Contribution of proliferation and DNA damage repair to alveolar epithelial type 2 cell recovery from hyperoxia

In this study, C57BL/6J mice were exposed to hyperoxia and allowed to recover in room air. The sublethal dose of hyperoxia for C57BL/6J was 48 h. Distal lung cellular isolates from treated animals were characterized as 98% epithelial, with minor fibroblast and endothelial cell contaminants. Cells were then verified as 95% pure alveolar epithelial type II cells (AEC2) by surfactant protein C (SP-C) expression. After hyperoxia exposure in vivo, fresh, uncultured AEC2 were analyzed for proliferation by cell yield, cell cycle, PCNA expression, and telomerase activity. DNA damage was assessed by TdT-dUTP nick-end labeling, whereas induction of DNA repair was evaluated by GADD-153 expression. A baseline level for proliferation and damage was observed in cells from control animals that did not alter significantly during acute hyperoxia exposure. However, a rise in these markers was observed 24 h into recovery. Over 72 h of recovery, markers for proliferation remained elevated, whereas those for DNA damage and repair peaked at 48 h and then returned back to baseline. The expression of GADD-153 followed a distinct course, rising significantly during acute exposure and peaking at 48 h recovery. These data demonstrate that in healthy, adult male C57BL/6J mice, AEC2 proliferation, damage, and repair follow separate courses during hyperoxia recovery and that both proliferation and efficient repair may be required to ensure AEC2 survival.     

Fate of intraperitoneally injected fluorescent microspheres in developing Ictalurus punctatus

Fluorescent microspheres (FMS) were injected intraperitoneally into channel catfish fry at 2 days post hatch (dph), 1, 2, 3, 4 and 8 weeks post hatch (wph). The FMS were observed in the vasculature almost immediately after injection in all age groups except 2 dph. Fluorescent microspheres were observed within mononuclear phagocytes in the vasculature after 0.16 dph in all age groups. Fluorescent microspheres were first phagocytized in the coelomic cavity immediately after injection, while the majority of coelomic FMS were phagocytized between 0.16 and 1 dph for all ages. Enzyme cytochemical staining indicated that both polymorphonuclear (neutrophilic granulocytes) and mononuclear phagocytes had phagocytized FMS in the coelomic cavity and organs, with a predominance of FMS found in mononuclear phagocytic cells in all age groups across all sample periods. The predominant organs associated with the observed cellular responses were the posterior kidney, spleen, and anterior kidney. Splenic organization and melanomacrophage development and activity were more pronounced as the fish aged from 2 wph on. Particulate clearance rates were faster in the 2 dph and 1 wph fish than the older ages of fish. These results suggest that to facilitate particulate retention, channel catfish should be vaccinated at 4 wph or older.     

Crystal structure of aurora-2, an oncogenic serine/threonine kinase

Aurora-2 is a key member of a closely related subgroup of serine/threonine kinases that plays important roles in the completion of essential mitotic events. Aurora-2 is oncogenic and amplified in various human cancers and could be an important therapeutic target for inhibitory molecules that would disrupt the cell cycle and block proliferation. We report the first crystal structure of Aurora-2 kinase in complex with adenosine. Analysis of residues in the active site suggests differences with structurally and biologically related protein kinases. The activation loop, which contains residues specific to the Aurora family of kinases, has a unique conformation. These results provide valuable insight into the design of selective and highly potent ATP-competitive inhibitors of the Aurora kinases.     

The DMPC lipid phase transition influences differently the first and the second electron transfer reactions in bacterial reaction centers

Photosynthetic reaction centers (RCs) from Rhodobacter sphaeroides were incorporated in dimyristoylphosphatidylcholine (DMPC) liposomes. The first and second electron transfer rates (k(AB)(1) and k(AB)(2), respectively) between the first and the second quinone electron acceptors have been measured as a function of temperature, across the phase transition of DMPC (23 degrees C). The Eyring plots of k(AB)(1) display straight lines. In contrast, the Eyring plots for k(AB)(2) in proteoliposomes show a break at about 23.5 degrees C. This physical discrimination between the two electron transfer reactions demonstrates that the stiffness of the lipid environment of the RCs and/or the protein-protein interactions influence the parameters governing k(AB)(2), but not the gating process limiting k(AB)(1).     

Functional screening of five PYPAF family members identifies PYPAF5 as a novel regulator of NF-kappaB and caspase-1

PYRIN-containing Apaf-1-like proteins (PYPAFs) are a recently identified family of proteins thought to function in apoptotic and inflammatory signaling pathways. PYPAF1 and PYPAF7 proteins have been found to assemble with the PYRIN–CARD protein ASC and coordinate the activation of NF-κB and pro-caspase-1. To determine if other PYPAF family members function in pro-inflammatory signaling pathways, we screened five other PYPAF proteins (PYPAF2, PYPAF3, PYPAF4, PYPAF5 and PYPAF6) for their ability to activate NF-κB and pro-caspase-1. Co-expression of PYPAF5 with ASC results in a synergistic activation of NF-κB and the recruitment of PYPAF5 to punctate structures in the cytoplasm. The expression of PYPAF5 is highly restricted to granulocytes and T-cells, indicating a role for this protein in inflammatory signaling. In contrast, PYPAF2, PYPAF3, PYPAF4 and PYPAF6 failed to colocalize with ASC and activate NF-κB. PYPAF5 also synergistically activated caspase-1-dependent cytokine processing when co-expressed with ASC. These findings suggest that PYPAF5 functions in immune cells to coordinate the transduction of pro-inflammatory signals to the activation of NF-κB and pro-caspase-1.     

Desensitization of the PDGFbeta receptor by modulation of the cytoskeleton: the role of p21(Ras) and Rho family GTPases

Ligand-induced PDGF-type beta receptor (PDGFbeta-R) autophosphorylation is profoundly suppressed in cells transformed by activated p21(Ras). We report here that the integrity of the actin cytoskeleton is a critical regulator of PDGFbeta-R function in the presence of p21(Ras). Morphological reversion of Balb cells expressing a constitutively activated p21(Ras), with re-formation of actin stress fibers and cytoskeletal architecture, rendering them phenotypically similar to untransformed fibroblasts, allowed recovery of ligand-dependent PDGFbeta-R autophosphorylation. Conversely, disruption of the actin cytoskeleton in Balb/c-3T3 cells obliterated the normal ligand-induced phosphorylation of the PDGFbeta-R. The Rho family GTPases Rac and Rho are activated by p21(Ras) and are critical mediators of cell motility and morphology via their influence on the actin cytoskeleton. Transient expression of wild-type or constitutively active mutant forms of RhoA suppressed ligand-dependent PDGFbeta-R autophosphorylation and downstream signal transduction. These studies demonstrate the necessary role of Rho in the inhibition of PDGFbeta-R autophosphorylation in cells containing activated p21(Ras) and also demonstrate the importance of cell context and the integrity of the actin cytoskeleton in the regulation of PDGFbeta-R ligand-induced autophosphorylation.     

Effect of binding of Cd2+ on bacterial reaction center mutants: proton-transfer uses interdependent pathways

In bacterial reaction center of Rhodobacter sphaeroides, Cd2+ binds in stoichiometric amount to the protein. In the wild type, this results into a notable decrease of the rates of electron-transfer between the two quinone acceptors after the first (kAB(1)) and second flash (kAB(2)). We have studied these effects in two single mutants, L209PY and L209PF. L209Pro is situated in a protein region rich in hydrogen-bond networks involving water molecules. We show that (1) the combined effects of Cd2+ binding and point mutations have a cumulative consequence in the two mutants, decreasing very substantially the observed rates of electron-transfer. Interestingly, the [Cd2+] titration curves of kAB(2) in the L209PY and L209PF mutants are nearly superimposable to those previously reported for the M17DN and L210DN mutants (Paddock, M. L., Feher, G., and Okamura, M. Y. (2000) Proc. Natl. Acad. Sci U.S.A. 97, 1548-1553). These observations suggest a common effect of all of these mutations (L209, M17, L210) on the protonation state of the histidine cluster to which Cd2+ binds; (2) in the L209PY mutant, the pH titration curves of kAB(1), kAB(2), and k(H)(+), the proton-transfer rate at the second flash, are systematically downshifted by 1.5-2 pH units in the presence of 300 microM Cd2+, similarly to the wild type RCs (Gerencser, L., and Maroti, P. (2001) Biochemistry 40, 1850-1860). We propose that Cd2+ binding influences the electrostatics of interdependent ways of proton penetration within the protein, involving at least, directly or indirectly, L209P, L210D, and M17D, probably in conjunction with hydrogen-bonded connected water molecules.     

Development during single IVP of bovine oocytes from dissected follicles: Interactive effects of estrous cycle stage,follicle size and atresia

Previous work suggests that a number of factors such as follicle size, day of estrous cycle, and level of atresia influence the developmental potential of bovine oocytes in vitro. To understand better the interactions of these factors, 1299 follicles ≥3 mm in diameter were dissected from ovaries of synchronized dairy cows on four days (d2, d7, d10, or d15) during the estrous cycle. The oocyte from each follicle was collected and matured, fertilized, and cultured singly to d8 (d0 of culture = IVF). Control follicles (302) were similarly dissected and processed from an ovary pair randomly collected from the abattoir on each slaughter day. Results showed that development to blastocyst was greater in oocytes collected during phases of follicular growth (d2 and d10) than those collected during phases of follicular dominance (d7 and d15; 44.8% vs. 36.0%, respectively: P < 0.001) over all follicle size categories (3–5 mm, 6–8 mm, 9–12 mm and ≥13 mm). Oocyte competence tended to increase with increasing follicle size (P < 0.1). Follicular cells from follicles containing an oocyte that developed to morula or greater by d8 (484 samples) were analyzed by flow cytometry to measure the level of apoptosis. Results showed an increase in mean percent apoptotic cells in subordinate follicles (18.65 ± 0.86 over all size categories), particularly those of medium size (25.55 ± 2.2 for 6–8 mm size follicles; P < 0.001), during the dominance phase compared to growth phase (9.25 ± 0.95 over all sizes; P < 0.05). These results show a significant affect of the stage of estrous cycle on both oocyte competence and levels of follicular atresia. Mol. Reprod. Dev. 53:451–458, 1999. © 1999 Wiley‐Liss, Inc.     

In vitro and early in vivo development of sheep gynogenones and putative androgenones

Genomic imprinting, where only one of the two parental genes is expressed, occurs in many phyla. In mammals, however, this phenomenon has been primarily studied in mice, and to a lesser extent, in humans. To understand how genomic imprinting may affect development in other species, particularly those with a different mode of placental development from mice and humans, 339 sheep zygotes were micromanipulated to contain either 2 large (presumptive male) or 2 small (presumptive female) pronuclei. One hundred and twenty-seven of these embryos and 86 manipulated and nonmanipulated control embryos were transferred to recipient ewes over 3 breeding seasons. Twenty-one control and 7 experimental conceptuses were recovered on day 21. Four of these conceptuses derived from zygotes with 2 small pronuclei were identified by karyotyping to be gynogenones (maternal-derived genome). While the gross morphology of the embryos appeared no different to those of normal controls, the extra-embryonic tissue from the conceptuses showed some hypertrophy and hypervascularization. Preliminary Northern blots of mRNA from allantoic and trophoblast tissue showed an overexpression of H19 and an underexpression of IGF2. Although the sheep gynogenetic phenotype contrasts with that seen in mice, these two genes appear to be similarly differentially expressed. Mol. Reprod. Dev. 50:154–162, 1998. © 1998 Wiley-Liss, Inc.