Exofacial photolabelling of the human erythrocyte glucose transporter with an azitrifluoroethylbenzoyl-substituted bismannose.

The synthesis of 2-N-[4-(1'-azitrifluoroethyl)benzoyl]-1,3-bis-(D-mannos-4-++ +yloxy)-2- propylamine (ATB-BMPA) is described. This compound was used as an exofacial probe for the human erythrocyte glucose-transport system. A new method is described for directly estimating the affinity for exofacial ligands which bind to the erythrocyte glucose transporter. By using this equilibrium-binding method, the Ki for ATB-BMPA was found to be 338 +/- 37 microM at 0 degrees C and 368 +/- 59 microM at 20 degrees C. This was similar to the concentration of ATB-BMPA required to half-maximally inhibit D-galactose uptake (Ki = 297 +/- 53 microM). The new photoaffinity reagent labelled the glucose transporter in intact cells but, because of its improved selectivity, was also used to label the glucose transporter in isolated erythrocyte membranes. The ATB-BMPA-labelled glucose transporter was 80% immunoprecipitated by anti-(GLUT1-C-terminal peptide) antibody, which shows that the GLUT1 glucose transporter is the major isoform present in erythrocytes. The labelling of the glucose transporter at its exofacial site, and the adoption of an outward-facing conformation, renders the transport system resistant to thermolysin and trypsin treatment. Trypsin treatment of the unlabelled glucose transporter in erythrocyte membranes produced an 18 kDa fragment which was subsequently labelled by ATB-BMPA, but had low affinity for this exofacial ligand. This suggests that the trypsin-treated transporter adopts an inward-facing conformation. The ability of D-glucose to displace ATB-BMPA from the native transporter and from the 18 kDa trypsin fragment have been compared. The D-glucose concentration which was required to obtain half-maximal inhibition of ATB-BMPA labelling was 6-fold lower for the 18 kDa tryptic fragment.     

Crystal and solution structures of the oligonucleotide d(ATGCGCAT)2: a combined X-ray and NMR study.

A combined crystal-structure determination and NMR analysis of the octanucleotide d(ATGCGCAT)2 is reported. The X-ray analysis shows that the structure is A-form duplex in crystal state. The NMR study shows that in solution this sequence is B-type. The conformational results from each technique are presented in detail. The implications of these findings in terms of conformational flexibility and ligand binding are discussed.     

Identification of functioning sweat pores and visualization of skin temperature patterns in X-linked hypohidrotic ectodermal dysplasia by whole body thermography

Summary In this preliminary study, non-invasive infrared thermography has been used to visualize individual sweat pores and whole body skin temperature patterns in subjects with X-linked hypohidrotic ectodermal dysplasia (XHED) and normal controls. The findings in eight obligate heterozygotes and four affected males were compared to six normal female controls and to six non-manifesting females at risk for carrier status. Sweat secretion from individual pores in circumscribed areas was imaged using a high spatial resolution SPRITE infrared detector system working in the 8–14 m band. In seven out of eight obligate heterozygotes, skin areas devoid of active sweat glands were found on the face, the hands or the trunk. Tear front movement over the cornea was also visualized and abnormal patterns were identified in obligate heterozygotes. Whole body skin temperature patterns, obtained with an Agema 780 Medical Thermovision system, identified abnormal skin temperature distributions, including characteristic aberrant cas-cade back patterns, in obligate carriers. Two out of six at risk females had skin temperature patterns comparable with obligate heterozygotes and we have tentatively concluded that they are carriers. Thermal imaging may be used for the examination of at risk non-manifesting females in families with a single affected male. The results of this study suggest that the random X-inactivation in females with XHED, as well as producing relatively large skin areas with sweat pore aplasia, is also associated with abnormal temperature patterns that are consistent with altered peripheral vascular perfusion.     

Production of somatic and germline chimeras in the chicken by transfer of early blastodermal cells

Cells were isolated from stage X embryos of a line of Barred Plymouth Rock chickens (that have black pigment in their feathers due to the recessive allele at the I locus) and injected into the subgerminal cavity of embryos from an inbred line of Dwarf White Leghorns (that have white feathers due to the dominant allele at the I locus). Of 53 Dwarf White Leghorn embryos that were injected with Barred Plymouth Rock blastodermal cells, 6 (11.3%) were phenotypically chimeric with respect to feather colour and one (a male) survived to hatching. The distribution of black feathers in the recipients was variable and not limited to a particular region although, in all but one case, the donor cell lineage was evident in the head. The male somatic chimera was mated to several Barred Plymouth Rock hens to determine the extent to which donor cells had been incorporated into his testes. Of 719 chicks hatched from these matings, 2 were phenotypically Barred Plymouth Rocks demonstrating that cells capable of incorporation into the germline had been transferred. Fingerprints of the blood and sperm DNA from the germline chimera indicated that both of these tissues were different from those of the inbred line of Dwarf White Leghorns. Bands that were present in fingerprints of blood DNA from the chimera and not present in those of the Dwarf White Leghorns were observed in those of the Barred Plymouth Rocks.(ABSTRACT TRUNCATED AT 250 WORDS)     

Electron microscopic identification of the yeast spliceosome.

We have partially purified the yeast spliceosome by differential sedimentation in glycerol gradients. By electron microscopy we have identified a particle in these fractions that is the spliceosome. In 100 mM KCl buffer, the yeast spliceosome is an ovoid disc with the dimensions of 20 x 23.5 nm with a central indentation. To verify that these ovoid particles were spliceosomes, specific labels were used to tag them. These tagged spliceosomes were then identified in the electron microscope. The salt dependent shift of sedimentation rate for the spliceosome can be explained by a change in size of the particle.     

Identification of the glucose transporter in mammalian cell membranes with a 125I-forskolin photoaffinity label.

The glucose transporter has been identified in a variety of mammalian cell membranes using a photoactivatable carrier-free radioiodinated derivative of forskolin, 3-[125I]iodo-4-azidophenethylamido-7-O-succinyldeacetylforskoli n ([125I]IAPS-forskolin) at 1-3 nM. The membranes that were photolabelled with [125I]IAPS-forskolin were human placental membranes, rat cortical and cerebellar synaptic membranes, rat cardiac sarcolemmal membranes, rat adipocyte plasma membranes, smooth-muscle membranes, and S49 wild-type (WT) lymphoma-cell membranes. The glucose transporter in plasma membranes prepared from the insulin-responsive rat cardiac sarcolemmal cells, rat adipocytes and smooth-muscle cells were determined to be approx. 45 kDa by SDS/polyacrylamide-gel electrophoresis (PAGE). Photolysis of human placental membranes, rat cortical and cerebellar synaptic membranes, and WT lymphoma membranes with [125I]-IAPS-forskolin, followed by SDS/PAGE, indicated specific derivatization of a broad band (43-55 kDa) in placental membranes and a narrower band (approx. 45 kDa) in synaptic membranes and WT lymphoma membranes. Digestion of the [125I]IAPS-forskolin-labelled placental and WT lymphoma membranes with endo-beta-galactosidase showed a reduction in the apparent molecular mass of the radiolabelled band to approx. 40 kDa. The membranes that were photolabelled with [125I]IAPS-forskolin and trypsin-treated produced a radiolabelled proteolytic fragment with an apparent molecular mass of 18 kDa. [125I]IAPS-forskolin is a highly effective probe for identifying low levels of glucose transporters in mammalian tissues.     

RNA11 protein is associated with the yeast spliceosome and is localized in the periphery of the cell nucleus.

The yeast rna mutations (rna2 through rna10/11) are a set of temperature-sensitive mutations that result in the accumulation of pre-mRNAs at the nonpermissive temperature. Most of the yeast RNA gene products are involved in and essential for mRNA splicing in vitro, suggesting that they code for components of the splicing machinery. We tested this proposal by using an in vitro-synthesized RNA11 protein to complement the temperature-sensitive defect of the rna11 extract. During the in vitro complementation, the input RNA11 protein was associated with the 40S spliceosome and a 30S complex, suggesting that the RNA11 protein is indeed a component of the spliceosome. The formation of the RNA11-associated 30S complex did not require any exogenous RNA substrate, suggesting that this 30S particle is likely to be a preassembled complex involved in splicing. The RNA11-specific antibody inhibited the mRNA splicing in vitro, confirming the essential role of the RNA11 protein in mRNA splicing. Finally, using the anti-RNA11 antibody, we localized the RNA11 protein to the periphery of the yeast nucleus.