Interactions between trophoblast cells and the maternal and fetal circulation in the mouse placenta

Mammalian embryos have an intimate relationship with their mothers, particularly with the placental vasculature from which embryos obtain nutrients essential for growth. It is an interesting vascular bed because maternal vessel number and diameter change dramatically during gestation and, in rodents and primates, the terminal blood space becomes lined by placental trophoblast cells rather than endothelial cells. Molecular genetic studies in mice aimed at identifying potential regulators of these processes have been hampered by lack of understanding of the anatomy of the vascular spaces in the placenta and the general nature of maternal-fetal vascular interactions. To address this problem, we examined the anatomy of the mouse placenta by preparing plastic vascular casts and serial histological sections of implantation sites from embryonic day (E) 10.5 to term. We found that each radial artery carrying maternal blood into the uterus branched into 5-10 dilated spiral arteries located within the metrial triangle, populated by uterine natural killer (uNK) cells, and the decidua basalis. The endothelial-lined spiral arteries converged together at the trophoblast giant cell layer and emptied into a few straight, trophoblast-lined "canals" that carried maternal blood to the base of the placenta. Maternal blood then percolated back through the intervillous space of the labyrinth toward the maternal side of the placenta in a direction that is countercurrent to the direction of the fetal capillary blood flow. Trophoblast cells were found invading the uterus in two patterns. Large cells that expressed the trophoblast giant cell-specific gene Plf (encoding Proliferin) invaded during the early postimplantation period in a pattern tightly associated with spiral arteries. These peri/endovascular trophoblast were detected only approximately 150-300 microm upstream of the main giant cell layer. A second type of widespread interstitial invasion in the decidua basalis by glycogen trophoblast cells was detected after E12.5. These cells did not express Plf, but rather expressed the spongiotrophoblast-specific gene Tpbp. Dilation of the spiral arteries was obvious between E10.5 and E14.5 and was associated with a lack of elastic lamina and smooth muscle cells. These features were apparent even in the metrial triangle, a site far away from the invading trophoblast cells. By contrast, the transition from endothelium-lined artery to trophoblast-lined (hemochorial) blood space was associated with trophoblast giant cells. Moreover, the shaping of the maternal blood spaces within the labyrinth was dependent on chorioallantoic morphogenesis and therefore disrupted in Gcm1 mutants. These studies provide important insights into how the fetoplacental unit interacts with the maternal intrauterine vascular system during pregnancy in mice.     

Structure and stability of the ankyrin domain of the Drosophila Notch receptor

The Notch receptor contains a conserved ankyrin repeat domain that is required for Notch-mediated signal transduction. The ankyrin domain of Drosophila Notch contains six ankyrin sequence repeats previously identified as closely matching the ankyrin repeat consensus sequence, and a putative seventh C-terminal sequence repeat that exhibits lower similarity to the consensus sequence. To better understand the role of the Notch ankyrin domain in Notch-mediated signaling and to examine how structure is distributed among the seven ankyrin sequence repeats, we have determined the crystal structure of this domain to 2.0 angstroms resolution. The seventh, C-terminal, ankyrin sequence repeat adopts a regular ankyrin fold, but the first, N-terminal ankyrin repeat, which contains a 15-residue insertion, appears to be largely disordered. The structure reveals a substantial interface between ankyrin polypeptides, showing a high degree of shape and charge complementarity, which may be related to homotypic interactions suggested from indirect studies. However, the Notch ankyrin domain remains largely monomeric in solution, demonstrating that this interface alone is not sufficient to promote tight association. Using the structure, we have classified reported mutations within the Notch ankyrin domain that are known to disrupt signaling into those that affect buried residues and those restricted to surface residues. We show that the buried substitutions greatly decrease protein stability, whereas the surface substitutions have only a marginal affect on stability. The surface substitutions are thus likely to interfere with Notch signaling by disrupting specific Notch-effector interactions and map the sites of these interactions.     

Retrovirus capsid protein assembly arrangements

During retrovirus particle assembly and morphogenesis, the retrovirus structural (Gag) proteins organize into two different arrangements: an immature form assembled by precursor Gag (PrGag) proteins; and a mature form, composed of proteins processed from PrGag. Central to both Gag protein arrangements is the capsid (CA) protein, a domain of PrGag, which is cleaved from the precursor to yield a mature Gag protein composed of an N-terminal domain (NTD), a flexible linker region, and a C-terminal domain (CTD). Because Gag interactions have proven difficult to examine in virions, a number of investigations have focused on the analysis of structures assembled in vitro. We have used electron microscope (EM) image reconstruction techniques to examine assembly products formed by two different CA variants of both human immunodeficiency virus type 1 (HIV-1) and the Moloney murine leukemia virus (M-MuLV). Interestingly, two types of hexameric protein arrangements were observed for each virus type. One organizational scheme featured hexamers composed of putative NTD dimer subunits, with sharing of subunits between neighbor hexamers. The second arrangement used apparent NTD monomers to coordinate hexamers, involved no subunit sharing, and employed putative CTD interactions to connect hexamers. Conversion between the two assembly forms may be achieved by making or breaking the proposed symmetric NTD dimer contacts in a process that appears to mimic viral morphogenesis.     

Identification and characterization of an adeno-associated virus integration site in CV-1 cells from the African green monkey

Adeno-associated virus (AAV) is a classification given to a group of nonpathogenic, single-stranded DNA viruses known to reside latently in primates. During latency in humans, AAV type 2 (AAV2) preferentially integrates at a site on chromosome 19q13.3ter by targeting a sequence composed of an AAV Rep binding element (RBE), a spacer, and a nicking site. Here, we report the DNA sequence of an African green monkey AAV integration site isolated from CV-1 cells. Overall, it has 98% homology to the analogous human site, including identical spacer and nicking sequences. However, the simian RBE is expanded, having five perfect directly repeated GAGC tetramers. We carried out a number of in vitro and in vivo assays to determine the effect of this expanded RBE sequence on the Rep-RBE interaction and AAV targeted integration. Using electromobility shift assays it was demonstrated that AAV4 Rep68 bound the expanded RBE with a sixfold-greater affinity than the human RBE. To determine the basis for the affinity increase, DNase I protection and methylation interference (MI) assays were performed. Comparison of footprints on both the human and simian RBEs revealed nearly identical protection; however, MI analysis suggested greater interaction with the guanine nucleotides of the expanded RBE, thus providing a biochemical basis for the increased binding activity. In vivo, integration targeted to the simian RBE was demonstrated by PCR analysis of latently infected Cos-7 cells. Interestingly, the frequency of site-specific integration was twofold greater in Cos-7 cells than in HeLa cells. Overall, these experiments establish that the simian RBE, identified in CV-1 cells, functions analogously to the human RBE and provide further evidence for a developing model that proposes individual roles for the RBE and the spacer and nicking site elements.     

Direct activation of cyclin-dependent kinase 2 by human papillomavirus E7

Addition of human papillomavirus (HPV) E7 CDK2/cyclin A or CDK2/cyclin E, purified from either insect cells or bacteria, dramatically upregulates histone H1 kinase activity. Activation is substrate specific, with a smaller effect noted for retinoblastoma protein (Rb). The CDK2 stimulatory activity is equivalent in high-risk (HPV type 16 [HPV16] and HPV31) and low-risk (HPV6b) E7. Mutational analyses of HPV16 E7 indicate that the major activity resides in amino acids 9 to 38, spanning CR1 and CR2, and does not require casein kinase II or Rb-binding domain functions. Synthetic peptides spanning HPV16 amino acid residues 9 to 38 also activate CDK2. Peptides containing this sequence that carry biotin on the carboxy terminus, as well as a photoactivated cross-linking group (benzophenone), also activate the complex and covalently associate with the CDK2/cyclin A complex in a specific manner requiring UV. Cross-linking studies that use protein monomers detect association of the E7 peptides with cyclin A but not CDK2. Together, our results indicate a novel mechanism whereby E7 promotes HPV replication by directly altering CDK2 activity and substrate specificity.     

Development of an intact cell reporter gene beta-lactamase assay for G protein-coupled receptors for high-throughput screening

G protein-coupled receptors (GPCRs) are involved in a large variety of physiological disorders, and are thus important pharmaceutical drug targets. Here, we describe the development and characterization of a beta-lactamase reporter gene assay as a functional readout for the ligand-induced activation of the human bradykinin B1 receptor, expressed recombinantly in CHO cells. The beta-lactamase reporter gene assay provides high sensitivity due to the absence of endogenous beta-lactamase activity in mammalian cells. The cell-permeable fluorogenic substrate allows single-cell cloning of cells expressing functional BK1 receptors. Pharmacological characterization reveals comparable sensitivity and potency of known BK1 receptor agonists and antagonists between the beta-lactamase assay, competition-binding assay, and other direct measurements of second messengers. The beta-lactamase assay has been optimized for cell density, time of agonist stimulation, and DMSO sensitivity. This CHO-hBK1-beta-lactamase assay is well suited to automation and miniaturization required for high-throughput screening.     

Structure-based kinetic modeling of excited-state transfer and trapping in histidine-tagged photosystem II core complexes from synechocystis

Chlorophyll fluorescence decay kinetics in photosynthesis are dependent on processes of excitation energy transfer, charge separation, and electron transfer in photosystem II (PSII). The interpretation of fluorescence decay kinetics and their accurate simulation by an appropriate kinetic model is highly dependent upon assumptions made concerning the homogeneity and activity of PSII preparations. While relatively simple kinetic models assuming sample heterogeneity have been used to model fluorescence decay in oxygen-evolving PSII core complexes, more complex models have been applied to the electron transport impaired but more highly purified D1-D2-cyt b(559) preparations. To gain more insight into the excited-state dynamics of PSII and to characterize the origins of multicomponent fluorescence decay, we modeled the emission kinetics of purified highly active His-tagged PSII core complexes with structure-based kinetic models. The fluorescence decay kinetics of PSII complexes contained a minimum of three exponential decay components at F(0) and four components at F(m). These kinetics were not described well with the single radical pair energy level model, and the introduction of either static disorder or a dynamic relaxation of the radical pair energy level was required to simulate the fluorescence decay adequately. An unreasonably low yield of charge stabilization and wide distribution of energy levels was required for the static disorder model, and we found the assumption of dynamic relaxation of the primary radical pair to be more suitable. Comparison modeling of the fluorescence decay kinetics from PSII core complexes and D1-D2-cyt b(559) reaction centers indicated that the rates of charge separation and relaxation of the radical pair are likely altered in isolated reaction centers.     

Facultative prioritization of wing growth in the Welcome Swallow Hirundo neoxena

Bird species adapted to variable environments tend to have slow lean tissue growth rates and high fat deposition rates, allowing survival during food shortages. This emphasis on fat deposition may be a fixed physiological trait. Alternatively, tissue allocation may be adjusted facultatively according to the proximate food supply. We consider two models of facultative adjustment that could account for the emphasis on fat deposition: (1) the fat-priority model, in which no lean growth occurs when food is scarce, and (2) the lean-priority model, in which a minimal level of lean growth always occurs but nutrients are otherwise allocated to fat deposition. We tested these two models using Welcome Swallows Hirundo neoxena , a species we show to have a variable food supply that is influenced by weather. We reduced food supply to chicks experimentally, by enlarging broods or excluding parents from chicks, and tested for reduction in wing growth (an indicator of lean growth) and mass growth (an indicator of fat deposition). Mass growth was retarded by both manipulations, but not wing growth, corroborating the lean-priority model. This growth strategy may function not to cope with violent variation in food supply, but to maintain development and symmetry of wings and feathers in the face of moderate variation in food supply. Our results contrast with those of a similar experiment on the Black Noddy Anous minutus , a species with more severe variation in food supply.