Creating a dynamic picture of the sliding clamp during T4 DNA polymerase holoenzyme assembly by using fluorescence resonance energy transfer

The coordinated assembly of the DNA polymerase (gp43), the sliding clamp (gp45), and the clamp loader (gp44/62) to form the bacteriophage T4 DNA polymerase holoenzyme is a multistep process. A partially opened toroid-shaped gp45 is loaded around DNA by gp44/62 in an ATP-dependent manner. Gp43 binds to this complex to generate the holoenzyme in which gp45 acts to topologically link gp43 to DNA, effectively increasing the processivity of DNA replication. Stopped-flow fluorescence resonance energy transfer was used to investigate the opening and closing of the gp45 ring during holoenzyme assembly. By using two site-specific mutants of gp45 along with a previously characterized gp45 mutant, we tracked changes in distances across the gp45 subunit interface through seven conformational changes associated with holoenzyme assembly. Initially, gp45 is partially open within the plane of the ring at one of the three subunit interfaces. On addition of gp44/62 and ATP, this interface of gp45 opens further in-plane through the hydrolysis of ATP. Addition of DNA and hydrolysis of ATP close gp45 in an out-of-plane conformation. The final holoenzyme is formed by the addition of gp43, which causes gp45 to close further in plane, leaving the subunit interface open slightly. This open interface of gp45 in the final holoenzyme state is proposed to interact with the C-terminal tail of gp43, providing a point of contact between gp45 and gp43. This study further defines the dynamic process of bacteriophage T4 polymerase holoenzyme assembly.

In vitro characterization of mutant yeast RNA polymerase II with reduced binding for elongation factor TFIIS.

We have reported previously the isolation and genetic characterization of mutations in the gene encoding the largest subunit of yeast RNA polymerase II (RNAPII), which lead to 6-azauracil (6AU)-sensitive growth. It was suggested that these mutations affect the functional interaction between RNAPII and transcription-elongation factor TFIIS because the 6AU-sensitive phenotype of the mutant strains was similar to that of a strain defective in the production of TFIIS and can be suppressed by increasing the dosage of the yeast TFIIS-encoding gene, PPR2, RNAPIIs were purified and characterized from two independent 6AU-sensitive yeast mutants and from wild-type (wt) cells. In vitro, in the absence of TFIIS, the purified wt polymerase and the two mutant polymerases showed similar specific activity in polymerization, readthrough at intrinsic transcriptional arrest sites and nascent RNA cleavage. In contrast to the wt polymerase, both mutant polymerases were not stimulated by the addition of a 3-fold molar excess of TFIIS in assays of promoter-independent transcription, readthrough or cleavage. However, stimulation of the ability of the mutant RNAPIIs to cleave nascent RNA and to read through intrinsic arrest sites was observed at TFIIS:RNAPII molar ratios greater than 600:1. Consistent with these findings, the binding affinity of the mutant polymerases for TFIIS was found to be reduced by more than 50-fold compared with that of the wt enzyme. These studies demonstrate that TFIIS has an important role in the regulation of transcription by yeast RNAPII and identify a possible binding site for TFIIS on RNAPII.

A dynamic assembly of diverse transcription factors integrates activation and cell-type information for interleukin 2 gene regulation.

The interleukin 2 (IL-2) gene is subject to two types of regulation: its expression is T-lymphocyte-specific and it is acutely dependent on specific activation signals. The IL-2 transcriptional apparatus integrates multiple types of biochemical information in determining whether or not the gene will be expressed, using multiple diverse transcription factors that are each optimally activated or inhibited by different signaling pathways. When activation of one or two of these factors is blocked IL-2 expression is completely inhibited. The inability of the other, unaffected factors to work is explained by the striking finding that none of the factors interacts stably with its target site in the IL-2 enhancer unless all the factors are present. Coordinate occupancy of all the sites in the minimal enhancer is apparently maintained by continuous assembly and disassembly cycles that respond to the instantaneous levels of each factor in the nuclear compartment. In addition, the minimal enhancer undergoes specific increases in DNase I accessibility, consistent with dramatic changes in chromatin structure upon activation. Still to be resolved is what interaction(s) conveys T-lineage specificity. In the absence of activating signals, the minimal IL-2 enhancer region in mature T cells is apparently unoccupied, exactly as in non-T lineage cells. However, in a conserved but poorly studied upstream region, we have now mapped several novel sites of DNase I hypersensitivity in vivo that constitutively distinguish IL-2 producer type T cells from cell types that cannot express IL-2. Thus a distinct domain of the IL-2 regulatory sequence may contain sites for competence- or lineage-marking protein contacts.

Overexpression of the transcription factor UBF1 is sufficient to increase ribosomal DNA transcription in neonatal cardiomyocytes: implications for cardiac hypertrophy.

The accelerated protein accumulation characteristic of cardiomyocyte hypertrophy results from increased cellular protein synthetic capacity (elevated ribosome content). The rate limiting step in ribosome accumulation is transcription of the rRNA genes. During neonatal cardiomyocyte hypertrophy induced by norepinephrine or spontaneous contraction, changes in the expression of a ribosomal DNA transcription factor, UBF, correlated with increased rates of ribosome biogenesis. We hypothesized that elevated expression of UBF was part of the mechanism by which these hypertrophic stimuli effected increases in the rate of transcription from the rDNA promoter. In this study, we have examined directly the effect of overexpressing UBF on rDNA transcription in neonatal cardiomyocytes in culture. In control experiments, a novel reporter construct for rDNA transcription (pSMECAT) showed similar increases in activity in response to hypertrophic stimuli (10(-4) M phenylephrine, 10(-7) M endothelin, and spontaneous contraction) as did the endogenous rRNA genes. When contraction-arrested cardiomyocytes were cotransfected with pSMECAT and increasing amounts of a UBF1 expression vector; a dose-dependent (3-5 fold) increase in rDNA transcription was observed. Western blot analysis confirmed that the overexpressed, FLAG-tagged UBF accumulated in the cardiomyocyte nuclei. The observation that overexpression of UBF1 is sufficient to increase rDNA transcription in neonatal cardiomyocytes provides evidence in support of the hypothesis that the regulation of UBF is a key component of the increased ribosome biogenesis and protein accumulation associated with cardiomyocyte hypertrophy.

Dynamic contrast-enhanced magnetic resonance imaging reveals stress-induced angiogenesis in MCF7 human breast tumors.

The mechanism of contrast enhancement of tumors using magnetic resonance imaging was investigated in MCF7 human breast cancer implanted in nude mice. Dynamic contrast-enhanced images recorded at high spatial resolution were analyzed by an image analysis method based on a physiological model, which included the blood circulation, the tumor, the remaining tissues, and clearance via the kidneys. This analysis enabled us to map in rapidly enhancing regions within the tumor, the capillary permeability factor (capillary permeability times surface area per voxel volume) and the fraction of leakage space. Correlation of these maps with T2-weighted spin echo images, with histopathology, and with immunohistochemical staining of endothelial cells demonstrated the presence of dense permeable microcapillaries in the tumor periphery and in intratumoral regions that surrounded necrotic loci. The high leakage from the intratumoral permeable capillaries indicated an induction of a specific angiogenic process associated with stress conditions that cause necrosis. This induction was augmented in tumors responding to tamoxifen treatment. Determination of the distribution and extent of this stress-induced angiogenic activity by contrast-enhanced MRI might be of diagnostic and of prognostic value.

Dynamic friction and the origin of the complexity of earthquake sources.

We study a simple antiplane fault of finite length embedded in a homogeneous isotropic elastic solid to understand the origin of seismic source heterogeneity in the presence of nonlinear rate- and state-dependent friction. All the mechanical properties of the medium and friction are assumed homogeneous. Friction includes a characteristic length that is longer than the grid size so that our models have a well-defined continuum limit. Starting from a heterogeneous initial stress distribution, we apply a slowly increasing uniform stress load far from the fault and we simulate the seismicity for a few 1000 events. The style of seismicity produced by this model is determined by a control parameter associated with the degree of rate dependence of friction. For classical friction models with rate-independent friction, no complexity appears and seismicity is perfectly periodic. For weakly rate-dependent friction, large ruptures are still periodic, but small seismicity becomes increasingly nonstationary. When friction is highly rate-dependent, seismicity becomes nonperiodic and ruptures of all sizes occur inside the fault. Highly rate-dependent friction destabilizes the healing process producing premature healing of slip and partial stress drop. Partial stress drop produces large variations in the state of stress that in turn produce earthquakes of different sizes. Similar results have been found by other authors using the Burridge and Knopoff model. We conjecture that all models in which static stress drop is only a fraction of the dynamic stress drop produce stress heterogeneity.

Ligand occupancy of the alpha-V-beta3 integrin is necessary for smooth muscle cells to migrate in response to insulin-like growth factor.

Smooth muscle cells (SMCs) have been shown to migrate in response to insulin-like growth factor I (IGF-I). However, the mechanism mediating this response has not been determined. The migration rates of porcine and human vascular SMCs were assessed in a monolayer wounding assay. IGF-I and IGF-II induced increases of 141% and 97%, respectively, in the number of cells that migrated in 4 days. The presence of 0.2% fetal bovine serum in the culture medium was necessary for the IGFs to stimulate migration over uncoated plastic surfaces. However, if vitronectin was used as the substratum, IGF-I stimulated migration by 162% even in the absence of serum. To determine the role of integrins in mediating this migration, SMC surface proteins were labeled with 125I and immunoprecipitated with specific anti-integrin antibodies. Integrins containing alpha-V (vitronectin receptor), alpha5 (fibronectin receptor), and alpha3 (collagen/laminin receptor) subunits were the most abundant. IGF-I treatment caused a 73% reduction in alpha5-integrin subunit protein and a 25% increase in alpha-V subunit. More importantly, ligand binding of alpha-V-beta3 was increased by 2.4-fold. We therefore examined whether the function of the alpha-V-beta3 integrin was important for IGF-I-mediated migration. The disintegrin kistrin was shown by affinity crosslinking to specifically bind with high affinity to alpha-V-beta3 and not to alpha5-beta1 or other abundant integrins. The related disintegrin echistatin specifically inhibited 125I-labeled kistrin binding to alpha-V-beta3, while a structurally distinct disintegrin, decorsin, had 1000-fold lower affinity. The addition of increasing concentrations of either kistrin or echistatin inhibited IGF-I-induced migration, whereas decorsin had a minimal effect. The potency of these disintegrins in inhibiting IGF-I-induced migration paralleled their apparent affinity for the alpha-V integrin. Furthermore, an alpha-V-beta3 blocking antibody inhibited SMC migration by 80%. In summary, vitronectin receptor activation is a necessary component of IGF-I-mediated stimulation of smooth muscle migration, and alpha-V-beta3 integrin antagonists appear to be important reagents for modulating this process.

Persistent elevations of cerebrospinal fluid concentrations of corticotropin-releasing factor in adult nonhuman primates exposed to early-life stressors: implications for the pathophysiology of mood and anxiety disorders.

There is increasing evidence for an important role of adverse early experience on the development of major psychiatric disorders in adulthood. Corticotropin-releasing factor (CRF), an endogenous neuropeptide, is the primary physiological regulator of the mammalian stress response. Grown nonhuman primates who were exposed as infants to adverse early rearing conditions were studied to determine if long-term alterations of CRF neuronal systems had occurred following the early stressor. In comparison to monkeys reared by mothers foraging under predictable conditions, infant monkeys raised by mothers foraging under unpredictable conditions exhibited persistently elevated cerebrospinal fluid (CSF) concentrations of CRF. Because hyperactivity of CRF-releasing neurons has been implicated in the pathophysiology of certain human affective and anxiety disorders, the present finding provides a potential neurobiological mechanism by which early-life stressors may contribute to adult psychopathology.

Insulin-like growth factor II mediates epidermal growth factor-induced mitogenesis in cervical cancer cells.

There is increasing evidence that activation of the insulin-like growth factor I (IGF-I) receptor plays a major role in the control of cellular proliferation of many cell types. We studied the mitogenic effects of IGF-I, IGF-II, and epidermal growth factor (EGF) on growth-arrested HT-3 cells, a human cervical cancer cell line. All three growth factors promoted dose-dependent increases in cell proliferation. In untransformed cells, EGF usually requires stimulation by a "progression" factor such as IGF-I, IGF-II, or insulin (in supraphysiologic concentrations) in order to exert a mitogenic effect. Accordingly, we investigated whether an autocrine pathway involving IGF-I or IGF-II participated in the EGF-induced mitogenesis of HT-3 cells. With the RNase protection assay, IGF-I mRNA was not detected. However, IGF-II mRNA increased in a time-dependent manner following EGF stimulation. The EGF-induced mitogenesis was abrogated in a dose-dependent manner by IGF-binding protein 5 (IGFBP-5), which binds to IGF-II and neutralizes it. An antisense oligonucleotide to IGF-II also inhibited the proliferative response to EGF. In addition, prolonged, but not short-term, stimulation with EGF resulted in autophosphorylation of the IGF-I receptor, and coincubations with both EGF and IGFBP-5 attenuated this effect. These data demonstrate that autocrine secretion of IGF-II in HT-3 cervical cancer cells can participate in EGF-induced mitogenesis and suggest that autocrine signals involving the IGF-I receptor occur "downstream" of competence growth factor receptors such as the EGF receptor.

Identification of a putative estrogen response element in the gene encoding brain-derived neurotrophic factor.

We have been studying the role and mechanism of estrogen action in the survival and differentiation of neurons in the basal forebrain and its targets in the cerebral cortex, hippocampus, and olfactory bulb. Previous work has shown that estrogen-target neurons in these regions widely coexpress the mRNAs for the neurotrophin ligands and their receptors, suggesting a potential substrate for estrogen-neurotrophin interactions. Subsequent work indicated that estrogen regulates the expression of two neurotrophin receptor mRNAs in prototypic peripheral neural targets of nerve growth factor. We report herein that the gene encoding the neurotophin brain-derived neurotrophic factor (BDNF) contains a sequence similar to the canonical estrogen response element found in estrogen-target genes. Gel shift and DNA footprinting assays indicate that estrogen receptor-ligand complexes bind to this sequence in the BDNF gene. In vivo, BDNF mRNA was rapidly up-regulated in the cerebral cortex and the olfactory bulb of ovariectomized animals exposed to estrogen. These data suggest that estrogen may regulate BDNF transcription, supporting our hypothesis that estrogen may be in a position to influence neurotrophin-mediated cell functioning, by increasing the availability of specific neurotrophins in forebrain neurons.

cAMP- and rapamycin-sensitive regulation of the association of eukaryotic initiation factor 4E and the translational regulator PHAS-I in aortic smooth muscle cells.

Incubating rat aortic smooth muscle cells with either platelet-derived growth factor BB (PDGF) or insulin-like growth factor I (IGF-I) increased the phosphorylation of PHAS-I, an inhibitor of the mRNA cap binding protein, eukaryotic initiation factor (eIF) 4E. Phosphorylation of PHAS-I promoted dissociation of the PHAS-I-eIF-4E complex, an effect that could partly explain the stimulation of protein synthesis by the two growth factors. Increasing cAMP with forskolin decreased PHAS-I phosphorylation and markedly increased the amount of eIF-4E bound to PHAS-I, effects consistent with an action of cAMP to inhibit protein synthesis. Both PDGF and IGF-I activated p70S6K, but only PDGF increased mitogen-activated protein kinase activity. Forskolin decreased by 50% the effect of PDGF on increasing p70S6K, and forskolin abolished the effect of IGF-I on the kinase. The effects of PDGF and IGF-I on increasing PHAS-I phosphorylation, on dissociating the PHAS-I-eIF-4E complex, and on increasing p70S6K were abolished by rapamycin. The results indicate that IGF-I and PDGF increase PHAS-I phosphorylation in smooth muscle cells by the same rapamycin-sensitive pathway that leads to activation of p70S6K.

Instabilidades MHD no Tokamak TCABR

Este trabalho descreve o estudo das instabilidades magneto-hidro-dinâmicas (MHD) comumente observadas nas descargas elétricas de plasma no tokamak TCABR, do Instituto de Física da USP. Dois diagnósticos principais foram empregados para observar essas instabilidades: um conjunto poloidal de 24 bobinas magnéticas (bobinas de Mirnov) colocadas próximas à borda do plasma e um medidor de emissões na faixa do Ultra Violeta e de raios X moles com 20 canais (sistema SXR), cujo circuito de condicionamento de sinais foi aprimorado como parte deste trabalho. Esses diagnósticos foram escolhidos porque fornecem informações complementares, uma vez que o sistema SXR observa a parte central da coluna de plasma, enquanto as bobinas de Mirnov detectam as instabilidades MHD na região mais externa da coluna. As informações coletadas por esses diagnósticos foram submetidas à análise espectral com resolução temporal e espacial, possibilitando determinar a evolução das características espectrais e espaciais das instabilidades MHD observadas. Essas análises revelaram que durante a etapa inicial da formação do plasma (quando a corrente de plasma ainda está aumentando) ilhas magnéticas com números de onda decrescente, identificadas como sendo modos kink de borda, são detectadas nas bobinas de Mirnov. Após a formação do plasma, quando os parâmetros de equilíbrio estão relativamente estáveis (platô), oscilações são detectadas tanto nas bobinas de Mirnov quanto no sistema de SXR, indicando a presença de instabilidades MHD em toda a coluna de plasma. Em geral as oscilações medidas nas bobinas de Mirnov tem baixa amplitude e correspondem a pequenas ilhas magnéticas que foram identificadas como sendo modos de ruptura (modos tearing). Por outro lado, as instabilidades na região central foram identificadas como dentes de serra, que correspondem a relaxações periódicas da região interna à superfície magnética com fator de segurança q=1 e que são acompanhadas de oscilações precursoras, cuja amplitude depende da fase do ciclo de relaxação. Devido à essa modulação de amplitude, aparecem picos de frequência satélite nos espectrogramas dos sinais do SXR. Além disso, devido ao fato dos ciclos de relaxação não serem sinusoidais, os harmônicos da frequência de relaxação também aparecem nesses espectrogramas. No entanto, em muitas descargas do TCABR, a intensidade das oscilações medidas nas bobinas de Mirnov aumentam significativamente durante o platô, com efeitos sobre a frequência de todas as instabilidades MHD, até mesmo sobre os dentes de serra localizados na região central da coluna. Em todos os casos, observou-se que durante o platô a frequência das ilhas magnéticas coincide com a frequência das oscilações precursoras do dente de serra, apesar de serem duas instabilidades distintas, localizadas em posições radiais muito diferentes. Essa coincidência de frequências possibilitou descrever a evolução em frequência de todas as oscilações detectadas em diversos diagnósticos com base em apenas duas frequências básicas: a dos ciclos de relaxação dente de serra e a das ilhas magnéticas.

Effects of static stretching following a dynamic warm-up on speed, agility and power

Static stretching prior to sport has been shown to decrease force production in comparison to the increasing popularity of dynamic warm-up methods. However some athletes continue to use a bout of static stretching following dynamic methods. The purpose of this study was to investigate the effects on speed, agility and power following a period of additional static stretching following a dynamic warm-up routine. Twenty-five male University students who participated in team sports performed two warm-up protocols concentrating on the lower body one week apart through a randomised cross over design. The dynamic warm-up (DW) protocol used a series of specific progressive exercises lasting 10 minutes over a distance of 20m. The dynamic warm-up plus static stretching (DWS) protocol used the same DW protocol followed by a 5 minute period during which 7 muscle groups were stretched. Following each warm-up the subjects performed a countermovement vertical jump, 20m sprint and Illinois agility test, 1 minute apart. The results demonstrated no significant differences in speed, agility and jump performance following the two protocols DW and DWS. The study concludes that performing static stretching following a dynamic warm-up prior to performance does not significantly affect speed, agility and vertical jump performance.

Wind Shear, Gust, and Yaw-Induced Dynamic Stall on Wind-Turbine Blades

This study examined the effect of a spanwise angle of attack gradient on the growth and stability of a dynamic stall vortex in a rotating system. It was found that a spanwise angle of attack gradient induces a corresponding spanwise vorticity gradient, which, in combination with spanwise flow, results in a redistribution of circulation along the blade. Specifically, when modelling the angle of attack gradient experienced by a wind turbine at the 30% span position during a gust event, the spanwise vorticity gradient was aligned such that circulation was transported from areas of high circulation to areas of low circulation, increasing the local dynamic stall vortex growth rate, which corresponds to an increase in the lift coefficient, and a decrease in the local vortex stability at this point. Reversing the relative alignment of the spanwise vorticity gradient and spanwise flow results in circulation transport from areas of low circulation generation to areas of high circulation generation, acting to reduce local circulation and stabilise the vortex. This circulation redistribution behaviour describes a mechanism by which the fluctuating loads on a wind turbine are magnified, which is detrimental to turbine lifetime and performance. Therefore, an understanding of this phenomenon has the potential to facilitate optimised wind turbine design.

Essays in dynamic optimal taxation: working experience, unemployment scarring and poverty

This thesis investigates the design of optimal tax systems in dynamic environments. The first essay characterizes the optimal tax system where wages depend on stochastic shocks and work experience. In addition to redistributive and efficiency motives, the taxation of inexperienced workers depends on a second-best requirement that encourages work experience, a social insurance motive and incentive effects. Calibrations using U.S. data yield higher expected optimal marginal income tax rates for experienced workers for most of the inexperienced workers. They confirm that the average marginal income tax rate increases (decreases) with age when shocks and work experience are substitutes (complements). Finally, more variability in experienced workers' earnings prospects leads to increasing tax rates since income taxation acts as a social insurance mechanism. In the second essay, the properties of an optimal tax system are investigated in a dynamic private information economy where labor market frictions create unemployment that destroys workers' human capital. A two-skill type model is considered where wages and employment are endogenous. I find that the optimal tax system distorts the first-period wages of all workers below their efficient levels which leads to more employment. The standard no-distortion-at-the-top result no longer holds due to the combination of private information and the destruction of human capital. I show this result analytically under the Maximin social welfare function and confirm it numerically for a general social welfare function. I also investigate the use of a training program and job creation subsidies. The final essay analyzes the optimal linear tax system when there is a population of individuals whose perceptions of savings are linked to their disposable income and their family background through family cultural transmission. Aside from the standard equity/efficiency trade-off, taxes account for the endogeneity of perceptions through two channels. First, taxing labor decreases income, which decreases the perception of savings through time. Second, taxation on savings corrects for the misperceptions of workers and thus savings and labor decisions. Numerical simulations confirm that behavioral issues push labor income taxes upward to finance saving subsidies. Government transfers to individuals are also decreased to finance those same subsidies.