ATP-induced calcium mobilization and inositol 1,4,5-trisphosphate formation in H-35 hepatoma cells

AbstractAddition of ATP (but not epinephrine, angiotensin II, vasopressin, or platelet-activating factor) to H-35 hepatoma cells whose cellular lipids have been pre-labelled with [3H]inositol, causes a rapid increase in [3H]inositol trisphosphate. In H-35 cells pre-incubated in the presence of 45Ca2+, ATP causes a similarly rapid release of 45Ca2+. The concentration-effect relationships for inositol trisphosphate formation and Ca2+ efflux are similar to those reported previously for differentiated hepatocytes. These results demonstrate that at least one of the Ca2+-mobilizing receptors normally found on hepatocytes is functionally retained in the H-35 hepatoma cell line and thus could provide a useful model for the study of these receptor mechanisms in liver

BABY BOOM-induced somatic embryogenesis in Arabidopsis

Under appropriate tissue culture conditions, somatic plant cells can be induced to form embryos in a process called somatic embryogenesis (SE). SE provides a way to clonally propagate desirable plants and is therefore an important plant breeding tool. SE has also fascinated scientists for decades as an expression of plant ‘totipotency’, the ability to regenerate a whole new individual through embryogenesis. This thesis aims to obtain a deeper understanding of somatic embryo induction in Arabidopsis by the transcription factor BABY BOOM (BBM), through identification and functional analysis of BBM-binding proteins and BBM target genes. Chapter 1 introduces the concept of somatic embryogenesis, describes the different SE systems in Arabidopsis, and discusses the role of the plant hormone auxin and chromatin modifying proteins in this process. An overview is presented on the current knowledge on SE-induction through ectopic overexpression of certain transcription factor genes. These include BBM, as well as other genes that are studied in this thesis in relation to BBM. BBM is part of the eight member AIL subfamily of AP2/ERF domain transcription factors. Chapter 2 reviews the role of AIL proteins during embryogenesis, stem cell niche specification, meristem maintenance and organ positioning and growth. We summarize the gene regulatory networks in which AILs function and describe how these transcription factors integrate multiple hormonal inputs, with special emphasis on the interactions between AILs and auxin. Finally, we conclude that although the functions of AILs in plant development are well described, knowledge on the molecular mode of action of AIL proteins and the identity of AIL target genes is still limited. Transcription factors function in protein complexes and in Chapter 3 we show that members of the HOMEODOMAIN GLABROUS (HDG) transcription factor family physically interact with BBM and other AILs. HDG genes are expressed in the epidermis, the outer cell layer of the plant, where they promote differentiation of cells into specialized epidermal cell types, such as trichomes or stomata. We show that ectopic overexpression of HDG1 leads to loss of root and shoot meristems, phenotypes that had previously been reported for loss-of-function ail mutants. Conversely, down-regulation of HDG genes led to reduced cell differentiation, enhanced cell proliferation and SE phenotypes, phenotypes that resemble those found in AIL overexpression lines. Moreover, we found that co-overexpression of BBM and HDG1 reduces the overexpression phenotypes of both proteins. These results suggest opposite functions of AIL and HDG transcription factors, with AILs stimulating cell proliferation and HDGs stimulating cell differentiation, with the ratio between the two proteins determining the developmental outcome. Finally, we show that HDGs and AILs regulate each other on a transcriptional level and that they share common target genes. A variety of AIL overexpression phenotypes has been described in the literature, with BBM and PLT5/AIL5 being the only known AILs that induce SE upon overexpression. We show in Chapter 4 that all AIL proteins except AIL1 and ANT are able to induce SE, but that this phenotype relies on a high AIL protein dosage. Using BBM and PLT2 as AIL representatives, we show that an intermediate AIL concentration induces organogenesis (ectopic root and shoot formation) and that a low concentration inhibits cellular differentiation. In addition, we show that BBM and PLT2 induce direct SE when activated at seed germination, while post-germination activation leads to indirect SE from callus. The LEAFY COTYLEDON (LEC)/LAFL genes, which also encode SE-inducing transcription factors, are direct targets of BBM/PLT2 during direct SE, showing that these two SE pathways are linked. Using LAFL gene mutants, we show that the LAFL pathway is an important downstream component of BBM-mediated SE. Chapter 5 presents the in vivo, genome-wide analysis of BBM DNA binding sites in somatic embryos using chromatin immunoprecipitation followed by sequencing (ChIP-seq). Our ChIP-seq and gene expression analysis reveal that BBM binds and positively regulates auxin biosynthesis genes and the recently discovered positive regulators of SE, the AT-HOOK MOTIF CONTAINING NUCLEAR LOCALIZED (AHL) genes. Knock-out of either pathway reduced BBM-mediated SE, showing that auxin biosynthesis and the AHL genes are important components of the BBM pathway. We also show that BBM binds to a consensus DNA motif that resembles the reported ANT binding motif. Chapter 6 reviews methods for identifying the direct target genes of a plant transcription factor using microarrays, as was done for HDG1 (Chapter 4). We describe which different systems can be used to control transcription factor activity, and how these can be combined with microarray analysis to identify target genes. In addition, we provide guidelines for the statistical analysis of microarray data and for the confirmation of candidate target genes. In plant biology, protein-protein interactions are often studied using bimolecular fluorescence complementation (BiFC) or split-YFP. In my BBM-HDG interaction studies I encountered problems using this method, which lead to the cautionary note on the use of BiFC presented in Chapter 7. BiFC is based on the restoration of fluorescence after the two non-fluorescent halves of a fluorescent protein are brought together by a protein-protein interaction event. However, because the fluorescent protein halves are prone to self-assembly, it is crucial to use proper controls and a quantitative read-out of fluorescence to avoid false positive interactions. We present a guideline for the setup of a BiFC experiment, discussing each step in the protocol. Chapter 8 discusses how the results presented in this thesis contribute to our knowledge on AIL transcription factors and somatic embryo induction, as well as the questions that still remain. An extended model of dose-dependent AIL function is proposed, as well as mechanisms by which the AIL-HDG interaction could function at the molecular level. Finally, an overview is provided of the molecular-genetic intersection between the different transcription factor-induced SE pathways. </p

Comparison Between Experiment and Prediction for a Transonic Turbulent Separated Flow

Solutions of the time-dependent, mass-averaged Navier-Stokes equations are compared In detail with experimental results obtained on an axisymmetric "bump" model at a transonic Mach number that produced an extensive separated now region. In addition, an inverse boundary method is evaluated for this type of flow. The Cebeci-Smith algebraic and the Wilcox-Rubesin two-equation turbulence models used in the Navier-Stokes calculations both predict the maximum boundary-layer displacement thickness generated by the interaction reasonably well, with the details of the now best described with the two-equation formulation. However, both models predict a shock location substantially farther aft on the bump than observed experimentally. This error in shock location was slightly less with the two-equation model (0.12 chord compared with 0.16 chord). In the vicinity of the shock, the calculations predict a more rapid increase in turbulent shear stress than observed in the experimental results; this more rapid increase is believed to be the cause or the poor predictions in shock position

Model versus nature: Hydrodynamics in mangrove pneumatophores

Water flows through submerged and emergent vegetation control the transport and deposition of sediment in coastal wetlands. Many past studies into the hydrodynamics of vegetation fields have used idealized vegetation mimics, mostly rigid dowels of uniform height. In this study, a canopy of real mangrove pneumatophores was reconstructed in a flume to quantify flow and turbulence within and above this canopy. At a constant flow forcing, an increase in pneumatophore density, from 71 m⁻² to 268 m⁻², was found to cause a reduction of the within-canopy flow velocities, whereas the over-canopy flows increased. Within-canopy velocities reduced to 46% and 27% of the free-stream velocities for the lowest and highest pneumatophore densities, respectively, resulting in stronger vertical shear and hence greater turbulence production around the top of the denser pneumatophore canopies. The maximum Reynolds stress was observed at 1.5 times the average pneumatophore height, in contrast to uniform-height canopies, in which the maximum occurs at approximately the height of the vegetation. The ratios of the within-canopy velocity to the free-stream velocity for the pneumatophores were found to be similar to previous observations with uniform-height vegetation mimics for the same vegetation densities. However, maxima of the scaled friction velocity were two times smaller over the real pneumatophore canopies than for idealized dowel canopies, due to the reduced velocity gradients over the variable-height pneumatophores compared to uniform-height dowels. These findings imply that results from previous studies with idealized and uniform vegetation mimics may have limited application when considering sediment transport and deposition in real vegetation, as the observed turbulence characteristics in nonuniform canopies deviate significantly from those in dowel canopies

Triazine Herbicide Exposure and Breast Cancer Incidence: An Ecologic Study of Kentucky Counties

The incidence of breast cancer in the United States has steadily increased for the past three decades. Exposure to excess estrogen, in both natural and synthetic forms, has been implicated as a risk factor for the development of this disease. Considerable interest has been focused on organochlorines, such as the triazine herbicides, and their possible role in the initiation or promotion of human breast cancer. To explore this relationship, an ecologic study of Kentucky counties was designed. Exposure to triazines was estimated by use of water contamination data, corn crop production, and pesticide use data. A summary index of triazine herbicide exposure was developed to classify counties into low, medium, or high exposure levels. Data on county breast cancer rates were obtained from the state registry. A Poisson regression analysis was performed, controlling for age, race, age at first live birth, income, and level of education. Results revealed a statistically significant increase in breast cancer risk with medium and high levels of triazine exposure [odds ratio (OR) = 1.14,p\u3c0.0001 and OR = 1.2, p\u3c0.0001, respectively]. The results suggest a relationship between exposure to triazine herbicides and increased breast cancer risk, but conclusions concerning causality cannot be drawn, due to the limitations inherent in ecologic study design

Evidence of platelet activation in multiple sclerosis

This is an Open Access article distributed under the terms of the Creative Commons Attribution Licens

US Air Force Materiel Command`s second round ECAMP results

The United States Air Force`s Environmental Compliance Assessment and Management Program (ECAMP) is a process to improve Air Force environmental compliance, management, and programmatic support. Midway into the third round of Command-initiated (external) ECAMP evaluations for its fourteen Air Logistics and Systems Centers, the Air Force Material Command (AFMC) is continuing to identify the root causes of instances of environmental noncompliance. The AFMC initiated an analysis of all negative findings identified during the second round of external ECAMP evaluations (June 1991 to January 1993). Presented here is a summary of the analysis with emphasis on trends and root causes