In vitro nuclear transport of ribosomal ribonucleoprotein: temperature affects quantity but not quality of exported particles.

The in vitro export of ribosomal ribonucleoprotein (rRNP) from Tetrahymena nuclei was investigated at the optimal growth temperature of 28 degrees C and at the nonlethal temperature of 8 degrees C. At both temperatures, nuclei exported ribosomal precursor particles that revealed the same physical qualities of size, appearance in negative-staining electron microscopy, sedimentation coefficient, buoyant density, and rRNA pattern. Surprisingly, fewer rRNP particles were exported at 8 than at 28 degrees C, as was revealed by a lower saturation plateau in the export kinetics from nuclei prelabeled with [3H]uridine. Upon a temperature increase from 8 to 28 degrees C, additional rRNP particles were exported. We conclude that nuclei export only a defined portion of rRNP particles at a given temperature, although enough potentially transportable rRNP particles are present in nuclei. Obviously, the reactivity of at least one of the reactants involved directly or indirectly in rRNP export changes with temperature.     

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.     

Growth and vitality of epiphytic lichens

 We tested the hypothesis that changed microclimate at induced forest edges causes reduced growth of epiphytic lichens. Two foliose, green algal lichens were transplanted to the lower canopy of a mature Picea abies forest at six distances (2, 6.25, 12.5, 25, 50 and 100 m) from a clearcut. The biomass growth in Platismatia glauca (6.2% in 16 months) was 41% higher than in Lobaria pulmonaria (4.4%). We found no growth reduction near the forest edge. In contrast, the highest growth in both species occurred within 12 m from the edge. Further, fluorescence and chlorophyll measurements showed that lichen vitality was unaffected by distance from edge. The light intensity was 4.3 times higher at the edge than in the interior during the growing season, but there were only minor differences in air temperature and relative humidity. Monitoring of thallus water content revealed clear differences in both number and length of wetting and drying cycles. However, the total time with water content sufficient for photosynthetic activity was only slightly higher at the edge. The data thus indicate that our gradient in microclimate was too small to significantly affect lichen growth, and that lichens are largely metabolically inactive when large edge-interior contrasts in microclimate occur. Lichen response to forest edge microclimate results from intricate interactions among several biotic and abiotic factors. Linking data on lichen growth, microclimate and thallus water content with physiological measurements provides a framework for future studies of the mechanisms behind abiotic edge effects. Received: 15 April 1996 / Accepted: 21 June 1996     

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.     

Pool sequencing of natural HLA-DR,DQ, and DP ligands reveals detailed peptide motifs,constraints of processing,and general rules

We have approached the problem of MHC class II ligand motifs by pool sequencing natural peptides eluted from HLA-DR, DQ, and DP molecules. The results indicate surprisingly clear patterns, although not quite as clear as with natural class I ligands. The most striking feature is a highly dominant Proline at position 2. We interpret this to be a consequence of aminopeptidase N-like activity in processing. Another general aspect is the existence of three to four hydrophobic or aromatic anchors, whereby the first and the last are separated by five to eight residues. The peptide motifs for HLA-DR1, DR5, DQ7, and DPw4 are allele-specific and differ by spacing and occupancy of anchors. The anchors tend to be flanked by clusters of charged residues, and small residues, especially Ala, are frequent in the motif centers. These detailed motifs allow one to interpret most previous (DR-) motifs as fitting one or more of the anchors or conserved clusters. The relative motif symmetry suggests the possibility of bidirectional binding of peptides in the class II groove.