Structural differences between the glucocorticoid, dioxin and oxysterol receptors from rat liver cytosol

The rat hepatic glucocorticoid, dioxin and oxysterol receptors were subjected to high performance liquid chromatography on size-exclusion and anion-exchange columns. Both the glucocorticoid receptor and the dioxin receptor had a Stokes radius Rs approximately 7.5 nm, expected value for heteromeric complexes containing a dimer of the Mr approximately 90,000 heat shock protein, hsp90 (Rs approximately 7.0 nm). The oxysterol receptor represented a much smaller entity (Rs approximately 6.0 nm). When analyzed on a Mono Q anion-exchange column, the molybdate-stabilized glucocorticoid receptor and dioxin receptor eluted as single peaks at approximately 0.30 M and 0.26-0.28 M NaCl, respectively, whereas the oxysterol receptor represented a less negatively charged species (0.11-0.14 M NaCl). Following washing of the Mono Q column with molybdate-free buffer, the activated monomeric glucocorticoid receptor was detected (0.10-0.12 M NaCl). In contrast, no modification in the elution pattern of the dioxin receptor and the oxysterol receptor was observed. These data demonstrate differences in the physico-chemical properties of the glucocorticoid, dioxin and oxysterol receptors, respectively, which might reflect structural differences.     

Suppression of protein kinase C and the stimulation of glucocorticoid receptor synthesis by dexamethasone in human fibroblasts derived from tumor tissue

Exposure of fibroblasts derived from keloid tissues, desmoid and dermal tissue from individuals with Gardner's syndrome (GS) to dexamethasone resulted in the suppression of protein kinase C (PKC) activity and [3H]thymidine incorporation into DNA, and a 20-fold induction of glutamine synthetase activity. Treatment of GS and keloid fibroblasts with 0.1 microM dexamethasone for 36 h increased glucocorticoid receptor (GR) synthesis, as determined by [35S]methionine labeling and immunoprecipitation with a monoclonal antibody to the human GR. The suppression of PKC activity by dexamethasone was shown to result from a loss of protein mass as determined by immunoblotting using an antibody to PKC type III. In contrast to these results, exposure of fibroblasts isolated from normal tissues to dexamethasone did not result in the suppression PKC and [3H]thymidine incorporation, there was only a sixfold induction of glutamine synthetase, and a decrease of GR synthesis. As no primary receptor binding defect could be detected, the altered response of tumor cells to steroid-occupied receptor indicates a partial post-receptor binding defect in GS and keloid cells.     

Neonatal screening for congenital adrenal hyperplasia using 17-hydroxyprogesterone assay in filter paper blood spots

Screening of infants for congenital adrenal hyperplasia (CAH) using filter paper blood samples collected on the 5th day of life was performed with a radioimmunoassay for 17-hydroxyprogesterone without extraction with organic solvents. A total of 153,000 newborns were screened and 12 cases of CAH were detected (1:12,800). With recall levels related to gestational age, the recall rate could be lowered to 0.05%.     

Suppression of growth defects of α-amylase secreting Escherichia coli by signal sequence fusion

Abstract In a previous study, we have described unusual cross-reactions among monoclonal antibodies (Mabs) to bacteria and in particular to the Inaba and Ogawa serotypes of Vibrio cholerae . In this study, the extent to which the binding sites of both antibodies and antigens overlap has been investigated by competitive binding and idiotypic analysis. The competitive binding data indicate that the cross-reactive binding of the Inaba Mabs to the Ogawa vibrios can be abolished by incubation with higher affinity Ogawa Mabs. However, rabbit antiserum raised against the Inaba series does not react with the Ogawa series, indicating that anti-Inaba Mabs do not share idiotypic determinants with anti-Ogawa Mabs. The results therefore suggest that the two sets of antibodies recognise different determinants which are closely related in spatial terms, and which consequently do not permit simultaneous binding of the two types of monoclonal antibody.     

Class II genes of miniature swine

Class II genes of miniature swine have been characterized by restriction fragment length polymorphism (RFLP) analysis and by analysis of a series of clones isolated from a lymphocyte genomic library. For RFLP analysis, DNA samples from three independent major histocompatibility complex homozygous lines and three intra-MHC recombinant lines were digested with a variety of restriction enzymes and analyzed in Southern blots using human cDNA probes for DP, DQ, DR, and DZ alpha genes, and DP, DQ, DR, and DO beta genes. One, or at most two, unique fragments were detected by hybridization with each of the human probes tested. In contrast, multiple bands (five to six for most enzymes examined) were detected by each of the human probes tested, the majority of which were found to cross-react with at least three of these probes under conditions of moderate stringency. Genomic DNA from the SLA ^c haplotype was cloned into an EMBL-3 bacteriophage vector, and the corresponding genomic library was screened with each of these human cDNA probes. The class II genes thereby isolated from this library showed characteristics consistent with those anticipated from the RFLP analysis. Thus, unique genes were obtained which showed no evidence of cross-hybridization, while genes showed extensive cross-hybridization and were frequently detected in the library by more than one human gene probe. These data are consistent with early evolutionary divergence of a genes, prior to mammalian speciation, and with continuing evolution of genes, with possible shared usage of these genes by different a loci. The data also imply that genes can readily be assigned to loci homologous to their human counterparts, but that genes will require further mapping and/or sequence analysis to confirm assignments.     

Lateral diffusion of the secretory component (SC) in the basolateral membrane of the human colon carcinoma cell line HT29 assessed with fluorescence recovery after photobleaching

The lateral diffusion of the secretory component (SC), acting as a receptor for dimeric IgA in the basolateral side of intestinal epithelial cells, was studied in the human colonic carcinoma cell line HT29. The HT29 cells were grown in Dulbecco's modified Eagle's medium in which galactose had been substituted for glucose to promote development of small intestine-like cells, with a distinct separation of the basolateral side from the apical surface. The SC was stained with rhodamine-labeled polyclonal anti-human SC rabbit antibodies (Ig) or Fab fragments, and the lateral mobility was assessed with the fluorescence recovery after photobleaching technique. The average lateral diffusion was consistent with a diffusion constant of 7.7 +/- 2.0 (mean value +/- SD; n = 29) and 7.1 +/- 2.3 (n = 30) x 10(-10) cm2s-1 for Ig-and Fab-labeled receptors, respectively, which is slower than lipid diffusion but is similar to that found for other membrane receptors. The corresponding values for the fraction of mobile receptors were 66 +/- 13% and 71 +/- 12%, respectively. Cells were labeled from the top of the culture plate, and cells adjacent to a mechanically made rift or a natural opening in the cell monolayer were labeled more strongly, confirming the microscope-based impression that the basolateral surface primarily harboured the SC receptor.     

Mutations in a herpes simplex virus type 1 origin that inhibit interaction with origin-binding protein also inhibit DNA replication.

The herpes simplex virus type 1 genome contains three origins of replication: OriL and a diploid OriS. The origin-binding protein, the product of the UL9 gene, interacts with two sites within OriS, box I and box II. A third site, box III, which is homologous to boxes I and II, may also be a binding site for the origin-binding protein. Mutations in these three sites significantly reduce OriS-directed plasmid replication measured in transient replication assays. The reduction in replication efficiency of the mutants correlates well with the decrease in the ability to bind to the origin-binding protein, as determined by Elias et al. (P. Elias, C. M. Gustafsson, and O. Hammarsten, J. Biol. Chem. 265: 17167-17173, 1990). The effect of multiple mutations in boxes I, II, and III on plasmid replication suggests that there are multiple binding sites in OriS for the origin-binding protein. These studies indicate that proper interaction of the origin-binding protein with the OriS sequence is essential for OriS-directed DNA replication.