-3- terminus or in which the beta-linked Man had been replaced with an alpha-linked Man were totally inactive. It was concluded that a terminal Man alpha 1-3Man beta 1-sequence is a minimal structural requirement for a GlcNAc-transferase I substrate. The only effective substrate for partially purified UDP-GlcNAc:alpha-D-mannoside (GlcNAc to Man alpha 1-6) beta 2-N-acetylglucosaminyltransferase II (GlcNAc-transferase II) from bovine colostrum was R1-GlcNAc beta 1-2Man alpha 1-3(Man alpha 1-6)Man beta 1-R2 where R1 = H-. The absence of a terminal GlcNAc beta 1-2- residue or masking this residue by making R1 = Gal beta 1-4-, both prevented enzyme activity, indicating that GlcNAc-transferase I action must precede GlcNAc-transferase II action during biosynthesis of complex Asn-linked oligosaccharides.