HBsAg, HBcAg and delta-Ag in liver tissue: simultaneous visualization in a single tissue section by triple immunostaining

The distribution pattern of HBsAg, HBcAg and Delta-Ag was investigated by immunohistochemistry in a series of paraffin embedded liver tissue specimens from 45 subjects with serum HBsAg and anti-Delta antibody positivity. An indirect immunoperoxidase technique was used. Stains for HBsAg, HBcAg and Delta-Ag were alternatively carried out on serial tissue sections and, for the first time, consecutively in a single section (triple immunostaining). Simultaneous presence of all antigens occurred in 7 out of 45 cases, and of two antigens (HBsAg and Delta-Ag) in the remaining 38. Two antigens (either HBsAg and HBcAg or HBcAg and Delta-Ag) could also be shown in the same cell. A series of new observations was forwarded by the successful application of triple immunostaining in the present series: 1) high frequence of simultaneous presence of HBcAg and Delta-Ag (7 out of 45 cases = 16%); 2) cytoplasmic localization of Delta-Ag; 3) localization of HBcAg on the cell membrane of HBsAg positive Ground-Glass (G-G) hepatocytes; 4) Ground-Glass appearance of hepatocytic cytoplasm associated with exclusive content of HBcAg (HBcAg-Ground-Glass: a new variant of G-G-hepatocytes).     

Defective dendritic cell migration and activation of adaptive immunity in PI3Kgamma-deficient mice

Gene-targeted mice were used to evaluate the role of the gamma isoform of phosphoinositide 3-kinase (PI3Kgamma) in dendritic cell (DC) migration and induction of specific T-cell-mediated immune responses. DC obtained from PI3Kgamma-/- mice showed a reduced ability to respond to chemokines in vitro and ex vivo and to travel to draining lymph nodes under inflammatory conditions. PI3Kgamma-/- mice had a selective defect in the number of skin Langerhans cells and in lymph node CD8alpha- DC. Furthermore, PI3Kgamma-/- mice showed a defective capacity to mount contact hypersensitivity and delayed-type hypersensitivity reactions. This defect was directly related to the reduced ability of antigen-loaded DC to migrate from the periphery to draining lymph nodes. Thus, PI3Kgamma plays a nonredundant role in DC trafficking and in the activation of specific immunity. Therefore, PI3Kgamma may be considered a new target to control exaggerated immune reactions.     

Immunoglobulins against gp273, the ligand for sperm-egg interaction in the mollusc bivalve Unio elongatulus,are directed against charged O-linked oligosaccharide chains bearing a Lewis-like structure and interact with epitopes of the human zona pellucida

In oocytes of the mollusc bivalve Unio elongatulus, gp273 is the ligand molecule for sperm-egg interaction and binding is mediated by its O-glycans. A serum raised against this protein enabled its localization in the crater region, the area of the vitelline coat where sperm recognition occurs, and showed that after cyanogen bromide fragmentation, the anti-gp273 epitope(s) was retained by a peptide where the O-glycans are localized. In this article, we utilized purified anti-gp273 immunoglobulins to characterize the corresponding epitope by: (i) immunoblotting analysis of the protein after removal of O- and N-glycans; (ii) solid phase binding analysis of anti-gp273 IgG to gp273 N- and O-glycans; and (iii) binding analysis of the same antibody to commercially available oligosaccharides. The results showed that the epitope consists of O-glycans and contains a Lewis-like structure with fucose as determinant. Anti-gp273 IgG were then used to investigate human zona pellucida by immunoelectronmicroscopy and immunoblotting. Epitopes recognized by the antibody were demonstrated on the outer surface of the zona pellucida and shown to belong to a zona pellucida protein having electrophoretic mobility similar to human ZP3. Since human sperm specifically bind to gp273, and anti-gp273 interferes with this binding a functional role for these epitopes is suggested.     

Biochemical characterization of a CDC6-like protein from the crenarchaeon Sulfolobus solfataricus

Cdc6 proteins play an essential role in the initiation of chromosomal DNA replication in Eukarya. Genes coding for putative homologs of Cdc6 have been also identified in the genomic sequence of Archaea, but the properties of the corresponding proteins have been poorly investigated so far. Herein, we report the biochemical characterization of one of the three putative Cdc6-like factors from the hyperthermophilic crenarchaeon Sulfolobus solfataricus (SsoCdc6-1). SsoCdc6-1 was overproduced in Escherichia coli as a His-tagged protein and purified to homogeneity. Gel filtration and glycerol gradient ultracentrifugation experiments indicated that this protein behaves as a monomer in solution (molecular mass of about 45 kDa). We demonstrated that SsoCdc6-1 binds single- and double-stranded DNA molecules by electrophoretic mobility shift assays. SsoCdc6-1 undergoes autophosphorylation in vitro and possesses a weak ATPase activity, whereas the protein with a mutation in the Walker A motif (Lys-59 --> Ala) is completely unable to hydrolyze ATP and does not autophosphorylate. We found that SsoCdc6-1 strongly inhibits the ATPase and DNA helicase activity of the S. solfataricus MCM protein. These findings provide the first in vitro biochemical evidence of a functional interaction between a MCM complex and a Cdc6 factor and have important implications for the understanding of the Cdc6 biological function.     

The Topology of a Discussion: The #Occupy Case

Introduction

We analyse a large sample of the Twitter activity that developed around the social movement ''Occupy Wall Street'', to study the complex interactions between the human communication activity and the semantic content of a debate.

Methods

We use a network approach based on the analysis of the bipartite graph @Users-#Hashtags and of its projections: the ''semantic network'', whose nodes are hashtags, and the ''users interest network'', whose nodes are users. In the first instance, we find out that discussion topics (#hashtags) present a high structural heterogeneity, with a relevant role played by the semantic hubs that are responsible to guarantee the continuity of the debate. In the users’ case, the self-organisation process of users’ activity, leads to the emergence of two classes of communicators: the ''professionals'' and the ''amateurs''.

Results

Both the networks present a strong community structure, based on the differentiation of the semantic topics, and a high level of structural robustness when certain sets of topics are censored and/or accounts are removed.

Conclusions

By analysing the characteristics of the dynamical networks we can distinguish three phases of the discussion about the movement. Each phase corresponds to a specific moment of the movement: from declaration of intent, organisation and development and the final phase of political reactions. Each phase is characterised by the presence of prototypical #hashtags in the discussion.