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At least 793 records · Page 44Linked to original sources
PubMed2005

Nrf2 controls constitutive and inducible expression of ARE-driven genes through a dynamic pathway involving nucleocytoplasmic shuttling by Keap1.

Nrf2 regulates the expression of genes encoding antioxidant proteins involved in cellular redox homeostasis. Previous studies have suggested that activation of Nrf2 is mediated by mechanisms promoting its dissociation from Keap1, a cytosolic repressor that acts to sequester the transcription factor in the cytoplasm. As a short-lived protein, Nrf2 is also activated by mechanisms leading to its stabilization in cells under stress, and recent evidence indicates that Keap1 has an active role in the control of its stability. In this report, using immunocytochemistry, cell fractionation, and chromatin immunoprecipitation analyses, we found that Nrf2 is primarily a nuclear protein and that it is expressed and recruited to the chromatin constitutively to drive basal gene expression. Furthermore, we found evidence indicating that Keap1 may repress Nrf2 activity by transiently shuttling into the nucleus to promote its ubiquitylation. The data suggested that the steady-state level of Nrf2 is maintained by a dynamic pathway that balances its constitutive expression with a Keap1-regulated degradation process downstream of its role as a transcriptional activator. We suggest that the stabilization of Nrf2 in cells under stress represents the central regulatory response mediated by mechanisms that interfere with its interaction with Keap1, leading to the induction of antioxidant enzymes important to maintain cellular redox homeostasis.

Active Transport, Cell Nucleus↗
PubMed2002

Sensitive and efficient detection of RB1 gene mutations enhances care for families with retinoblastoma.

Timely molecular diagnosis of RB1 mutations enables earlier treatment, lower risk, and better health outcomes for patients with retinoblastoma; empowers families to make informed family-planning decisions; and costs less than conventional surveillance. However, complexity has hindered clinical implementation of molecular diagnosis. The majority of RB1 mutations are unique and distributed throughout the RB1 gene, with no real hot spots. We devised a sensitive and efficient strategy to identify RB1 mutations that combines quantitative multiplex polymerase chain reaction (QM-PCR), double-exon sequencing, and promoter-targeted methylation-sensitive PCR. Optimization of test order by stochastic dynamic programming and the development of allele-specific PCR for four recurrent point mutations decreased the estimated turnaround time to <3 wk and decreased direct costs by one-third. The multistep method reported here detected 89% (199/224) of mutations in bilaterally affected probands and both mutant alleles in 84% (112/134) of tumors from unilaterally affected probands. For 23 of 27 exons and the promoter region, QM-PCR was a highly accurate measure of deletions and insertions (accuracy 95%). By revealing those family members who did not carry the mutation found in the related proband, molecular analysis enabled 97 at-risk children from 20 representative families to avoid 313 surveillance examinations under anesthetic and 852 clinic visits. The average savings in direct costs from clinical examinations avoided by children in these families substantially exceeded the cost of molecular testing. Moreover, health care savings continue to accrue, as children in succeeding generations avoid unnecessary repeated anaesthetics and examinations.

Algorithms↗
PubMed1998

Dynamics of the cell cycle engine: Cdk2-kinase and the transition into mitosis.

The autonomous cell divisions during the early development of Xenopus laevis believed to comprise a universal cell cycle engine. Recent experimental data indicates that the Cdk2-cyclin E kinase is required for the rapid divisions during Xenopus embryogenesis and that the complex is crucial for the transition into mitosis. In the present paper, the activity of Cdk2-cyclin E is incorporated into an existing comprehensive model of the cell cycle engine as an activity operating in parallel with the mitosis promotion factor (MPF) on the phosphatase Cdc25. This introduces interesting regulatory and dynamic properties for the transition into mitosis that reveals new insight into the mechanisms of the cell division process. It is shown that the Cdk2-cyclin E complex can act as an effective modulator of the threshold MPF activity needed to initiate mitosis. When the Cdk2-cyclin E activity is below a critical value, the cell cycle arrests in a well-defined state of low MPF activity corresponding to G2 arrest. In agreement with experiments a single mitotic event occurs following injection of free cyclin B. Above a critical activity, the presence of Cdk2-cyclin E allows for sustained oscillations corresponding to repeated cell divisions and the Cdk2-cyclin E may be the cause for the suppressed G2 checkpoint in the early embryonic cell cycles. A detailed bifurcation analysis reveals that the transition from steady to oscillatory behavior involves a homoclinic orbit of infinite period through an omega explosion. The general properties of the omega explosion explain the bifurcation as a dynamic mechanism well-suited for the G2 checkpoint and suggest a plausible explanation for the elongation of the cell cycle as observed at the mid-blastula transition. The proposed mechanism also suggests a plausible explanation of G2 checkpoint failure following DNA damage in human cells overexpressing Cdk2 and we suggest that the onset of mitosis in the mammalian cell occurs as the result of a slow passage through a critical point.

Animals↗
PubMed1989

Binding of phospholipase A2 to zwitterionic bilayers is promoted by lateral segregation of anionic amphiphiles.

Catalytic action of phospholipase A2 is appreciably influenced by the organization and dynamics of bilayers of glycerophosphocholines (Apitz-Castro et al. (1988) Biochim. Biophys. Acta 688, 341-348). However, such effects of the quality of the interface are not observed with bilayers of glycerophosphoryl methanol and other anionic phospholipids (Jain et al. (1986) Biochim. Biophys. Acta 860, 435-447). Such differences between the catalytic susceptibility of zwitterionic versus anionic bilayers are due to a large difference in the affinity of the enzyme for these interfaces. Binding to phospholipase A2 to zwitterionic interfaces can be promoted in the presence of certain anionic additives. For example in the pre-steady-state phase of hydrolysis, segregation of the nacently produced products of hydrolysis could promote binding of phospholipase A2 to regions of higher anionic charge density in the zwitterionic interface. In this paper we show that the dynamics of segregation of the nacently produced products of hydrolysis in zwitterionic bilayers can be readily followed by monitoring the fluorescence intensity of the cationic dye NK-529 (Yu and Jain (1989) Biochim. Biophys. Acta 980, 15-22). The fluorescence emission characteristics of NK-529 change appreciably due to self-quenching of the bound dye molecules as the fatty acid molecules segregate in the bilayer. The kinetics of segregation of fatty acids during the course of hydrolysis of bilayers of zwitterionic phospholipids by phospholipase A2 exhibits an unequivocal correlation with a variety of phenomena that are observed during the transition from the pre-steady-state phase to the steady-state phase of hydrolysis in the reaction progress curves as a function of temperature and in the presence of lipophilic additives.

Anions↗
PubMed2005

Transcription factor KLF7 is important for neuronal morphogenesis in selected regions of the nervous system.

The Krüppel-like transcription factors (KLFs) are important regulators of cell proliferation and differentiation in several different organ systems. The mouse Klf7 gene is strongly active in postmitotic neuroblasts of the developing nervous system, and the corresponding protein stimulates transcription of the cyclin-dependent kinase inhibitor p21waf/cip gene. Here we report that loss of KLF7 activity in mice leads to neonatal lethality and a complex phenotype which is associated with deficits in neurite outgrowth and axonal misprojection at selected anatomical locations of the nervous system. Affected axon pathways include those of the olfactory and visual systems, the cerebral cortex, and the hippocampus. In situ hybridizations and immunoblots correlated loss of KLF7 activity in the olfactory epithelium with significant downregulation of the p21waf/cip and p27kip1 genes. Cotransfection experiments extended the last finding by documenting KLF7's ability to transactivate a reporter gene construct driven by the proximal promoter of p27kip1. Consistent with emerging evidence for a role of Cip/Kip proteins in cytoskeletal dynamics, we also documented p21waf/cip and p27kip1 accumulation in the cytoplasm of differentiating olfactory sensory neurons. KLF7 activity might therefore control neuronal morphogenesis in part by optimizing the levels of molecules that promote axon outgrowth.

Animals↗
PubMed2006

Pharmaceutical promotion and GP prescription behaviour.

The aim of this paper is to empirically analyse the responses by general practitioners to promotional activities for ethical drugs by pharmaceutical companies. Promotion can be beneficial as a means of providing information, but it can also be harmful in the sense that it lowers price sensitivity of doctors and it merely is a means of maintaining market share, even when cheaper, therapeutically equivalent drugs are available. A model is estimated that includes interactions of promotion expenditures and prices and that explicitly exploits the panel structure of the data, allowing for drug specific effects and dynamic adjustments, or habit persistence. The data used are aggregate monthly GP prescriptions per drug together with monthly outlays on drug promotion for the period 1994-1999 for 11 therapeutic markets, covering more than half of the total prescription drug market in the Netherlands. Identification of price effects is aided by the introduction of the Pharmaceutical Prices Act, which established that Dutch drugs prices became a weighted average of the prices in surrounding countries after June 1996. We conclude that GP drug price sensitivity is small, but adversely affected by promotion. Ltd.

Drug Costs↗
PubMed2004

A beta-tubulin mutation selectively uncouples nuclear division and cytokinesis in Tetrahymena thermophila.

The ciliated protozoan Tetrahymena thermophila contains two distinct nuclei within a single cell-the mitotic micronucleus and the amitotic macronucleus. Although microtubules are required for proper division of both nuclei, macronuclear chromosomes lack centromeres and the role of microtubules in macronuclear division has not been established. Here we describe nuclear division defects in cells expressing a mutant beta-tubulin allele that confers hypersensitivity to the microtubule-stabilizing drug paclitaxel. Macronuclear division is profoundly affected by the btu1-1 (K350M) mutation, producing cells with widely variable DNA contents, including cells that lack macronuclei entirely. Protein expressed by the btu1-1 allele is dominant over wild-type protein expressed by the BTU2 locus. Normal macronuclear division is restored when the btu1-1 allele is inactivated by targeted disruption or expressed as a truncated protein. Immunofluorescence studies reveal elongated microtubular structures that surround macronuclei that fail to migrate to the cleavage furrows. In contrast, other cytoplasmic microtubule-dependent processes, such as cytokinesis, cortical patterning, and oral apparatus assembly, appear to be unaffected in the mutant. Micronuclear division is also perturbed in the K350M mutant, producing nuclei with elongated early-anaphase spindle configurations that persist well after the initiation of cytokinesis. The K350M mutation affects tubulin dynamics, as the macronuclear division defect is exacerbated by three treatments that promote microtubule polymerization: (i) elevated temperatures, (ii) sublethal concentrations of paclitaxel, and (iii) high concentrations of dimethyl sulfoxide. Inhibition of phosphatidylinositol 3-kinase (PI 3-kinase) with 3-methyladenine or wortmannin also induces amacronucleate cell formation in a btu1-1-dependent manner. Conversely, the myosin light chain kinase inhibitor ML-7 has no effect on nuclear division in the btu1-1 mutant strain. These findings provide new insights into microtubule dynamics and link the evolutionarily conserved PI 3-kinase signaling pathway to nuclear migration and/or division in Tetrahymena.

Alleles↗
PubMedDate not supplied

Short-term cell/substrate contact dynamics of subconfluent endothelial cells following exposure to laminar flow.

The manner in which fluid stresses are transmitted from the apical to the basal surface of the endothelium will influence the dynamics of cell/substrate contacts. Such dynamics could be important in the design of synthetic vascular grafts to promote endothelial cell adhesion. To examine whether the initial response of cell/substrate contact sites to flow depends on the magnitude of the applied shear stress, subconfluent monolayers of endothelial cells were exposed to flow at 10, 20, and 30 dyn cm-2 wall shear stresses for 20 min. Cell/substrate contact sites were visualized with total internal reflection fluorescence microscopy. Flow induced a rapid fluctuation in the membrane topography, which was reflected in dynamic changes in cell/substrate contacts. Exposure to flow caused marked changes in contact area. Contact movement occurred normal and parallel to the direction of flow. Contact sites demonstrated significant variability in contact area at 30 dyn cm-2 during the experiment but no significant movement of the contact sites in flow direction after 20 min of flow. Mean square displacements of the contact center of mass were described in terms of a directed diffusion model. Prior to onset of flow, contact movement was random. Flow induced a significant convective component to contact movement for 300-600 s, followed by reestablishment of diffusive growth and movement of contacts. These results suggest that fluid stresses are rapidly transmitted from the apical to the basal surface of the cell via the cytoskeleton.

Animals↗
PubMed2002

Dynamics of global histone acetylation and deacetylation in vivo: rapid restoration of normal histone acetylation status upon removal of activators and repressors.

DNA-binding activators and repressors recruit histone acetylases and deacetylases to promoters, thereby generating localized domains of modified histones that influence transcriptional activity. At the end of a transcriptional response, alterations in histone acetylation status are reversed, but the dynamics of this process are poorly understood. Here, we recruit histone deacetylases and acetylases to a well-defined yeast promoter in a regulated manner. Following dissociation of the recruiting protein from the promoter, targeted deacetylation and acetylation are reversed with rapid, yet distinct, kinetics. Reversal of targeted deacetylation occurs within 5-8 min, whereas reversal of targeted acetylation is more rapid, taking 1.5 min. These findings imply that untargeted, globally acting enzymes generate a highly dynamic equilibrium of histone acetylation and deacetylation reactions across chromatin. Targeted acetylases and deacetylases can locally perturb this equilibrium, yet once they are removed, the global activities mediate a rapid return to the steady-state level of histone acetylation. Our results also indicate that TBP occupancy depends on the presence of the activator, not histone acetylation status.

Acetylation↗
PubMed2000

Methylation patterns of the E-cadherin 5' CpG island are unstable and reflect the dynamic, heterogeneous loss of E-cadherin expression during metastatic progression.

Metastatic progression of most common epithelial tumors involves a heterogeneous, transient loss of expression of the homotypic cell adhesion protein, E-cadherin, rather than the uniform loss of a functional protein resulting from coding region mutation. Indeed, whereas E-cadherin loss may promote invasion, reexpression may facilitate cell survival within metastatic deposits. The mechanisms underlying such plasticity are unclear. We now show that the heterogeneous loss of E-cadherin expression in primary human breast cancers reflects a heterogeneous pattern of promoter region methylation, which begins early prior to invasion. In cultured human tumor cells, such heterogeneous methylation is dynamic, varying from allele to allele and shifting in relation to the tumor microenvironment. Following invasion in vitro, which favors diminished E-cadherin expression, the density of promoter methylation markedly increased. When these cells were cultured as spheroids, which requires homotypic cell adhesion, promoter methylation decreased dramatically, and E-cadherin was reexpressed. These data show that the methylation associated with E-cadherin loss in human breast cancer is heterogeneous and unstable and suggest that such epigenetic plasticity may contribute to the dynamic, phenotypic heterogeneity that drives metastatic progression.

Alleles↗
PubMedDate not supplied

Prevention and health promotion on the international scene: the need for a more effective and comprehensive approach.

The last decade has shown a boom in international networks and projects in health promotion and prevention. The growth of international collaboration is considered to be of crucial importance for the future development of the field. Several arguments are discussed in support of a further expansion of collaboration across countries and regions. The author criticizes the current lack of effectiveness in international collaboration in health promotion and prevention and identifies main barriers to effective collaboration. To make significant progress in research, evidence-based programs and policies, and their implementation worldwide, the creation of a comprehensive system of functional and effective linkages is needed across the core task areas, across disciplines, across system levels, and across countries and cultures. The quality of prevention and health promotion research as well as its impact on the health of communities depends on the degree to which research institutes and other stakeholders are embedded in an effective network of interorganizational and international relationships capable of performing a broad range of essential and functionally related tasks. It is critical that the dynamics of effective international collaboration become a legitimate subject of prevention and health promotion science.

Health Policy↗
PubMed2005

The in vitro regulation of ovarian follicle development using alginate-extracellular matrix gels.

The extracellular matrix (ECM) provides a three-dimensional structure that promotes and regulates cell adhesion and provides signals that direct the cellular processes leading to tissue development. In this report, synthetic matrices that present defined ECM components were employed to investigate these signaling effects on tissue formation using ovarian follicle maturation as a model system. In vitro systems for follicle culture are being developed to preserve fertility for women, and cultures were performed to test the hypothesis that the ECM regulates follicle maturation in a manner that is dependent on both the ECM identity and the stage of follicle development. Immature mouse follicles were cultured within alginate-based matrices that were modified with specific ECM components (e.g., laminin) or RGD peptides. The matrix maintains the in vivo like morphology of the follicle and provides an environment that supports follicle development. The ECM components signal the somatic cells of the follicle, affecting their growth and differentiation, and unexpectedly also affect the meiotic competence of the oocyte. These effects depend upon both the identity of the ECM components and the initial stage of the follicle, indicating that the ECM is a dynamic regulator of follicle development. The development of synthetic matrices that promote follicle maturation to produce meiotically competent oocytes may provide a mechanism to preserve fertility, or more generally, provide design principles for scaffold-based approaches to tissue engineering.

Alginates↗
PubMed2006

[Evaluation and health promotion in Brittany].

A specific context combined with national devices and mechanisms, which are developed on a regional level, has proven to be favourable to the development of health promotion evaluation. The study presented here retraces the evolution of how this question has been approached in Brittany through a review of the regional literature. This work offered a means by which project funders, decision-makers and grass-roots workers and professionals in the field could obtain a better perception of what issues were at stake, the challenges posed and perspectives to which the process adheres within the timeframe of commitment. Certain steps mark this process: the difficulty in creating awareness of the need for evaluation of health promotion following the whims upon which the development and formulation of projects are based; projects coming forward slowly but surely in support of health promotion development, of its effectiveness and which support the regional health policy dynamic. The expression of strong expectations which have been incessantly repeated emphasises the necessity and readiness for action today, and above all a joint willingness and responsibility of the professionals on the ground along with the decision-makers.

Community Networks↗
PubMed2001

Developing an ecosystem perspective from experimental monitoring programs: I. Demographic responses of a rare geothermal grass to soil temperature.

The geysers panic grass [Dichanthelium lanuginosum Spellenberg var. thermale (Bol.) Spellenberg or DILA] is exclusively associated with surface geothermal manifestations in Sonoma County, California, USA (38 degrees 46'N, 122 degrees 38'W). Steam extraction by power plants could alter the subsurface distribution of heat and water to the site, potentially impacting subpopulations of this rare plant. The purpose of this study was to use demographic monitoring to determine: (1) temporal and spatial patterns of soil temperature in relation to the distribution of established DILA individuals at Little Geysers, (2) in situ response of experimental populations of DILA to spatial variations in soil temperature, and (3) habitat requirements of DILA as an indicator of its tolerance to variations in surficial geothermal features. Thermocouple transects and a datalogger provided data for characterizing the spatial and temporal patterns of soil temperature in four microhabitats (fumarole, DILA stand, Andropogon stand, and cleared). Experimental populations were established by precisely sowing and monitoring DILA seeds in these microhabitats. The results indicated that spatial and temporal variations in soil temperature had significant effects on the processes of germination, growth, survivorship, and reproduction, thus producing a readily observed metapopulation patch dynamic in relation to geothermal activity. Seasonal depressions of soil temperature near the fumaroles by cold air and prolonged rainfall events also promoted the emergence and survival of DILA seedlings in a microhabitat that was previously too hot to occupy. Over longer periods of time, DILA metapopulation dynamism reflected climatic and geothermal variation. Drought years inhibited germination for lack of water, but more importantly for the lack of requisite soil temperature depressions in the fumarole microhabitat. Wet years promoted subpopulation expansion into transition areas that were once too hot and dry. There have also been shifts in the underground distribution of steam into areas distant from known geothermal features. The demographic responses of DILA to spatial and temporal variations in soil temperature indicate that heat is an absolutely essential component of the steam resource. In its absence, germination, seeding survivorship, growth, and maturation are significantly inhibited even if soil conditions are favorable and potential competitors are controlled. Ultimately, persistence of the species depends on maintaining the ecosystem dynamic of colonization and extirpation in response to variations in surficial geothermal features over long spatial and temporal scales. This should shift management perspective from its narrow focus on individual plants to a wider focus on monitoring the essential habitat component of steam.

Conservation of Natural Resources↗
PubMed2006

Regulation of cartilaginous ECM gene transcription by chondrocytes and MSCs in 3D culture in response to dynamic loading.

This study explored the biologic response of chondrocytes and mesenchymal stem cells (MSCs) to a dynamic mechanical loading regime. We developed a time-efficient methodology for monitoring regional changes in extracellular matrix gene transcription using reporter promoter constructs. Specifically, transfected cells were homogenously distributed throughout agarose hydrogel constructs, and spatial and temporal gene expression and the ability to form functional ECM were analyzed in response to dynamic mechanical stimuli. Theoretical analyses were used to predict the physical signals generated within the gel in response to these loading regimes. Using a custom compression bioreactor system, changes in aggrecan and type II collagen promoter activity in transfected chondrocyte-laden cylindrical constructs were evaluated in response to a range of loading frequencies and durations. In general, aggrecan promoter activity increased with increasing duration of loading, particularly in the outer annulus region. Interestingly, type II collagen promoter activity decreased in this annular region under identical loading conditions. In addition, we explored the role of mechanical compression in directing chondrogenic differentiation of MSCs by monitoring short-term aggrecan promoter activity. As an example of long-term utility, a specific loading protocol was applied to MSC-laden constructs for 5 days, and the resultant changes in glycosaminoglycan (GAG) production were evaluated over a 4-week period. This dynamic loading regime increased not only short-term aggrecan transcriptional activity but also GAG deposition in long-term culture. These results demonstrate the utility of a new reporter promoter system for optimizing loading protocols to improve the outcome of engineered chondrocyte- and MSC-laden cartilaginous constructs.

Aggrecans↗
PubMed2006

Dynamic tradeoffs in the raft-mediated entry of human immunodeficiency virus type 1 into cells.

To initiate an infection human immunodeficiency virus type 1 (HIV-1) particles must first bind to receptors on the surface of their host cells, a process that eventually leads to fusion of viral and cellular membranes and release of the viral genome into the cytoplasm. Understanding the molecular mechanisms of these processes may enable the development of new anti-HIV strategies. Disagreement currently prevails on the role in virus entry of microdomains within the cellular plasma membrane known as lipid rafts. Experiments have suggested that lipid rafts, in their interactions with cellular receptors and viral particles, either promote or have minimal effect on viral entry. Here we develop a dynamic model for HIV-1 entry that enables us to identify and quantitatively assess tradeoffs that can arise from the clustering of receptors in rafts. Specifically, receptor clustering can be detrimental to the initiation of viral infection by reducing the probability that a virus particle finds its primary receptor, CD4. However, receptor clustering can also enable a virus particle, once bound, to rapidly form multivalent interactions with receptors and co-receptors that are required for virus-cell membrane fusion. We show how the resolution of such tradeoffs hinges on the level and spatial distribution of receptors and co-receptors on the cell surface, and we discuss implications of these effects for the design of therapeutics that inhibit HIV-1 entry.

CD4 Antigens↗
PubMed2006

Role for CED-9 and Egl-1 as regulators of mitochondrial fission and fusion dynamics.

Bcl-2 family proteins play central roles in apoptosis by regulating the release of mitochondrial intermembrane space proteins such as cytochrome c. Death-promoting Bcl-2 family members, such as Bax, can promote cytochrome c release and fragmentation of the mitochondrial network, whereas apoptosis-inhibitory members, such as Bcl-2 and Bcl-xL, can antagonize these events. It remains unclear whether CED-9, the worm Bcl-2 relative, can regulate mitochondrial fission/fusion dynamics or the release of proteins from the mitochondrial intermembrane space. Here, we show that CED-9 interacts with Mitofusin-2/fuzzy onions and can promote mitochondrial clustering and dramatic reorganization of mitochondrial networks. Consistent with its ability to neutralize CED-9 function, EGL-1 antagonized CED-9-dependent remodeling of the mitochondrial network. However, CED-9 failed to inhibit mitochondrial cytochrome c release or apoptosis induced by diverse triggers in mammalian cells. These data suggest that the ability to regulate mitochondrial fission/fusion dynamics is an evolutionarily conserved property of the Bcl-2 family.

Animals↗