Deviations in thymocyte development induced by divalent and monovalent ligation of the alpha/beta T cell receptor and by its cross-linking to CD8

Fetal thymic organ cultures (FTOC) were tested as a model system to induce, in a polyclonal fashion, negative and positive thymic selection events. By flow cytometry, thymocytes developed in FTOC differed in several parameters from their in vivo differentiated counterparts. In particular, no clear distinction was possible between CD4+CD8+ immature cells with low TCR expression and mature CD4+ or CD8+ cells with high TCR expression. Thymocyte development in FTOC was manipulated with three different antibody reagents: anti-V beta 8 (F23.1), anti-Lyt-2.2 (19/178) and the quadroma derived bifunctional antibody HPHT-2, carrying one binding site of each. This antibody served also as a monovalent anti-V beta 8 reagent in FTOC from Lyt-2.1 mouse strains. Antibody 19/178 suppressed the development of single positive CD8+ cells, but only at very high concentrations. F23.1 and HPHT-2 suppressed the development of CD4+V beta 8+ and CD8+V beta 8+ thymocytes at relatively low concentrations giving rise to V beta 8 occupancies from about 2% upwards. Suppression was equally pronounced in cells with low and high TCR densities. Moreover, V beta 8 suppression occurred upon divalent and monovalent V beta 8 binding and was not significantly influenced by V beta 8-CD8 cross-linking. This suggests that ligation of the TCR alone is sufficient for clonal deletion. The data do not exclude a role for CD8 as an accessory adhesion molecule but suggest that exogenous cross-linking of CD8 to the TCR is not essential in transmembrane signaling for clonal deletion. At lower antibody concentrations giving rise to V beta 8 occupancies below detection, V beta 8-CD8 cross-linking by HPHT-2, but no divalent and monovalent V beta 8 ligation, induced an increase of CD8+V beta 8+ cells at the expense of CD4+ V beta 8+ cells with no change in the proportion of total V beta 8+ thymocytes. The latter effect was quantitatively of borderline significance but reproducible. These latter results are compatible with the hypothesis that cross-linking of the alpha beta TCR and CD8 on the thymocyte surface provides a maturation signal resulting in loss of CD4 from CD4+ CD8+ double positive immature thymocytes.     

Cytochemical properties of osteoblast cell membrane domains

The interactions of osteoblasts with one another and with the extracellular milieu are of vital importance for cell function. These interactions are mediated by cell membrane-associated components. In the present work, we studied the distribution of several mediators known to be associated with the cell surface, using ultrastructural cytochemistry, to characterize the three cell membrane domains (osteoid, lateral, and vascular) of osteoblasts. Osteoblasts in neonatal rat calvariae were studied for cell surface distribution of alkaline phosphatase (APase), calcium-activated adenosine triphosphatase (Ca2+-ATPase), calcium, soybean agglutinin (SBA)-reactive sites, and peanut agglutinin (PNA)-reactive sites. APase was absent in the osteoid domain but was evenly distributed in the other domains. Ca2+-ATPase was found to be concentrated mainly in the lateral domains. In contrast, calcium was present in all cell membrane domains. Using lectins conjugated to horseradish peroxidase, we demonstrated that SBA binding sites were evenly distributed along the osteoblast cell membrane, whereas PNA binding sites were absent or minimally present in the osteoid and lateral domains but were evenly distributed in the vascular domain. These results suggest that the various functions of osteoblasts may be facilitated by specialized cell membrane domains which are cytochemically distinct. Previous reports have failed to demonstrate the cytochemical differences between the three domains of the osteoblast cell membrane.     

Nonsense mutations of the von Willebrand factor gene in patients with von Willebrand disease type III and type I.

von Willebrand disease (vWD) is the most common inherited bleeding disorder in humans. The disease is caused by qualitative and quantitative abnormalities of the von Willebrand factor (vWF). Genomic DNA from 25 patients with vWD type III, the most severe form of the disease, was studied using PCR followed by restriction-enzyme analysis and direct sequencing of the products. Nonsense mutations (CGA----TGA) were detected in exons 28, 32, and 45 by screening of all the 11 CGA arginine codons of the vWF gene. Two patients were found to be homozygous and five heterozygous for the mutation. Both parents and some of the relatives of the homozygous patients carry the mutation. These are the first reported examples of homozygous point mutations associated with the severe form of vWD. In the three heterozygous probands, one of the parents carried the mutation and had vWD type I. Family studies including parents and family members with or without vWD type I indicated that these three heterozygous patients are likely to be compound heterozygous. Twenty-one individuals from these seven families with vWD type I were found to be heterozygous for the mutation.     

Linker insertion analysis of the FimH adhesin of type 1 fimbriae in an Escherichia coli fimH-null background

Abstract The gene encoding the Escherichia coli FimH adhesin of type 1 fimbriae has been subjected to linker insertion mutagenesis. Amino acid changes were introduced at a number of positions spanning the entire sequence in order to probe the structure-function relationship of the FimH protein. The effect of these mutations on the ability of bacteria to express a D-mannose binding phenotype was assessed in a fimH null mutant (MS4) constructed by allelic exchange in the E. coli K-12 strain PC31. Mutations mapping at amino acid residues 36, 58 and 279 of the mature FimH protein were shown to completely abolish binding to D-mannose receptors. Differences in the level of fimbriation were also observed as a result of some of the mutations in the fimH gene. These mutants may prove useful in dissecting receptor-ligand interactions by defining regions of the FimH protein that are important in erythrocyte binding.     

Genomic organization of a mouse MHC class II region including theH2-M andLmp2 loci

The region encompassing theMa, Mb1, Mb2, andLmp2 genes of the mouse class II major histocompatibility complex (MHC) was sequenced. Since this region contains clusters of genes required for efficient class I and class II antigen presentation, it was interesting to search for putative additional genes in the 21 kilobase gap between theMb1 andLmp2 genes. Computer predictions of coding regions and CpG islands, exon trapping experiments, and cross-species comparison with the corresponding human sequence indicate that no additional functional gene is present in that stretch. However, computer analysis revealed the possible existence of an alternative 3 exon forMb1. Except for the fact that the mouse MHC contains twoMb genes, the genomic organization of theH2-M loci was found to be almost identical to the organization of the humanHLA-DM genes. The promoter regions of theMa andMb genes also resemble classical class II promoters, containing typical S, X, and Y boxes. Like the human genes, the threeH2-M genes displayed very limited polymorphism when we compared the cDNA sequences from six haplotypes. Finally, comparison ofDMB withMb1 andMb2, both at the genomic level and in their coding regions, suggests that theMb gene was recently duplicated, probably only in certain rodents.     

Power and work produced in different leg muscle groups when rising from a chair

The aim of this study was to determine the power output and work done by different muscle groups at the hip and knee joints during a rising movement, to be able to tell the degree of activation of the muscle groups and the relationship between concentric and eccentric work. Nine healthy male subjects rose from a chair with the seat at knee level. The moments of force about the hip and knee joints were calculated semidynamically. The power output (P) and work in the different muscle groups surrounding the joints was calculated as moment of force times joint angular velocity. Work was calculated as: work = f Pdt. The mean peak concentric power output was for the hip extensors 49.9 W, hip flexors 7.9 W and knee extensor 89.5 W. This power output corresponded to a net concentric work of 20.7 J, 1.0 J and 55.6 J, respectively. There was no concentric power output from the knee flexor muscles. Energy absorption through eccentric muscle action was produced by the hip extensors and hip flexors with a mean peak power output of 4.8 W and 7.4 W, respectively. It was concluded that during rising, the hip and knee muscles mainly worked concentrically and that the greatest power output and work were produced during concentric contraction of the knee and hip extensor muscles. There was however also a demand for eccentric work by the hip extensors as well as both concentric and eccentric work by the hip flexors. The knee flexor muscles were unloaded.     

Linkage disequilibrium between the FES, D15S127, and BLM loci in Ashkenazi Jews with Bloom syndrome.

Bloom syndrome (BS) is more common in the Ashkenazi Jewish than in any other population. Approximately 1 in 110 Ashkenazi Jews carries blm, the BS mutation. The locus mutated in BS, BLM, maps to chromosome subband 15q26.1, tightly linked to the proto-oncogene FES. We have investigated the basis for the increased frequency of blm in the Ashkenazim by genotyping polymorphic microsatellite loci tightly linked to BLM in affected and unaffected individuals from Ashkenazi Jewish and non-Ashkenazi populations. A striking association of the C3 allele at FES with blm (delta = .422; p = 5.52 x 10(-7)) and of the 145-bp and 147-bp alleles at D15S127 with blm (delta = .392 and delta = .483, respectively; p = 2.8 x 10(-5) and p = 5.4 x 10(-7), respectively) was detected in Ashkenazi Jews with BS. This linkage disequilibrium constitutes strong support for a founder-effect hypothesis: the chromosome in the hypothetical founder who carried blm also carried the C3 allele at FES and either the 145-bp or the 147-bp allele at D15S127.     

Essential role of phosphatidylinositol 3-kinase in insulin-induced activation and phosphorylation of the cGMP-inhibited cAMP phosphodiesterase in rat adipocytes studies using the selective inhibitor wortmannin

Incubation of rat adipocytes with wortmannin, a potent and selective phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor, completely blocked the antilipolytic action of insulin (IC₅₀≈ 100 nM), the insulin-induced activation and phosphorylation of cGMP-inhibited cAMP phosphodiesterase (cGI-PDE) as well as the activation of the insulin-stimulated cGI-PDE kinase (IC₅₀≈ 10–30 nM). No direct effects of the inhibitor on the insulin-stimulated cGI-PDE kinase, the cGI-PDE and the hormone-sensitive lipase were observed. These data suggest that activation of PI 3-kinase upstream of the insulin-stimulated cGI-PDE kinase in the antilipolytic insulin signalchain has an essential role for insulin-induced cGI-PDE activation/ phosphorylation and anti-lipolysis.