Membrane fusion tropism and heterotypic functional activities of the Nipah virus and Hendra virus envelope glycoproteins

Nipah virus (NiV) and Hendra virus (HeV) are novel paramyxoviruses from pigs and horses, respectively, that are responsible for fatal zoonotic infections of humans. The unique genetic and biological characteristics of these emerging agents has led to their classification as the prototypic members of a new genus within the Paramyxovirinae subfamily called HENIPAVIRUS: These viruses are most closely related to members of the genus Morbillivirus and infect cells through a pH-independent membrane fusion event mediated by the actions of their attachment (G) and fusion (F) glycoproteins. Understanding their cell biological features and exploring the functional characteristics of the NiV and HeV glycoproteins will help define important properties of these emerging viruses and may provide new insights into paramyxovirus membrane fusion mechanisms. Using a recombinant vaccinia virus system and a quantitative assay for fusion, we demonstrate NiV glycoprotein function and the same pattern of cellular tropism recently reported for HeV-mediated fusion, suggesting that NiV likely uses the same cellular receptor for infection. Fusion specificity was verified by inhibition with a specific antiserum or peptides derived from the alpha-helical heptads of NiV or HeV F. Like that of HeV, NiV-mediated fusion also requires both F and G. Finally, interactions between the glycoproteins of the paramyxoviruses have not been well defined, but here we show that the NiV and HeV glycoproteins are capable of highly efficient heterotypic functional activity with each other. However, no heterotypic activity was observed with envelope glycoproteins of the morbilliviruses Measles virus and Canine distemper virus.     

S100B proteins that lack one or both cysteine residues can induce inflammatory responses in astrocytes and microglia

The astrocytic protein S100B stimulates neurite outgrowth and neuronal survival during CNS development. S100B can also stimulate glial activation, leading to induction of pro-inflammatory molecules like interleukin-1 beta (IL-1 beta) and inducible nitric oxide synthase (iNOS). Although it is known that S100B's neurotrophic activity requires a disulfide-linked dimeric form of the protein, the structural features of S100B that are important for glial activation have not been defined. As an initial step towards understanding the structural features of S100B required for its action on glia and to determine if these features are different from those required for its action on neurons, we tested two mutants of S100B for their ability to activate glia. The C68VC84S mutant lacks S100B's two cysteine residues (cys68, cys84) and lacks neurotrophic activity (Winningham-Major et al., 1989, J. Cell Biol. 109 3063-3071), and the truncation mutant S100B83stop lacks the C-terminal nine residues (including cys84) that have been shown to be important for some S100B:target protein interactions. We report here that both C68VC84S and S100B83stop stimulate glial activation, as determined by induction of iNOS and IL-1 beta in rat primary astrocyte and microglial cultures. C68VC84S showed activation profiles similar to those of wild-type S100B, demonstrating that a disulfide-linked dimer is not required for glial activation. S100B83stop also stimulated both iNOS and IL-1 beta, although S100B83stop was significantly less effective than wild-type S100B in inducing iNOS. These results indicate that the C-terminal region of S100B is not required for glial activation; however, its presence may influence the degree of activation by the protein. Altogether, these studies demonstrate that the structural features required for S100B's neurotrophic activity are distinct from those affecting its glial activation activity.     

Sequence analysis of a fish vitellogenin cDNA with a large phosvitin domain

Vitellogenins (Vtg) are egg-yolk precursor proteins crucial for reproductive success in oviparous animals. We have cloned the first complete cichlid Vtg cDNA from the tilapia fish, Oreochromis aureus. This cDNA has the largest phosvitin (PV) domain amongst piscine Vtgs, being comparable to those of lamprey, Xenopus, and chicken. Thus, the size of PV is independent of the evolutionary advancement of a species. The closer interspecific relationship between O. aureus Vtg1 and Fundulus VtgII than the intraspecific relationship between Fundulus VtgI and II isoforms suggests that teleost ancestors had at least two Vtg isoforms. Contrary to the results of previous phylogenetic inference using Vtgs which indicate that insect lineage is most diverged and nematodes are closer to vertebrate lineage, our results show that nematodes and hexapods form two monophyletic sister groups. Another arthropod taxon, represented by a malacostracan crustacean, Penaeus japonicus, appears to be more closely related to the vertebrates than the hexapods.     

The adaptor protein BLNK is required for b cell antigen receptor-induced activation of nuclear factor-kappa B and cell cycle entry and survival of B lymphocytes

B lymphocytes lacking the adaptor protein B cell linker (BLNK) do not proliferate in response to B cell antigen receptor (BCR) engagement. We demonstrate here that BCR-activated BLNK(-)/- B cells fail to enter the cell cycle, and this is due to their inability to induce the expression of the cell cycle regulatory proteins such as cyclin D2 and cyclin-dependent kinase 4. BCR-stimulated BLNK(-)/- B cells also do not up-regulate the cell survival protein Bcl-x(L), which may be necessary for the cells to complete the cell cycle. In addition, BLNK(-)/- B cells exhibit a high rate of spontaneous apoptosis in culture. Examination of the various BCR-activated signaling pathways in mouse BLNK(-)/- B cells reveals the intact activation of Akt and mitogen-activated protein kinases but the impaired activation of nuclear factor (NF)-kappaB that is known to regulate genes involved in cell proliferation and survival. The inability to activate NF-kappaB in BCR-stimulated BLNK(-)/- B cells is due to a failure to induce the degradation of the inhibitory kappaB protein. In all these aspects, BLNK(-)/- B cells resemble xid B cells that have a mutation in Bruton's tyrosine kinase (Btk). Recently, phospholipase C (PLC)-gamma2 has also been demonstrated to be essential for NF-kappaB activation. Since BLNK has been shown separately to interact with both Btk and PLC-gamma2, our finding of normal Btk but impaired PLC-gamma2 activation in BCR-stimulated BLNK(-)/- B cells strongly suggests that BLNK orchestrates the formation of a Btk-PLC-gamma2 signaling axis that regulates NF-kappaB activation. Taken together, the NF-kappaB activation defect may be sufficient to explain the similar defects in BCR-induced B cell proliferation and T cell-independent immune responses in BLNK(-)/-, Btk(-)/-, and PLC-gamma2(-)/- mice.     

Adhesion of epithelial cells to fibronectin or collagen I induces alterations in gene expression via a protein kinase C-dependent mechanism

Adhesion of human salivary gland (HSG) epithelial cells to fibronectin- or collagen I gel-coated substrates, mediated by beta1 integrins, has been shown to upregulate the expression of more than 30 genes within 3-6 h. Adhesion of HSG cells to fibronectin or collagen I for 6 h also enhanced total protein kinase C (PKC) activity by 1.8-2.3-fold. HSG cells expressed PKC-alpha, gamma, delta, epsilon, mu, and zeta. Adhesion of HSG cells to fibronectin or collagen I specifically activated PKC-gamma and PKC-delta. Cytoplasmic PKC-gamma and PKC-delta became membrane-associated, and immunoprecipitated PKC-gamma and PKC-delta kinase activities were enhanced 2.5-4.0-fold in HSG cells adherent to fibronectin or collagen I. In addition, adhesion of fibronectin-coated beads to HSG monolayers co-aggregated beta1 integrin and PKC-gamma and PKC-delta but not other PKC isoforms. Thus, integrin-dependent adhesion of HSG cells to fibronectin or collagen I activated PKC-gamma and PKC-delta. The role of this PKC upregulation on adhesion-responsive gene expression was then tested. HSG cells were treated with the specific PKC inhibitor bisindolylmaleimide I, cultured on non-precoated, fibronectin- or collagen I-coated substrates, and analyzed for changes in adhesion-responsive gene expression. Bisindolylmaleimide I strongly inhibited the expression of seven adhesion-responsive genes including calnexin, decorin, S-adenosylmethionine decarboxylase, steroid sulfatase, and 3 mitochondrial genes. However, the expression of two adhesion-responsive genes was not affected by bisindolylmaleimide I. Treatment with bisindolylmaleimide I did not affect cell spreading and did not significantly affect the actin cytoskeleton. These data suggest that adhesion of HSG cells to fibronectin or collagen I induces PKC activity and that this induction contributes to the upregulation of a variety of adhesion-responsive genes.     

The miracle of microarray data analysis

A report on the tenth Annual Bioinformatics and Genome Research meeting of the Cambridge Healthtech Institute's Beyond Genome 2001 series, San Francisco, USA, 17-19 June 2001.     

Detection of chromosomes tagged with green fluorescent protein in live Arabidopsis thaliana plants

Background

Structural and dynamic studies of chromosomes tagged with green fluorescent protein (GFP) in yeast and cultured animal cells have revealed some surprises. Although this technology can be very powerful, only a few studies using this approach with developed multicellular systems have been reported for the study of chromatin behavior in situ.

Results

We established vectors and conditions to visualize tagged loci stably inserted in the Arabidopsis genome via GFP fused to a bacterial DNA-binding protein. Using this system, three-dimensional coordinates for tagged loci within nuclei from cells of a live plant can be directly determined with concomitant visualization of the position of the nucleolus. Chromosome polyploidization in epidermal cells at the elongation zone of the root in transgenic plants can be visualized in situ using this technique.

Conclusion

We have established that GFP fusion with DNA-binding proteins can be used in conjunction with concatameric binding-site arrays to track genomic loci in living Arabidopsis plants. It should now be feasible to study the mechanisms of organization and dynamics of chromatin in specific cell types during various times of plant development, taking advantage of the well developed genetic systems and resources available for Arabidopsis.     

Characterization of sex chromosomes in rainbow trout and coho salmon using fluorescence in situ hybridization (FISH)

With the aim of characterizing the sex chromosomes of rainbow trout (Oncorhynchus mykiss) and to identify the sex chromosomes of coho salmon (O. kisutch), we used molecular markers OmyP9, 5S rDNA, and a growth hormone gene fragment (GH2), as FISH probes. Metaphase chromosomes were obtained from lymphocyte cultures from farm specimens of rainbow trout and coho salmon. Rainbow trout sex marker OmyP9 hybridizes on the sex chromosomes of rainbow trout, while in coho salmon, fluorescent signals were localized in the medial region of the long arm of one subtelocentric chromosome pair. This hybridization pattern together with the hybridization of a GH2 intron probe on a chromosome pair having the same morphology, suggests that a subtelocentric pair could be the sex chromosomes in this species. We confirm that in rainbow trout, one of the two loci for 5S rDNA genes is on the X chromosome. In males of this species that lack a heteromorphic sex pair (XX males), the 5S rDNA probe hybridized to both subtelocentrics This finding is discussed in relation to the hypothesis of intraspecific polymorphism of sex chromosomes in rainbow trout.