Analysis of TAF II Function in the Yeast Saccharomyces Cerevisiae

Transcription by RNA polymerase II is a highly regulated process requiring a number of general and promoter specific transcription factors. Although many of the factors involved in the transcription reaction are known, exactly how they function to stimulate or repress transcription is not well understood. Central to understanding gene regulation is understanding the mechanism by which promoter specific transcription activators (activators) stimulate transcription. A group of factors called coactivators have been shown to be required for activator function in vitro. The best characterized coactivators to date are members of the TFIID complex. TFIID is a multisubunit complex composed of the TATA box binding protein (TBP) and 8-12 TBP associated factors (TAFIIs). Results from numerous in vitro experiments indicate that TAFIIs function by binding to activators and forming a bridge between the activator and the basal transcription machinery. In order to gain insight into the mechanism by which activators stimulate transcription, we chose to analyze the in vivo function of TAFIIs, their proposed targets. Results from the genetic disruption of a number of TAFIIs in the yeast Saccharomyces cerevisiae showed that most are encoded by essential genes. In order to study their function, temperature-sensitive and conditional alleles were constructed. Cells depleted of individual TAFIIs by either of these two methods displayed no defect in global transcription activation. Inactivation of yTAFII17, however, resulted in a promoter specific defect. In addition, inactivation of yTAFII145, yTAFII90, or TSM1, resulted in an inability of cells to progress through the cell-cycle. In an attempt to identify genes whose expression required yTAFII90, we performed subtractive hybridization on strains containing wild-type and temperature-sensitive alleles. Although this technique successfully identified genes differentially expressed in the two strains, it failed to identify genes whose expression required yTAFII90. These results indicate that TAFIIs are not the obligatory targets of activators, and that other factors must provide this role in vivo. Furthermore, that many of TAFIIs are required for cell-cycle progression

Identification and partial characterization of cAMP-phosphodiesterases in the ciliate Euplotes raikovi.

In the ciliate Euplotes raikovi, two specific isoforms of cAMP- dependent phosphodiesterases were identified, one in the soluble and the other in the particulate fraction of the cell. Their activity was shown to be stimulated by Mg2+, insensitive to Ca2+ and cGMP, and scarcely inhibited by theophylline and 3-isobutyl-1-methyl-xanthine. They appear to be related to some phosphodiesterases of class II of other unicellular organisms in their biochemical features, and their enzymatic activity is up-regulated by elevation of intracellular cAMP level similarly to PDE-4 isoforms of mammals

Cross-talk between the autocrine (mitogenic) pheromone loop of the ciliate Euplotes raikovi and the intracellular cyclic AMP concentration

Cell type-specific protein signals, called pheromones, are constitutively secreted by Euplotes raikovi and bound back in autocrine fashion, with a positive effect on the vegetative (mitotic) cell growth. In cells growing suspended with their secreted pheromone, it was found that any interruption of this autocrine signaling loop was immediately followed by an effective enhancement of the basal intracellular cyclic AMP (cAMP) level. To establish a cause-effect relationship between these pheromone-induced variations in the cytoplasmic cAMP level and cell growth, cells ready to pass from a resting stage to a new growth cycle were conditioned either to incorporate a cAMP analog resistant to phosphodiesterase degradation, or to utilize cAMP released (following cell irradiation) from incorporated “caged” cAMP. Cells responded at every induced increase in their basal cAMP level by markedly decreasing their commitment to start a new growth cycle. It was deduced that the autocrine signaling of E. raikovi pheromones involves cAMP as inhibitor of its mitogenic activity

Ichthyofaunistic composition of the Quilombo river, tributary of the Mogi-Guaçu river, upper Paraná river basin, southeastern Brazil

Um estudo sobre a composição ictiofaunística do rio Quilombo foi realizado com o intuito de identificar quais espécies de peixes habitam a bacia, com que freqüência tais espécies são encontradas e verificar variações na distribuição longitudinal desta ictiofauna. Foram demarcados quatro pontos de coletas distribuídos na bacia, os quais foram visitados 21 vezes ao longo de um ano e dez meses (entre setembro de 2003 e junho de 2005), abrangendo os períodos seco e úmido que ocorrem anualmente na região estudada. Para coleta dos peixes foram utilizadas diferentes artes de pesca: tarrafas, redes de espera, rede de arrasto, peneiras, linha e anzol. Os peixes foram fixados em formalina 10%, conservados em etanol 70%, identificados e encontram-se depositados na coleção de peixes do Laboratório de Ictiologia Sistemática do Departamento de Ecologia e Biologia Evolutiva da UFSCar. Foram coletados 2982 exemplares, os quais estão divididos em 6 ordens, 19 famílias, 52 gêneros e 68 espécies. As ordens Characiformes (57,3%) e Siluriformes (30,9%) tiveram maior participação no total de espécies em relação às ordens Gymnotiformes, Cyprinodontiformes, Perciformes e Synbranchiformes, que juntas somaram 11,8% da riqueza. A análise da constância permitiu verificar que a composição da ictiofauna desse rio variou ao longo do período, principalmente nos trechos médio e inferior. O índice de similaridade (Jaccard) evidenciou que os conjuntos de espécies são diferentes entre os pontos de coleta, mostrando particularidades em cada um deles.A study about fish composition in the Quilombo river, of the upper Paraná hydrographic system, is presented. We aimed to identify which species inhabit this small river, to verify the frequency they occur and to study the longitudinal distribution of the ichthyofauna. Fish were sampled for a period of one year and ten months (September 2003 to June 2005) at four collection sites defined through the river basin, comprising dry and wet seasonal periods. Trawlnet, gillnets, seine net, sieves and hooks were used for fish sample. Fish collected were immediately fixed in 10% formalin solution. In the laboratory specimens were preserved in ethanol 70%, identified and deposited in the fish collection of the Laboratório de Ictiologia Sistemática (LISDEBE) of the Departamento de Ecologia e Biologia Evolutiva of the Universidade Federal de São Carlos. An amount of 2982 specimens belonging to 6 orders, 19 families, 52 genera and 68 species were collected. The orders Characiformes (57.3%) and Siluriformes (30.9%) predominated in terms of species richness. The orders Gymnotiformes, Cyprinodontiformes, Perciformes and Synbranchiformes summed 11.8% of total fish richness. The analysis of constancy revealed that the ichthyofaunistic composition in the middle and lower sampled stretches suffered higher temporal variability in comparison to upper stretches of Quilombo river basin. The similarity (Jaccard index) among samples showed that each collection site have distinct assemblages of fish

Targeting the Diuretic Hormone Receptor to Control the Cotton Leafworm,<i>Spodoptera littoralis</i>

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An Extract from Ficus carica Cell Cultures Works as an Anti-Stress Ingredient for the Skin

Psychological stress activates catecholamine production, determines oxidation processes, and alters the lipid barrier functions in the skin. Scientific evidence associated with the detoxifying effect of fruits and vegetables, the growing awareness of the long-term issues related to the use of chemical-filled cosmetics, the aging of the population, and the increase in living standards are the factors responsible for the growth of food-derived ingredients in the cosmetics market. A Ficus carica cell suspension culture extract (FcHEx) was tested in vitro (on keratinocytes cells) and in vivo to evaluate its ability to manage the stress-hormone-induced damage in skin. The FcHEx reduced the epinephrine (−43% and −24% at the concentrations of 0.002% and 0.006%, respectively), interleukin 6 (−38% and −36% at the concentrations of 0.002% and 0.006%, respectively), lipid peroxide (−25%), and protein carbonylation (−50%) productions; FcHEx also induced ceramide synthesis (+150%) and ameliorated the lipid barrier performance. The in vivo experiments confirmed the in vitro test results. Transepidermal water loss (TEWL; −12.2%), sebum flow (−46.6% after two weeks and −73.8% after four weeks; on the forehead −56.4% after two weeks and −80.1% after four weeks), and skin lightness (+1.9% after two weeks and +2.7% after four weeks) defined the extract’s effects on the skin barrier. The extract of the Ficus carica cell suspension cultures reduced the transepidermal water loss, the sebum production, the desquamation, and facial skin turning to a pale color from acute stress, suggesting its role as an ingredient to fight the signs of psychological stress in the skin

NF-Y recruitment of TFIID, multiple interactions with histone fold TAF(II)s

The nuclear factor y (NF-Y) trimer and TFIID contain histone fold subunits, and their binding to the CCAAT and Initiator elements of the major histocompatibility complex class II Ea promoter is required for transcriptional activation. Using agarose-electrophoretic mobility shift assay we found that NF-Y increases the affinity of holo-TFIID for Ea in a CCAAT- and Inr-dependent manner. We began to dissect the interplay between NF-Y- and TBP-associated factors PO1II (TAF(II)s)-containing histone fold domains in protein-protein interactions and transfections. hTAF(II)20, hTAF(II)28, and hTAF(II)18-hTAF(II)28 bind to the NF-Y B-NF-YC histone fold dimer; hTAF(II)80 and hTAF(II)31-hTAF(II)80 interact with the trimer but not with the NF-YB-NF-YC dimer. The histone fold alpha2 helix of hTAF(II)80 is not required for NF-Y association, as determined by interactions with the naturally occurring splice variant hTAF(II)80delta. Expression of hTAF(II)28 and hTAF(II)18 in mouse cells significantly and specifically reduced NF-Y activation in GAL4-based experiments, whereas hTAF,120 and hTAF(II)135 increased it. These results indicate that NF-Y (i) recruits purified holo-TFIID in vitro and (ii) can associate multiple TAF(II)s, potentially accommodating different core promoter architectures

Hibiscus syriacus extract from an established cell culture stimulates skin wound healing

Higher plants are the source of a wide array of bioactive compounds that support skin integrity and health. Hibiscus syriacus, family Malvaceae, is a plant of Chinese origin known for its antipyretic, anthelmintic, and antifungal properties. The aim of the present study was to assess the healing and hydration properties of an H. syriacus ethanolic extract (HSEE). We established a cell suspension culture from Hibiscus syriacus leaves and obtained an ethanol soluble extract from the cultured cells. The properties of the extract were tested by gene expression and functional analyses on human keratinocytes, dermal fibroblasts and human skin explants. HSEE treatment increased the healing potential of fibroblasts and keratinocytes. Specifically, HSEE stimulated the synthesis of fibronectin and collagen in fibroblasts and enhanced their contractility. The obtained results were confirmed on skin explants, where HSEE accelerated the wound healing activity in terms of epithelium formation and fibronectin production. Moreover, HSEE increased the expression of aquaporin 3 and filaggrin genes, both involved in skin hydration and homeostasis. Our data show that HSEE contains compounds capable of stimulating expression of biomarkers which are relevant for skin regeneration and hydration thereby counteracting molecular pathways leading to skin damage and aging

An E box comprises a positional sensor for regional differences in skeletal muscle gene expression and methylation

AbstractTo dissect the molecular mechanisms conferring positional information in skeletal muscles, we characterized the control elements responsible for the positionally restricted expression patterns of a muscle-specific transgene reporter, driven by regulatory sequences from the MLC1/3 locus. These sequences have previously been shown to generate graded transgene expression in the segmented axial muscles and their myotomal precursors, fortuitously marking their positional address. An evolutionarily conserved E box in the MLC enhancer core, not recognized by MyoD, is a target for a nuclear protein complex, present in a variety of tissues, which includes Hox proteins and Zbu1, a DNA-binding member of the SW12/SNF2 gene family. Mutation of this E box in the MLC enhancer has only a modest positive effect on linked CAT gene expression in transfected muscle cells, but when introduced into transgenic mice the same mutation elevates CAT transgene expression in skeletal muscles, specifically releasing the rostral restriction on MLC-CAT transgene expression in the segmented axial musculature. Increased transgene activity resulting from the E box mutation in the MLC enhancer correlates with reduced DNA methylation of the distal transgenic MLC1 promoter as well as in the enhancer itself. These results identify an E box and the proteins that bind to it as a positional sensor responsible for regional differences in axial skeletal muscle gene expression and accessibility

Production of Plant Secondary Metabolites: Examples, Tips and Suggestions for Biotechnologists

Plants are sessile organisms and, in order to defend themselves against exogenous (a)biotic constraints, they synthesize an array of secondary metabolites which have important physiological and ecological effects. Plant secondary metabolites can be classified into four major classes: terpenoids, phenolic compounds, alkaloids and sulphur-containing compounds. These phytochemicals can be antimicrobial, act as attractants/repellents, or as deterrents against herbivores. The synthesis of such a rich variety of phytochemicals is also observed in undifferentiated plant cells under laboratory conditions and can be further induced with elicitors or by feeding precursors. In this review, we discuss the recent literature on the production of representatives of three plant secondary metabolite classes: artemisinin (a sesquiterpene), lignans (phenolic compounds) and caffeine (an alkaloid). Their respective production in well-known plants, i.e., Artemisia, Coffea arabica L., as well as neglected species, like the fibre-producing plant Urtica dioica L., will be surveyed. The production of artemisinin and caffeine in heterologous hosts will also be discussed. Additionally, metabolic engineering strategies to increase the bioactivity and stability of plant secondary metabolites will be surveyed, by focusing on glycosyltransferases (GTs). We end our review by proposing strategies to enhance the production of plant secondary metabolites in cell cultures by inducing cell wall modifications with chemicals/drugs, or with altered concentrations of the micronutrient boron and the quasi-essential element silicon