scholarly journals FGF1 Promotes Xenopus laevis Lens Regeneration

2018 ◽  
Author(s):  
Lisa Moore ◽  
Kimberly J. Perry ◽  
Cindy Sun ◽  
Jonathan J. Henry
Keyword(s):  
Xenopus Laevis ◽  
Culture Method ◽  
Dominant Negative ◽  
Dependent Manner ◽  
Regeneration Rate ◽  
Culture Techniques ◽  
Lens Regeneration ◽  
Fgfr Signaling ◽  
Lens Formation

AbstractBackgroundThe frog Xenopus laevis has notable regenerative capabilities, including that of the lens. The neural retina provides the factors that trigger lens regeneration from the cornea, but the identity of these factors is largely unknown. In contrast to the cornea, fibroblast growth factors FGF1, 8, and 9 are highly expressed within the retina, and are potential candidates for those factors. The purpose of this study is to determine whether specific FGF proteins can induce lens formation, and if perturbation of FGFR signaling inhibits lens regeneration.MethodsA novel cornea epithelial culture method was developed to investigate the sufficiency of FGFs in lens regeneration. Additionally, transgenic larvae expressing dominant negative FGFR1 were used to investigate the necessity of FGFR signaling in lens regeneration.ResultsTreatment of cultured corneas with FGF1 induced lens regeneration in a dose-dependent manner, whereas treatment with FGF2, FGF8, or FGF9 did not result in significant lens regeneration. Inhibition of FGFR signaling decreased the lens regeneration rate for in vitro eye cultures.ConclusionThe culture techniques developed here, and elsewhere, have provided reliable methods for examining the necessity of various factors that may be involved in lens regeneration. Based on the results demonstrated in this study, we found that FGF1 signaling and FGFR activation are key factors for lens regeneration in Xenopus.

scholarly journals Extracellular Signal-Regulated Kinase Binds to TFII-I and Regulates Its Activation of the c-fosPromoter

2000 ◽  
Vol 20 (4) ◽  
pp. 1140-1148 ◽  
Author(s):  
Dae-Won Kim ◽  
Brent H. Cochran
Keyword(s):  
Map Kinase ◽  
Transcriptional Activation ◽  
Dominant Negative ◽  
Binding Motif ◽  
Dependent Manner ◽  
Consensus Map ◽  
Interaction Domain ◽  
Extracellular Signal

ABSTRACT We have previously shown that TFII-I enhances transcriptional activation of the c-fos promoter through interactions with upstream elements in a signal-dependent manner. Here we demonstrate that activated Ras and RhoA synergize with TFII-I for c-fospromoter activation, whereas dominant-negative Ras and RhoA inhibit these effects of TFII-I. The Mek1 inhibitor, PD98059 abrogates the enhancement of the c-fos promoter by TFII-I, indicating that TFII-I function is dependent on an active mitogen-activated protein (MAP) kinase pathway. Analysis of the TFII-I protein sequence revealed that TFII-I contains a consensus MAP kinase interaction domain (D box). Consistent with this, we have found that TFII-I forms an in vivo complex with extracellular signal-related kinase (ERK). Point mutations within the consensus MAP kinase binding motif of TFII-I inhibit its ability to bind ERK and its ability to enhance the c-fos promoter. Therefore, the D box of TFII-I is required for its activity on the c-fos promoter. Moreover, the interaction between TFII-I and ERK can be regulated. Serum stimulation enhances complex formation between TFII-I and ERK, and dominant-negative Ras abrogates this interaction. In addition, TFII-I can be phosphorylated in vitro by ERK and mutation of consensus MAP kinase substrate sites at serines 627 and 633 impairs the phosphorylation of TFII-I by ERK and its activity on the c-fos promoter. These results suggest that ERK regulates the activity of TFII-I by direct phosphorylation.

scholarly journals Micropropagation of Tarenaya rosea (Cleomaceae) from leaf explants

Rodriguésia ◽  
2021 ◽  
Vol 72 ◽  
Author(s):  
Claudia Simões-Gurgel ◽  
Tatiana Carvalho de Castro ◽  
Cátia Henriques Callado ◽  
Lívia da Silva Cordeiro ◽  
Norma Albarello
Keyword(s):  
Large Scale ◽  
Plant Conservation ◽  
Leaf Explants ◽  
Plant Production ◽  
Anthocyanin Content ◽  
Alternative Source ◽  
Regeneration Rate ◽  
Culture Techniques ◽  
Regenerated Plants

Abstract In vitro culture techniques are recognized as efficient strategies for large-scale plant production, as well as providing alternatives for plant conservation. In this study the micropropagation of Tarenaya rosea was established using petiole and foliar blade segments cultivated on MS medium with 6-benzyladenine (BA) and/or 6-furfurylaminopurine (KIN). The regeneration rate from explants was evaluated after 30-days in culture, as well as the proliferation rate from explant-derived shoots, reached after four subcultures performed at 30-days in culture. In vitro propagation occurred by both direct (DO) and indirect (IO) organogenesis. The highest regeneration rates by DO (50% to 100%) were reached on media containing only BA, while morphogenic calluses (IO) were mainly formed with BA+KIN. Explants on media with BA showed the presence of small black nodules on their surface, and histological analysis revealed the presence of trichomes with anthocyanin content. Elongation and rooting were reached on growth regulator-free MS. Acclimatization rates around 80% were achieved and the in vitro-regenerated plants were successfully maintained under field conditions. Results show significant morphogenetic potential of T. rosea from leaf explants, mainly when cultivated in the presence of 4.4 µM BA, providing a new alternative source of plant material for biotechnological and in vitro conservation studies.

scholarly journals Phosphorylation and SCF-mediated degradation regulate CREB-H transcription of metabolic targets

2015 ◽  
Vol 26 (16) ◽  
pp. 2939-2954 ◽  
Author(s):  
Sónia Barbosa ◽  
Suzanne Carreira ◽  
Daniel Bailey ◽  
Fernando Abaitua ◽  
Peter O'Hare
Keyword(s):  
Target Gene ◽  
Active Form ◽  
Dominant Negative ◽  
Dependent Manner ◽  
Critical Determinant ◽  
Phosphatase Treatment ◽  
Steady State Levels ◽  
Treatment Use

CREB‑H, an endoplasmic reticulum–anchored transcription factor, plays a key role in regulating secretion and in metabolic and inflammatory pathways, but how its activity is modulated remains unclear. We examined processing of the nuclear active form and identified a motif around S87–S90 with homology to DSG-type phosphodegrons. We show that this region is subject to multiple phosphorylations, which regulate CREB-H stability by targeting it to the SCFFbw1aE3 ubiquitin ligase. Data from phosphatase treatment, use of phosophospecific antibody, and substitution of serine residues demonstrate phosphorylation of candidate serines in the region, with the core S87/S90 motif representing a critical determinant promoting proteasome-mediated degradation. Candidate kinases CKII and GSK-3b phosphorylate CREB-H in vitro with specificities for different serines. Prior phosphorylation with GSK-3 at one or more of the adjacent serines substantially increases S87/S90-dependent phosphorylation by CKII. In vivo expression of a dominant-negative Cul1 enhances steady-state levels of CREB‑H, an effect augmented by Fbw1a. CREB-H directly interacts with Fbw1a in a phosphorylation-dependent manner. Finally, mutations within the phosphodegron, when incorporated into the full-length protein, result in increased levels of constitutively cleaved nuclear protein and increased transcription and secretion of a key endogenous target gene, apolipoprotein A IV.

scholarly journals The 36-kilodalton embryonic-type cytoplasmic polyadenylation element-binding protein in Xenopus laevis is ElrA, a member of the ELAV family of RNA-binding proteins.

1997 ◽  
Vol 17 (11) ◽  
pp. 6402-6409 ◽  
Author(s):  
L Wu ◽  
P J Good ◽  
J D Richter
Keyword(s):  
Xenopus Laevis ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Binding Protein ◽  
Dominant Negative ◽  
Cytoplasmic Polyadenylation ◽  
Element Binding Protein

The translational activation of several maternal mRNAs in Xenopus laevis is dependent on cytoplasmic poly(A) elongation. Messages harboring the UUUUUAU-type cytoplasmic polyadenylation element (CPE) in their 3' untranslated regions (UTRs) undergo polyadenylation and translation during oocyte maturation. This CPE is bound by the protein CPEB, which is essential for polyadenylation. mRNAs that have the poly(U)12-27 embryonic-type CPE (eCPE) in their 3' UTRs undergo polyadenylation and translation during the early cleavage and blastula stages. A 36-kDa eCPE-binding protein in oocytes and embryos has been identified by UV cross-linking. We now report that this 36-kDa protein is ElrA, a member of the ELAV family of RNA-binding proteins. The proteins are identical in size, antibody directed against ElrA immunoprecipitates the 36-kDa protein, and the two proteins have the same RNA binding specificity in vitro. C12 and activin receptor mRNAs, both of which contain eCPEs, are detected in immunoprecipitated ElrA-mRNP complexes from eggs and embryos. In addition, this in vivo interaction requires the eCPE. Although a number of experiments failed to define a role for ElrA in cytoplasmic polyadenylation, the expression of a dominant negative ElrA protein in embryos results in an exogastrulation phenotype. The possible functions of ElrA in gastrulation are discussed.

scholarly journals Study of Seaweed Kappaphycus alvarezii Explants Growth in the Different Salinity Concentrations

2021 ◽  
Vol 13 (1) ◽  
pp. 97
Author(s):  
Muhammad Aris ◽  
Fatma Muchdar ◽  
Rusmawati Labenua
Keyword(s):  
Tissue Culture ◽  
Kappaphycus Alvarezii ◽  
Environmental Parameters ◽  
Culture Method ◽  
Salinity Treatment ◽  
Culture Methods ◽  
Culture Techniques ◽  
Randomized Design ◽  
Salinity Levels

HighlightThe best salinity for the thallus growth of K. alvarezii is 32 mg/LThe salinity indicates the osmotic balance of K. alvarezii seaweedThis osmoregulation process affects the nutrient absorption of K. alvarezii seaweedThis study aims to develop the availibility of superior seeds of K. alvareziiAbstract Kappaphycus alvarezii is one of the leading commodities in Indonesian waters. Demand for this commodity is quite high, as reflected in the increasing volume of exports each year. Fulfillment of these demands is obtained from the production of cultivation. Generally farmers get natural seaweed seedlings, namely cuttings from existing seaweed. The continuous use of seeds from nature can cause deterioration in the quality and quantity. Handling the problem of quality deterioration from seaweed seeds originating from nature, can be overcome by multiplying the seeds through tissue culture methods in vitro. In term of tissue culture techniques method, the most important thing to note is environmental parameters. An environment parameter that changes suddenly such as salinity can inhibit the growth of seaweed. Thus, this work is attampting the different salinity treatment on seaweed explants K. alvarezii. This study aims at determining (weight) the explants of K. alvarezii with a comparison of the different salinity levels in the in vitro tissue culture method. The method used in this study was a completely randomized design (CRD) with the different salinity treatments namely 30, 31, 32, 33, and 34 ppt. The results showed that the different salinities influenced the growth rate of K. alvarezii seaweed explants with the best explant growth at the salinity of 31 ppt, while the lowest growth value was obtained at 34 ppt

scholarly journals The Candida albicans ATO Gene Family Promotes Neutralization of the Macrophage Phagolysosome

2015 ◽  
Vol 83 (11) ◽  
pp. 4416-4426 ◽  
Author(s):  
Heather A. Danhof ◽  
Michael C. Lorenz
Keyword(s):  
Amino Acids ◽  
Candida Albicans ◽  
Hyphal Growth ◽  
Dominant Negative ◽  
Dependent Manner ◽  
Ph Change ◽  
Content Type ◽  
Ammonia Release ◽  
Hyphal Formation

ABSTRACTCandida albicansis an opportunistic human fungal pathogen that causes a variety of diseases, ranging from superficial mucosal to life-threatening systemic infections, the latter particularly in patients with defects in innate immune function.C. albicanscells phagocytosed by macrophages undergo a dramatic change in their metabolism in which amino acids are a key nutrient. We have shown that amino acid catabolism allows the cell to neutralize the phagolysosome and initiate hyphal growth. We show here that members of the 10-geneATOfamily, which are induced by phagocytosis or the presence of amino acids in an Stp2-dependent manner and encode putative acetate or ammonia transporters, are important effectors of this pH changein vitroand in macrophages. When grown with amino acids as the sole carbon source, the deletion ofATO5or the expression of a dominant-negativeATO1G53Dallele results in a delay in alkalinization, a defect in hyphal formation, and a reduction in the amount of ammonia released from the cell. These strains also form fewer hyphae after phagocytosis, have a reduced ability to escape macrophages, and reside in more acidic phagolysosomal compartments than wild-type cells. Furthermore, overexpression of many of the 10ATOgenes accelerates ammonia release, and anato5Δ ATO1G53Ddouble mutant strain has additive alkalinization and ammonia release defects. Taken together, these results indicate that the Ato protein family is a key mediator of the metabolic changes that allowC. albicansto overcome the macrophage innate immunity barrier.

scholarly journals Insulin down-regulates insulin receptor substrate-2 expression through the phosphatidylinositol 3-kinase/Akt pathway

2003 ◽  
Vol 179 (2) ◽  
pp. 253-266 ◽  
Author(s):  
Y Hirashima ◽  
K Tsuruzoe ◽  
S Kodama ◽  
M Igata ◽  
T Toyonaga ◽  
...  
Keyword(s):  
Insulin Receptor ◽  
Kinase Inhibitor ◽  
Insulin Response ◽  
Dominant Negative ◽  
Akt Pathway ◽  
Insulin Receptor Substrate ◽  
Dependent Manner ◽  
Gene Promoters ◽  
Gene Assay

Insulin receptor substrate (IRS)-1 and IRS-2 are the major substrates that mediate insulin action. Insulin itself regulates the expression of the IRS protein in the liver, but the underlying mechanisms of IRS-1 and IRS-2 regulation are not fully understood. Here we report that insulin suppressed the expression of both IRS-1 and IRS-2 proteins in Fao hepatoma cells. The decrease in IRS-1 protein occurred via proteasomal degradation without any change in IRS-1 mRNA, whereas the insulin-induced suppression of IRS-2 protein was associated with a parallel decrease in IRS-2 mRNA without changing IRS-2 mRNA half-life. The insulin-induced suppression of IRS-2 mRNA and protein was blocked by the phosphatidylinositol (PI) 3-kinase inhibitor, LY294002, but not by the MAP kinase-ERK kinase (MEK) inhibitor, PD098059. Inhibition of Akt by overexpression of dominant-negative Akt also caused complete attenuation of the insulin-induced decrease in IRS-2 protein and partial attenuation of its mRNA down-regulation. Some nuclear proteins bound to the insulin response element (IRE) sequence on the IRS-2 gene in an insulin-dependent manner in vitro, and the binding was also blocked by the PI 3-kinase inhibitor. Reporter gene assay showed that insulin suppressed the activity of both human and rat IRS-2 gene promoters through the IRE in a PI 3-kinase-dependent manner. Our results indicate that insulin regulates IRS-1 and IRS-2 through different mechanisms and that insulin represses IRS-2 gene expression via a PI 3-kinase/Akt pathway.

scholarly journals The pro-apoptotic protein death-associated protein 3 (DAP3) interacts with the glucocorticoid receptor and affects the receptor function

2000 ◽  
Vol 349 (3) ◽  
pp. 885-893 ◽  
Author(s):  
Sanna M. HULKKO ◽  
Hideki WAKUI ◽  
Johanna ZILLIACUS
Keyword(s):  
Glucocorticoid Receptor ◽  
Transcriptional Activation ◽  
Heat Shock Protein 90 ◽  
Dominant Negative ◽  
Dependent Manner ◽  
Interaction Domain ◽  
Helix Loop Helix ◽  
Aryl Hydrocarbon ◽  
Apoptotic Protein

The yeast two-hybrid system was used to isolate cDNAs encoding proteins that interact with the glucocorticoid receptor (GR) ligand-binding domain in a ligand-dependent manner. One isolated cDNA encoded a fragment of death-associated protein 3 (DAP3), which has been implicated as a positive mediator of apoptosis. In vitro experiments showed that the full-length DAP3 also interacted with GR. The main interaction domain was mapped to the N-terminal region of DAP3 that had previously been shown to function in a dominant-negative fashion, protecting cells from apoptosis. Co-transfection experiments in COS-7 cells showed that DAP3 had a stimulatory effect on the ligand-induced transcriptional activation by GR and also increased the steroid-sensitivity. Furthermore, DAP3 formed a complex with several other nuclear receptors and some basic helix–loop–helix/Per–Arnt–Sim proteins, as well as with heat-shock protein 90 (hsp90) (Arnt is the aryl-hydrocarbon-receptor nuclear translocator, and Per and Sim are the Drosophila proteins Period and Single-minded). The results suggest that DAP3 could have an important role in GR action, possibly by modulating the cytoplasmic GR–hsp90 complex. Since glucocorticoids can induce apoptosis, the pro-apoptotic DAP3 protein may be involved in this function of GR.

scholarly journals ATR-Dependent Phosphorylation of DNA-Dependent Protein Kinase Catalytic Subunit in Response to UV-Induced Replication Stress

2006 ◽  
Vol 26 (20) ◽  
pp. 7520-7528 ◽  
Author(s):  
Hirohiko Yajima ◽  
Kyung-Jong Lee ◽  
Benjamin P. C. Chen
Keyword(s):  
Protein Kinase ◽  
Uv Irradiation ◽  
Cellular Response ◽  
Replication Stress ◽  
Dominant Negative ◽  
Catalytic Subunit ◽  
Dependent Manner ◽  
Dependent Protein Kinase ◽  
Dependent Protein

ABSTRACT Phosphorylation of the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) upon ionizing radiation (IR) is essential for cellular radioresistance and nonhomologous-end-joining-mediated DNA double-strand break repair. In addition to IR induction, we have previously shown that DNA-PKcs phosphorylation is increased upon camptothecin treatment, which induces replication stress and replication-associated double-strand breaks. To clarify the involvement of DNA-PKcs in this process, we analyzed DNA-PKcs phosphorylation in response to UV irradiation, which causes replication stress and activates ATR (ATM-Rad3-related)/ATM (ataxia-telangiectasia mutated) kinases in a replication-dependent manner. Upon UV irradiation, we observed a rapid DNA-PKcs phosphorylation at T2609 and T2647, but not at S2056, distinct from that induced by IR. UV-induced DNA-PKcs phosphorylation occurs specifically only in replicating cells and is dependent on ATR kinase. Inhibition of ATR activity via caffeine, a dominant-negative kinase-dead mutant, or RNA interference led to the attenuation of UV-induced DNA-PKcs phosphorylation. Furthermore, DNA-PKcs associates with ATR in vivo and is phosphorylated by ATR in vitro, suggesting that DNA-PKcs could be the direct downstream target of ATR. Taken together, these results strongly suggest that DNA-PKcs is required for the cellular response to replication stress and might play an important role in the repair of stalled replication forks.

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