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.

Ultrasound-induced temperature increase in the guinea-pig fetal brain in vitro.

The temperature of the brain of fetal guinea pigs was measured in vitro during exposure to an unscanned beam of pulsed ultrasound at intensity ISPTA 2.8 W/cm2. A mean temperature increase of 5.1 degrees C recorded after 2 min of insonation confirms results of an earlier similar study. The water-bath exposure system provided enhanced cooling of superficial tissue by acoustic streaming. When the scalp was removed, the ultrasound-induced temperature increase was substantially reduced (by 35%) due to cooling through radiation force-induced bulk fluid streaming along the direction of propagation in the water bath. The measured temperature increase in guinea pig fetal brain correlated with a modified cranial thermal index.

In vivo heating of the guinea-pig fetal brain by pulsed ultrasound and estimates of thermal index.

Temperature was measured in the brain in live near-term fetal guinea pigs (62-66 d gestational age), during in utero exposure to a fixed beam of pulsed ultrasound at intensity ISPTA 2.82 W/cm2. Mean temperature increases of 4.3 degrees C close to parietal bone and 1.1 degrees C in the mid-brain were recorded after 2-min exposures. These values were lower (12%) than those obtained for ultrasound-induced heating near the bone in dead fetuses insonated in utero. A significant cooling effect of vascular perfusion was observed only when guinea pig fetuses reached late gestation, near term, when the cerebral vessels were well developed. The estimated value for the thermal index (TIB), as used in AIUM/NEMA output display standard, underestimated the measured temperature increase at the bone-brain interface. The ratio of measured temperature to the TIB is 1.3. A modification of the cranial thermal index provided a more reasonable, conservative, estimate of the temperature increase at a biologically significant point of interest at the brain-bone interface.

Ultrasound-induced temperature increase in guinea-pig fetal brain in utero: third-trimester gestation.

Temperature increase was measured at various depths in the brain of living fetal guinea pigs during in utero exposure to unscanned pulsed ultrasound at ISPTA 2.8 W/cm2. Mean temperature increases of 4.9 degrees C close to parietal bone and 1.2 degrees C in the midbrain were recorded after 2-min exposures. When exposures were repeated on the same sites in each fetus after death, the corresponding mean temperature increases were 4.9 degrees C and 1.3 degrees C, respectively. Cerebral blood perfusion had little cooling effect on ultrasound-induced heating in the guinea pig fetus of 57-61 days gestational age.

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.

New family of high-resolution ion exchangers for protein and nucleic acid purifications from laboratory to process scales.

A new family of polymer-based ion exchangers was tested for the purification of acidic and basic proteins on both the analytical and preparative scales. Protein-Pak HR series packings are available as strong cation (SP) and weak anion (DEAE) exchangers, allowing the development of a purification method regardless of the isoelectric point of protein. Three particle sizes, 8, 15 and 40 microns, are offered in scalable Advanced Purification (AP) glass columns or as bulk packings. The lower back pressures of the 15- and 40-microns packings compared to that of the 8-microns material allow rapid throughput of large volumes without exceeding the pressure limitations of the resin or the column. The capacity of the AP1 (100 mm x 10 mm) glass columns, containing these ion-exchange packings, is comparable to other ion-exchange columns. The resolution of mouse serum, plasmids, and a standard protein mixture was demonstrated and compared with the results obtained with other resin-based ion exchangers of similar particle size. Proteins were purified without significant loss of biological activity or mass.

Interaction between human myelin basic protein and lipophilin.

The interaction of human myelin basic protein with lipophilin has been demonstrated by affinity chromatography. The interaction was specific since neither basic protein, nor albumin bound to an affinity column consisting of BP bound to agarose. Conversely an albumin affinity column failed to bind BP. The pH dependency of the interaction correlated with the known pK for histidine. By the use of large peptides formed by tryptophanyl cleavage by BNPS-skatole, peptide 1-117 bound to the BP affinity column while neither the smaller peptide, 118-170, nor the synthetic nonapeptide bound. The large fragment contains 9 of the 10 histidines in the molecule which may explain the binding of this fragment. The result of such protein-protein interactions makes available a large number of new antigenic sites and extends considerably the range of encephalitogens for disease induction.

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.

Oligosaccharide branching of glycoproteins: biosynthetic mechanisms and possible biological functions.

One of the most striking features of N- and O-glycosyl oligosaccharides and of lipid-linked oligosaccharides is the high degree of branching of these complex structures. Both proteins and nucleic acids are essentially linear structures and are synthesized by template mechanisms. The branched nature of complex carbohydrates dictates a totally different mechanism of biosynthetic control. Although there are undoubtedly many factors controlling this assembly (e.g. subcellular compartmentation, availability of substrates, cations), our laboratory has studied primarily the enzymatic factors that control the assembly of branched N-glycosyl (Asn-GlcNAc type) and O-glycosyl (Ser[Thr]-GalNAc type) oligosaccharides. There are three basic types of control points that appear to direct biosynthesis. (a) There may be two or more enzymes capable of acting on a single common substrate. Control at this juncture is exerted by the relative activities of these enzymes in a particular tissue. (b) Addition of a specific sugar to the growing oligosaccharide may shut off one or more subsequent enzyme steps, thereby 'freezing' the structure at a certain stage in its synthesis. (c) Progression of the pathway may be impossible until a certain key sugar residue is inserted into the growing oligosaccharide chain. Examples of all three types of control occur in the assembly of both N- and O-glycosyl oligosaccharides. This paper discusses our work on the N-acetylglucosaminyltransferases, which initiate branches in N-glycosyl oligosaccharides, as well as some studies on glycosyltransferases that control the assembly of the four basic Ser(Thr)-GalNAc cores. Important features at all stages of control are the three-dimensional shape of the oligosaccharide, the effect of certain key sugar residues on this three-dimensional shape and the stereochemistry of the interaction of oligosaccharides with proteins. From a functional point of view, protein-oligosaccharide interaction is of vital importance not only to enzyme control mechanisms but to a variety of biological problems such as malignancy and cell-cell interactions, differentiation and development, and susceptibility of cells to hormones, drugs and toxins.

The status of glycolaldehyde in the biosynthesis of vitamin B6.

Competition experiments, employing 14C-labeled samples of glycerol and glycolaldehyde, indicate that in Escherichia coli B there are two independent pathways leading to pyridoxal. In mutant WG2 (and therefore presumably also in the wild strain) the major pathway utilizes glycerol and related trioses as the sole carbon source in the construction of the C8N skeleton of pyridoxol: C-1, -3 of glycerol yields C-2', -3, -4', -5' and -6, C-2 of glycerol yields C-2, -4, and -5 of the vitamin. In the minor pathway glycolaldehyde and not glycerol supplies C-5 and C-5' of pyridoxol, while glycerol is the source of the other 6 carbon atoms. In mutant WG3 the major route is blocked and the "glycolaldehyde pathway" becomes the sole source of vitamin B6.