Ceramide-induced intracellular oxidant formation, iron signaling, and apoptosis in endothelial cells: protective role of endogenous nitric oxide

Sphingolipid ceramide (N-acetylsphingosine), a bioactive second messenger lipid, was shown to activate reactive oxygen species (ROS), mitochondrial oxidative damage, and apoptosis in neuronal and vascular cells. The proapoptotic effects of tumor necrosis factor-alpha, hypoxia, and chemotherapeutic drugs were attributed to increased ceramide formation. Here we investigated the protective role of nitric oxide (.NO) during hydrogen peroxide (H(2)O(2))-mediated transferrin receptor (TfR)-dependent iron signaling and apoptosis in C(2)-ceramide (C(2)-cer)-treated bovine aortic endothelial cells (BAECs). Addition of C(2)-cer (5-20 microm) to BAECs enhanced .NO generation. However, at higher concentrations of C(2)-cer (> or =20 microm), .NO generation did not increase proportionately. C(2)-cer (20-50 microm) also resulted in H(2)O(2)-mediated dichlorodihydrofluorescein oxidation, reduced glutathione depletion, aconitase inactivation, TfR overexpression, TfR-dependent uptake of (55)Fe, release of cytochrome c from mitochondria into cytosol, caspase-3 activation, and DNA fragmentation. N(w)-Nitro-l-arginine methyl ester (l-NAME), a nonspecific inhibitor of nitricoxide synthases, augmented these effects in BAECs at much lower (i.e. nonapoptotic) concentrations of C(2)-cer. The 26 S proteasomal activity in BAECs was slightly elevated at lower concentrations of C(2)-cer (< or =10 microm) but was greatly suppressed at higher concentrations (>10 microm). Intracellular scavengers of H(2)O(2), cell-permeable iron chelators, anti-TfR receptor antibody, or mitochondria-targeted antioxidant greatly abrogated C(2)-cer- and/or l-NAME-induced oxidative damage, iron signaling, and apoptosis. We conclude that C(2)-cer-induced H(2)O(2) and TfR-dependent iron signaling are responsible for its prooxidant and proapoptotic effects and that .NO exerts an antioxidative and cytoprotective role.     

Mechanical properties of shock-absorbing pylons used in transtibial prostheses

Prosthetic manufacturers have developed shock-absorbing pylons to attenuate the transient forces of foot-ground contact in order to supplement the residual capacity of lower limb amputees. The purpose of this study was to measure the elastic and damping properties of two frequently prescribed pylons (the ICON Shock Pylon and the Mercury TT Pyramid Pylon) at frequencies enveloping those observed during gait using pseudo-static compressive and dynamic cyclic testing methods. Results showed that the spring constants were linear functions of deformation (ranging from 74 to 110 N/mm and 91 to 157 N/mm for the ICON and the TT Pylons, respectively) while the damping force was a function of the square root of velocity combined with a coulomb element (1.6x0.5 + 21 and 7.4x0.5 + 102 N for the ICON and the TT Pylon, respectively).     

Aurora-A Kinase Is Essential for Bipolar Spindle Formation and Early Development

Aurora-A is a conserved kinase implicated in mitotic regulation and carcinogenesis. Aurora-A was previously implicated in mitotic entry and spindle assembly, although contradictory results prevented a clear understanding of the roles of Aurora-A in mammals. We developed a conditional null mutation in the mouse Aurora-A gene to investigate Aurora-A functions in primary cells ex vivo and in vivo. We show here that conditional Aurora-A ablation in cultured embryonic fibroblasts causes impaired mitotic entry and mitotic arrest with a profound defect in bipolar spindle formation. Germ line Aurora-A deficiency causes embryonic death at the blastocyst stage with pronounced cell proliferation failure, mitotic arrest, and monopolar spindle formation. Aurora-A deletion in mid-gestation embryos causes an increase in mitotic and apoptotic cells. These results indicate that murine Aurora-A facilitates, but is not absolutely required for, mitotic entry in murine embryonic fibroblasts and is essential for centrosome separation and bipolar spindle formation in vitro and in vivo. Aurora-A deletion increases apoptosis, suggesting that molecular therapies targeting Aurora-A may be effective in inducing tumor cell apoptosis. Aurora-A conditional mutant mice provide a valuable system for further defining Aurora-A functions and for predicting effects of Aurora-A therapeutic intervention.The equal partitioning of chromosomes at mitosis is critical for avoiding aneuploidy, a condition associated with spontaneous miscarriage, developmental disorders, and cancer (50). Mitosis requires coordinated completion of multiple events including nuclear envelope breakdown, chromosome condensation and congression to the metaphase plate, centrosome separation, spindle formation, chromosome-spindle attachment and error correction, sister chromatid separation, and cytokinesis. Multiple regulators, many of which are kinases, are required to ensure that each event is completed in a timely fashion and in the proper order (reviewed in reference 46). Although a number of mitotic kinases have been identified, their targets and the intricacies of mitotic signal transduction pathways are just beginning to be understood.The Aurora kinases are key mitotic regulators in eukaryotes (reviewed in reference 45). The Aurora family includes a single member in yeasts (Saccharomyces cerevisiae Ipl1p, Schizosaccharomyces pombe Ark1), two members each in Caenorhabditis elegans and Drosophila, and two or three members in vertebrates. Although originally given a variety of names, Aurora kinases in multicellular eukaryotes have subsequently been classified into A, B, and C groups based on patterns of mitotic subcellular localization and homology, which also appear to reflect functional distinctions (8, 46). Aurora-A kinases are observed at centrosomes and adjacent spindle fibers, and current evidence supports key roles in regulating protein localization and function at centrosomes, as well as regulation of the assembly, stability, and function of the mitotic spindle (reviewed in reference 43). Aurora-B kinases display “chromosomal passenger” localization, residing on mitotic chromosomes and subsequently moving to the spindle midzone after separation of sister chromatids. Aurora-B family members have been implicated in the regulation of kinetochore-spindle attachment, the spindle checkpoint, and cytokinesis (reviewed in references 1 and 8). Aurora-C kinases, which have only been identified in mammals, have a limited expression pattern and appear to have functions that overlap those of Aurora-B (7, 53).The human Aurora-A kinase (hAurA) was first identified because of its overexpression in cancer cell lines (5, 58). The hAurA gene (stk15) resides on chromosome 20q13, a region frequently amplified in human cancers (5, 58). hAurA has been dubbed an oncogene because of the fact that its overexpression transforms immortalized rodent fibroblasts (5, 70). Polymorphisms in hAurA are associated with an increased risk of colon cancer, while murine AurA (mAurA) polymorphisms confer increased susceptibility to experimentally induced skin tumors (14). The mAurA gene is frequently amplified in radiation-induced lymphomas from p53 heterozygous mice, while loss of one mAurA allele has been observed in lymphomas from p53-null mice (41). Thus, aberrant AurA expression is associated with tumorigenesis, suggesting that insight into AurA functions will lead to a better understanding of tumorigenesis mechanisms.A number of experimental observations suggest that AurA kinases are required for normal centrosome maturation and bipolar spindle assembly. The AurA ortholog in Drosophila melanogaster (Aurora) was identified in a screen for mutations that impact the centrosome cycle (21). Syncytial embryos from hypomorphic Aurora mutant females display a variety of mitotic abnormalities resulting from a failure to separate centrosomes. Aurora-null flies die at the larval stage with characteristic monopolar spindles and circular chromosome arrays in larval neuroblasts. Such monopolar spindles arise from failed centrosome separation (21). Subsequent studies of Drosophila Aurora mutant alleles revealed additional defects in centrosome maturation (including a failure to localize transforming acidic coiled-coil protein, centrosomin, and γ-tubulin at centrosomes) and in asymmetric localization of Numb protein in sensory organ precursor cells (3, 17). Similar to the case in Drosophila, disruption of the C. elegans AurA ortholog AIR-1 by RNA interference (RNAi) or mutation causes defects in centrosome maturation and monopolar spindle formation. Centrosomes undergo normal separation but collapse, leading to monopolar spindle formation (16, 24, 56). Studies of the Xenopus AurA homolog pEg2 revealed similar phenotypes after overexpression of kinase-dead mutants, antibody-mediated inhibition, or immunodepletion (18, 19, 38, 52). Furthermore, Xenopus AurA has been shown to interact with and phosphorylate Eg5, a mitotic kinesin required for bipolar spindle formation, suggesting a possible mechanism by which AurA could influence bipolar spindle formation and/or stabilization (19). Thus, existing reports from these systems are quite consistent in implicating AurA in centrosome separation and function.In contrast to the systems described above, published reports of RNAi-mediated reduction of AurA expression in mammalian cell lines have contained conflicting results about the role of AurA in mitotic entry, bipolar spindle formation, and mitotic progression. AurA RNAi in HeLa cells was reported to block or delay mitotic entry, prompting the conclusion that AurA is essential for mitotic commitment in mammalian cells (27, 36). In contrast, other AurA RNAi studies showed accumulation of mitotic cells with monopolar spindles (12, 20, 67). These discrepancies call into question the functional conservation of AurA in mammals and highlight a need for additional studies to definitively address the roles of AurA. This is particularly critical for understanding the roles of AurA in cancer and for projecting possible effects of AurA inhibitors currently in development as anticancer agents. We used gene targeting in mouse embryonic stem (ES) cells to produce a conditional null allele at the AurA locus. Here we describe cellular phenotypes of AurA deletion in primary cells in vitro and developmental phenotypes of AurA mutant mice. We show that AurA deletion in primary embryonic fibroblasts causes delayed mitotic entry with accumulation of cells in early prophase, consistent with a role for AurA in mitotic entry. Nevertheless, AurA-deficient cells that enter prometaphase arrest with monopolar spindles and eventually exit mitosis without segregating their chromosomes. Prolonged culture of AurA-deficient cells leads to polyploidy with abnormal nuclear structure. Germ line AurA deficiency causes embryonic death at the blastocyst stage with mitotic arrest and monopolar spindle formation, while AurA deletion in mid-gestation embryos causes an increased mitotic index and increased apoptosis. Together, our findings indicate that AurA is required for timely mitotic entry and bipolar spindle formation in vitro and in vivo.     

POT1 proteins in green algae and land plants: DNA-binding properties and evidence of co-evolution with telomeric DNA

Telomeric DNA terminates with a single-stranded 3′ G-overhang that in vertebrates and fission yeast is bound by POT1 (Protection Of Telomeres). However, no in vitro telomeric DNA binding is associated with Arabidopsis POT1 paralogs. To further investigate POT1–DNA interaction in plants, we cloned POT1 genes from 11 plant species representing major branches of plant kingdom. Telomeric DNA binding was associated with POT1 proteins from the green alga Ostreococcus lucimarinus and two flowering plants, maize and Asparagus. Site-directed mutagenesis revealed that several residues critical for telomeric DNA recognition in vertebrates are functionally conserved in plant POT1 proteins. However, the plant proteins varied in their minimal DNA-binding sites and nucleotide recognition properties. Green alga POT1 exhibited a strong preference for the canonical plant telomere repeat sequence TTTAGGG with no detectable binding to hexanucleotide telomere repeat TTAGGG found in vertebrates and some plants, including Asparagus. In contrast, POT1 proteins from maize and Asparagus bound TTAGGG repeats with only slightly reduced affinity relative to the TTTAGGG sequence. We conclude that the nucleic acid binding site in plant POT1 proteins is evolving rapidly, and that the recent acquisition of TTAGGG telomere repeats in Asparagus appears to have co-evolved with changes in POT1 DNA sequence recognition.     

5-Aminomethylbenzimidazoles as potent ITK antagonists

Benzamide 1 demonstrated good potency as a selective ITK inhibitor, however the amide moiety was found to be hydrolytically labile in vivo, resulting in low oral exposure and the generation of mutagenic aromatic amine metabolites. Replacing the benzamide with a benzylamine linker not only addressed the toxicity issue, but also improved the cellular and functional potency as well as the drug-like properties. SAR studies around the benzylamines and the identification of 10n and 10o as excellent tools for proof-of-concept studies are described.     

Forecasting of environmental risk maps of coastal algal blooms

We present the elements of an algal bloom risk forecast system which aims to provide a scientific prognosis of the likelihood of an algal bloom occurrence as a function of: (a) the nutrient concentration; and (b) the forecasted wind and tide-induced vertical mixing relative to the critical value defined by the environmental and algal growth conditions. The model is validated with high frequency field observations of algal blooms in recent years and only requires the input of readily available field measurements of water column transparency, nutrient concentration, representative maximum algal growth rate, and a simple estimate of vertical mixing as a function of tidal range, wind speed, and density stratification. The forecasted region-wide risk maps successfully predicted the observed algal bloom occurrences in Hong Kong waters over the past 20 years, with a correct prognosis rate of 87%. It is shown that algal blooms are to a large extent controlled by the interaction of physical and biological processes. This work provides a general framework to interpret the complex spatial and temporal dynamics of observed algal blooms, and paves the way for the development of real time algal bloom risk forecast systems.     

Beef cattle pasture to wetland reconversion: Impact on soil organic carbon and phosphorus dynamics

There is a major need to understand the historical condition and chemical/biological functions of the ecosystems following a conversion of wetlands to agricultural functions. To better understand the dynamics of soil total organic carbon (TOC) and phosphorus (P) during beef cattle pastures to wetland reconversion, soil core samples were collected from the beef cattle pasture and from the natural wetland at Plant City, FL, during five summer seasons (2002–2007). The levels of TOC and soil P were significantly affected by changing land use and hydrology. Draining natural wetlands to grazed pastures resulted in very pronounced reduction of TOC from 180.1 to 5.4 g g^?1. Cumulative concentrations of total phosphorus (TP) in soils (1134 mg kg^?1) under drained condition are two to three times lower than those in soils (2752 mg kg^?1) under flooded condition over the periods of land use reconversion. There was a declining trend (r = 0.82**; p ≤ 0.01) in total soil P from natural wetland (763 mg kg^?1) to altered pastures (340 mg kg^?1), largely as organic-bound P (natural wetland, 48%; grazed pastures, 44%; altered pastures, 29%). These results are important in establishing baseline information on soil properties in pasture and wetland prior to restoring and reconverting pasture back to wetland conditions. The results further suggest that changes in soil properties due to changing land use and hydrologic conditions (drying and re-wetting) could be long lasting.     

Rho kinase regulates the intracellular micromechanical response of adherent cells to rho activation

Local sol-gel transitions of the cytoskeleton modulate cell shape changes, which are required for essential cellular functions, including motility and adhesion. In vitro studies using purified cytoskeletal proteins have suggested molecular mechanisms of regulation of cytoskeleton mechanics; however, the mechanical behavior of living cells and the signaling pathways by which it is regulated remains largely unknown. To address this issue, we used a nanoscale sensing method, intracellular microrheology, to examine the mechanical response of the cell to activation of the small GTPase Rho. We observe that the cytoplasmic stiffness and viscosity of serum-starved Swiss 3T3 cells transiently and locally enhances upon treatment with lysophosphatidic acid, and this mechanical behavior follows a trend similar to Rho activity. Furthermore, the time-dependent activation of Rho decreases the degree of microheterogeneity of the cytoplasm. Our results reveal fundamental differences between intracellular elasticity and cellular tension and suggest a critical role for Rho kinase in the regulation of intracellular mechanics.     

Elements in the N-terminal signaling sequence that determine cytosolic topology of short-chain dehydrogenases/reductases. Studies with retinol dehydrogenase type 1 and cis-retinol/androgen dehydrogenase type 1

High affinity, retinoid-specific binding proteins chaperone retinoids to manage their transport and metabolism. Proposing mechanisms of retinoid transfer between these binding proteins and membrane-associated retinoid-metabolizing enzymes requires insight into enzyme topology. We therefore determined the topology of mouse retinol dehydrogenase type 1 (Rdh1) and cis-retinoid androgen dehydrogenase type 1 (Crad1) in the endoplasmic reticulum of intact mammalian cells. The properties of Rdh1 were compared with a chimera with a luminal signaling sequence (11beta-hydroxysteroid dehydrogenase (11beta-HSD1)(1-41)/Rdh1(23-317); the green fluorescent protein (GFP) fusion proteins Rdh1(1-22)/GFP, Crad1(1-22)/GFP, and 11beta-HSD1(1-41)/GFP; and signaling sequence charge difference mutants using confocal immunofluorescence, antibody access, proteinase K sensitivity, and deglycosylation assays. An N-terminal signaling sequence of 22 residues, consisting of a hydrophobic helix ending in a net positive charge, anchors Rdh1 and Crad1 in the endoplasmic reticulum facing the cytoplasm. Mutating arginine to glutamine in the signaling sequence did not affect topology. Inserting one or two arginine residues near the N terminus of the signaling sequence caused 28-95% inversion from cytoplasmic to luminal, depending on the net positive charge remaining at the C terminus of the signaling sequence; e.g. the mutant L3R,L5R,R16Q,R19Q,R21Q faced the lumen. Experiments with N- and C-terminal epitope-tagged Rdh1 and molecular modeling indicated that a hydrophobic helix-turn-helix near the C terminus of Rdh1 (residues 289-311) projects into the cytoplasm. These data provide insight into the features necessary to orient type III (reverse signal-anchor) proteins and demonstrate that Rdh1, Crad1, and other short-chain dehydrogenases/reductases, which share similar N-terminal signaling sequences such as human Rdh5 and mouse Rdh4, orient with their catalytic domains facing the cytoplasm.     

Mitochondrial DNA and prehistoric settlements: native migrations on the western edge of North America

We analyzed previously reported mtDNA haplogroup frequencies of 577 individuals and hypervariable segment 1 (HVS1) sequences of 265 individuals from Native American tribes in western North America to test hypotheses regarding the settlement of this region. These data were analyzed to determine whether Hokan and Penutian, two hypothesized ancient linguistic stocks, represent biological units as a result of shared ancestry within these respective groups. Although the pattern of mtDNA variation suggests regional continuity and although gene flow between populations has contributed much to the genetic landscape of western North America, some evidence supports the existence of both the Hokan and Penutian phyla. In addition, a comparison between coastal and inland populations along the west coast of North America suggests an ancient coastal migration to the New World. Similarly high levels of haplogroup A among coastal populations in the Northwest and along the California coast as well as shared HVS1 sequences indicate that early migrants to the New World settled along the coast with little gene flow into the interior valleys.