New human transforming genes detected by a tumorigenicity assay.

We have developed a sensitive bioassay for transforming genes based on the tumorigenicity of cotransfected NIH3T3 cells in nude mice. The assay differs substantially from the NIH3T3 focus assay. Using it, we have detected the transfer of three transforming genes from the DNA of MCF-7, a human mammary carcinoma cell line. One of these is N-ras, which is amplified in MCF-7 DNA. The other two, which we have called mcf2 and mcf3, do not appear to be related to known oncogenes. We cannot detect their transfer by using the NIH3T3 focus assay. We do not yet know whether either mcf2 or mcf3 is associated with genetic abnormalities in MCF-7 cells.     

A genome-wide study of gene activity reveals developmental signaling pathways in the preimplantation mouse embryo

The preimplantation development of the mammalian embryo encompasses a series of critical events: the transition from oocyte to embryo, the first cell divisions, the establishment of cellular contacts, the first lineage differentiation-all the first subtle steps toward a future body plan. Here, we use microarrays to explore gene activity during preimplantation development. We reveal robust and dynamic patterns of stage-specific gene activity that fall into two major phases, one up to the 2-cell stage (oocyte-to-embryo transition) and one after the 4-cell stage (cellular differentiation). The mouse oocyte and early embryo express components of multiple signaling pathways including those downstream of Wnt, BMP, and Notch, indicating that conserved regulators of cell fate and pattern formation are likely to function at the earliest embryonic stages. Overall, these data provide a detailed temporal profile of gene expression that reveals the richness of signaling processes in early mammalian development.     

Dynamics of alpha-actinin in focal adhesions and stress fibers visualized with alpha-actinin-green fluorescent protein

Motile cells undergo changes in cell adhesion, behavior, and shape that are mediated by small-scale cytoskeletal rearrangements. These rearrangements have proven difficult to follow quantitatively in living cells, without disrupting the very structures and delicate protein balances under study. We have expressed a prominent cytoskeletal protein, alpha-actinin, as a fusion with green fluorescent protein (alpha AGFP), and have followed this construct's movements within transfected mouse Swiss 3T3 and BALB/c fibroblasts. alpha AGFP was expressed at low levels to avoid overexpression artifacts. alpha AGFP localized to cellular structures, including stress fibers, focal adhesions, microspikes, and lamellipodia. High-resolution video-microscopy revealed that the alpha AGFP construct could be seen relocating to focal adhesions early in their formation and shortly thereafter to stress-fiber dense bodies. By Fluorescent Recovery After Photo-bleaching (FRAP) techniques, alpha AGFP was found to have similar exchange rates and protein stability in focal adhesions and stress fibers (despite the known differences in protein composition in these two structures). This raises the possibility that the two structures share common key regulatory factors and may not be as affected by protein-protein binding interactions as previously suggested. Additionally, the exchange rates revealed by video-microscopy and FRAP analysis of alpha AGFP are more rapid than those reported previously, which were obtained using microinjection of large excesses of fluorescently-tagged protein.     

Recreating a functional ancestral archosaur visual pigment

The ancestors of the archosaurs, a major branch of the diapsid reptiles, originated more than 240 MYA near the dawn of the Triassic Period. We used maximum likelihood phylogenetic ancestral reconstruction methods and explored different models of evolution for inferring the amino acid sequence of a putative ancestral archosaur visual pigment. Three different types of maximum likelihood models were used: nucleotide-based, amino acid-based, and codon-based models. Where possible, within each type of model, likelihood ratio tests were used to determine which model best fit the data. Ancestral reconstructions of the ancestral archosaur node using the best-fitting models of each type were found to be in agreement, except for three amino acid residues at which one reconstruction differed from the other two. To determine if these ancestral pigments would be functionally active, the corresponding genes were chemically synthesized and then expressed in a mammalian cell line in tissue culture. The expressed artificial genes were all found to bind to 11-cis-retinal to yield stable photoactive pigments with lambda(max) values of about 508 nm, which is slightly redshifted relative to that of extant vertebrate pigments. The ancestral archosaur pigments also activated the retinal G protein transducin, as measured in a fluorescence assay. Our results show that ancestral genes from ancient organisms can be reconstructed de novo and tested for function using a combination of phylogenetic and biochemical methods.     

Sperm entry position provides a surface marker for the first cleavage plane of the mouse zygote

The sperm entry position (SEP) of the mouse egg, labelled by placing a bead at the fertilisation cone, tends to be associated with the first cleavage plane (Piotrowska and Zernicka-Goetz: Nature 409:517-521, 2001). Nevertheless, in up to one-fourth of embryos the cleavage furrow did not pass close to the bead, and following the division the bead marked the cleavage plane in only 60% of cases. This raised the question of whether such variability arose from the labelling itself or had a biological basis. The zona pellucida was not responsible for this effect because similar results were obtained in its presence or absence. However, this variability could be attributable to the large size of the fertilisation cone relative to the SEP. Therefore, we have developed a means of fluorescently labelling sperm that can record the exact site of its penetration when the label transfers to the egg surface. This approach indicates that the SEP marks the first cleavage in the great majority (88%) of embryos. In conclusion, direct sperm labelling shows precisely the correlation between the SEP and the first cleavage, although there is natural variability in this process.     

Pollen tube growth and guidance is regulated by POP2, an Arabidopsis gene that controls GABA levels

During angiosperm reproduction, pollen grains form a tube that navigates through female tissues to the micropyle, delivering sperm to the egg; the signals that mediate this process are poorly understood. Here, we describe a role for gamma-amino butyric acid (GABA) in pollen tube growth and guidance. In vitro, GABA stimulates pollen tube growth, although vast excesses are inhibitory. The Arabidopsis POP2 gene encodes a transaminase that degrades GABA and contributes to the formation of a gradient leading up to the micropyle. pop2 flowers accumulate GABA, and the growth of many pop2 pollen tubes is arrested, consistent with their in vitro GABA hypersensitivity. Some pop2 tubes continue to grow toward ovules, yet they are misguided, presumably because they target ectopic GABA on the ovule surface. Interestingly, wild-type tubes exhibit normal growth and guidance in pop2 pistils, perhaps by degrading excess GABA and sharpening the gradient leading to the micropyle.     

Automated nano-electrospray mass spectrometry for protein-ligand screening by noncovalent interaction applied to human H-FABP and A-FABP

A method for ligand screening by automated nano-electrospray ionization mass spectrometry (nano-ESI/MS) is described. The core of the system consisted of a chip-based platform for automated sample delivery from a 96-well plate and subsequent analysis based on noncovalent interactions. Human fatty acid binding protein, H-FABP (heart) and A-FABP (adipose), with small potential ligands was analyzed. The technique has been compared with a previously reported method based on nuclear magnetic resonance (NMR), and excellent correlation with the found hits was obtained. In the current MS screening method, the cycle time per sample was 1.1 min, which is approximately 50 times faster than NMR for single compounds and approximately 5 times faster for compound mixtures. High reproducibility was achieved, and the protein consumption was in the range of 88 to 100 picomoles per sample. Futhermore, a novel protocol for preparation of A-FABP without the natural ligand is presented. The described screening approach is suitable for ligand screening very early in the drug discovery process before conventional high-throughput screens (HTS) are developed and/or used as a secondary screening for ligands identified by HTS.     

Fibroblast growth factor signaling is required for the proliferation and patterning of progenitor cells in the developing anterior pituitary

The proliferation and patterning of progenitor cells in the anterior pituitary require signals derived from the neuroepithelium of the juxtaposed infundibulum. The infundibulum expresses Fibroblast growth factor (Fgf) 8 and Fgf 18, and FGFs can mimic some of the activities of the infundibulum. The requirement for FGF signaling during growth and patterning of the anterior pituitary has not, however, been established. By blocking FGF receptor signaling in explants of the anterior pituitary cultured in vitro we provide evidence that FGF signaling derived from the infundibulum is required for the proliferation and patterning of progenitor cells in the anterior pituitary.     

Potent neutralizing anti-CD1d antibody reduces lung cytokine release in primate asthma model

CD1d is a receptor on antigen-presenting cells involved in triggering cell populations, particularly natural killer T (NKT) cells, to release high levels of cytokines. NKT cells are implicated in asthma pathology and blockade of the CD1d/NKT cell pathway may have therapeutic potential. We developed a potent anti-human CD1d antibody (NIB.2) that possesses high affinity for human and cynomolgus macaque CD1d (K_D ∼100 pM) and strong neutralizing activity in human primary cell-based assays (IC₅₀ typically <100 pM). By epitope mapping experiments, we showed that NIB.2 binds to CD1d in close proximity to the interface of CD1d and the Type 1 NKT cell receptor β-chain. Together with data showing that NIB.2 inhibited stimulation via CD1d loaded with different glycolipids, this supports a mechanism whereby NIB.2 inhibits NKT cell activation by inhibiting Type 1 NKT cell receptor β-chain interactions with CD1d, independent of the lipid antigen in the CD1d antigen-binding cleft. The strong in vitro potency of NIB.2 was reflected in vivo in an Ascaris suum cynomolgus macaque asthma model. Compared with vehicle control, NIB.2 treatment significantly reduced bronchoalveolar lavage (BAL) levels of Ascaris-induced cytokines IL-5, IL-8 and IL-1 receptor antagonist, and significantly reduced baseline levels of GM-CSF, IL-6, IL-15, IL-12/23p40, MIP-1α, MIP-1β, and VEGF. At a cellular population level NIB.2 also reduced numbers of BAL lymphocytes and macrophages, and blood eosinophils and basophils. We demonstrate that anti-CD1d antibody blockade of the CD1d/NKT pathway modulates inflammatory parameters in vivo in a primate inflammation model, with therapeutic potential for diseases where the local cytokine milieu is critical.