The extracellular architecture of adherens junctions revealed by crystal structures of type I cadherins

Adherens junctions, which play a central role in intercellular adhesion, comprise clusters of type I classical cadherins that bind via extracellular domains extended from opposing cell surfaces. We show that a molecular layer seen in crystal structures of E- and N-cadherin ectodomains reported here and in a previous C-cadherin structure corresponds to the extracellular architecture of adherens junctions. In all three ectodomain crystals, cadherins dimerize through a trans adhesive interface and are connected by a second, cis, interface. Assemblies formed by E-cadherin ectodomains coated on liposomes also appear to adopt this structure. Fluorescent imaging of junctions formed from wild-type and mutant E-cadherins in cultured cells confirm conclusions derived from structural evidence. Mutations that interfere with the trans interface ablate adhesion, whereas cis interface mutations disrupt stable junction formation. Our observations are consistent with a model for junction assembly involving strong trans and weak cis interactions localized in the ectodomain.     

Individual and synergistic cytokine effects controlling the expansion of cord blood CD34(+) cells and megakaryocyte progenitors in culture

Background aimsExpansion of hematopoietic progenitors ex vivo is currently investigated as a means of reducing cytopenia following stem cell transplantation. The principal objective of this study was to develop a new cytokine cocktail that would maximize the expansion of megakaryocyte (Mk) progenitors that could be used to reduce periods of thrombocytopenia.MethodsWe measured the individual and synergistic effects of six cytokines [stem cell factor (SCF), FLT-3 ligand (FL), interleukin (IL)-3, IL-6, IL-9 and IL-11] commonly used to expand cord blood (CB) CD34⁺ cells on the expansion of CB Mk progenitors and major myeloid populations by factorial design.ResultsThese results revealed an elaborate array of cytokine individual effects complemented by a large number of synergistic and antagonistic interaction effects. Notably, strong interactions with SCF were observed with most cytokines and its concentration level was the most influential factor for the expansion and differentiation kinetics of CB CD34⁺ cells. A response surface methodology was then applied to optimize the concentrations of the selected cytokines. The newly developed cocktail composed of SCF, thrombopoietin (TPO) and FL increased the expansion of Mk progenitors and maintained efficient expansion of clonogenic progenitors and CD34⁺ cells. CB cells expanded with the new cocktail were shown to provide good short- and long-term human platelet recovery and lymphomyeloid reconstitution in NOD/SCID mice.ConclusionsCollectively, these results define a complex cytokine network that regulates the growth and differentiation of immature and committed hematopoietic cells in culture, and confirm that cytokine interactions have major influences on the fate of hematopoietic cells.     

The E3 ubiquitin-ligase HACE1 catalyzes the ubiquitylation of active Rac1

Rac1 small GTPase controls essential aspects of cell biology and is a direct target of numerous bacterial virulence factors. The CNF1 toxin of pathogenic Escherichia coli addresses Rac1 to ubiquitin-proteasome system (UPS). We report the essential role of the tumor suppressor HACE1, a HECT-domain containing E3 ubiquitin-ligase, in the targeting of Rac1 to UPS. HACE1 binds preferentially GTP-bound Rac1 and catalyzes its polyubiquitylation. HACE1 expression increases the ubiquitylation of Rac1, when the GTPase is activated by point mutations or by the GEF-domain of Dbl. RNAi-mediated depletion of HACE1 blocks the ubiquitylation of active Rac1 and increases GTP-bound Rac1 cellular levels. HACE1 antagonizes cell isotropic spreading, a hallmark of Rac1 activation, and is required for endothelial cell monolayer invasion by bacteria. Together, these data establish the role of the HACE1 E3 ubiquitin-ligase in controlling Rac1 ubiquitylation and activity.     

All hematopoietic cells develop from hematopoietic stem cells through Flk2/Flt3-positive progenitor cells

While it is clear that a single hematopoietic stem cell?(HSC) is capable of giving rise to all other hematopoietic cell types, the differentiation paths beyond HSC remain controversial. Contradictory reports on?the lineage potential of progenitor populations have questioned their physiological contribution of progenitor populations to multilineage differentiation. Here, we established a lineage tracing mouse model that enabled direct assessment of differentiation pathways in?vivo. We provide definitive evidence that differentiation into all hematopoietic lineages, including megakaryocyte/erythroid cell types, involves Flk2-expressing non-self-renewing progenitors. A Flk2+ stage was used during steady-state hematopoiesis, after irradiation-induced stress and upon HSC transplantation. In contrast, HSC origin and maintenance do not include a Flk2+ stage. These data demonstrate that HSC specification and maintenance are Flk2 independent, and that hematopoietic lineage separation occurs downstream of Flk2 upregulation.     

New insights into one of nature's remarkable catalysts, the ATP synthase.

In the August 10, 2001 issue of Cell, Menz et al. report the crystal structure of a novel inhibited form of the bovine mitochondrial F(1)-ATPase at 2 A resolution, with all three catalytic sites occupied by nucleotide, one of which binds ADP in preference to ATP.     

The genus Cyphophthalmus (Arachnida, Opiliones, Cyphophthalmi) in Europe: a phylogenetic approach to Balkan Peninsula biogeography

In this study, we present phylogenetic data to characterize the relationships among sironids centered in the Balkan region, and use these results to discuss biogeographical aspects of sironid evolution. Analysis of ca. 4.5 kb of sequence data from three nuclear and two mitochondrial genes reveals monophyly of a Balkan clade for which we resurrect the name Cyphophthalmus, considered a junior synonym of Siro for over a century. This clade diversified into several groups, and at least three of them--the duricorius group, the serbicus group, and the minutus group--are well corroborated by the data as monophyletic lineages. The members of the different groups, mostly living in troglobitic environments, have diversified in overlapping geographic regions, with evidence of an eastern origin for the group. Our data also suggest that mitochondrial and nuclear genes are all contributing towards the final resolution of the combined analysis of the data.     

Synthesis of modified proanthocyanidins: introduction of acyl substituents at C-8 of catechin. Selective synthesis of a C-4-->O-->C-3 ether-linked procyanidin-like dimer

The regioselective introduction of substituents at C-8 of (+)-catechin is described, leading to the synthesis of several catechin derivatives with various substitution patterns to be used for the further synthesis of modified proanthocyanidins. Thereafter, a new 3-O-4 ether-linked procyanidin-like derivative was synthesized. Its formation was selectively achieved through TiCl(4)-catalyzed condensation of 4-(2-hydroxyethoxy)tetra-O-benzyl catechin with the 8-trifluoroacetyl adduct of tetra-O-benzyl catechin.     

Synthesis of modified proanthocyanidins: easy and general introduction of a hydroxy group at C-6 of catechin; efficient synthesis of elephantorrhizol

A general procedure for the oxidation of catechin derivatives is described, leading to the introduction of a new hydroxy group at C-6. This procedure has been applied for the synthesis of elephantorrhizol, a natural flavan-3-ol exhibiting a fully substituted cycle A.     

Modeling the effect of an associated single-nucleotide polymorphism in linkage studies

For linkage analysis in affected sibling pairs, we propose a regression model to incorporate information from a disease-associated single-nucleotide polymorphism located under the linkage peak. This model can be used to study if the associated single-nucleotide polymorphism marker partly explains the original linkage peak. Two sources of information are used for performing this task, namely the genotypes of the parents and the genotypes of the siblings. We applied the methods to three significantly disease-associated single-nucleotide polymorphisms and five microsatellite markers at the end of chromosome 3 of replicate 1 of Aipotu population. Two out of five of the microsatellite markers showed a LOD score higher than 3. The question to be answered was whether one of the single-nucleotide polymorphisms partly explains these high LOD scores. We did not have the answers when we analyzed the data.