Expression cloning of a Xenopus T-related gene (Xombi) involved in mesodermal patterning and blastopore lip formation

Development ◽  
1996 ◽  
Vol 122 (12) ◽  
pp. 4001-4012 ◽  
Author(s):  
K.D. Lustig ◽  
K.L. Kroll ◽  
E.E. Sun ◽  
M.W. Kirschner
Keyword(s):  
Time Course ◽  
Biological Activities ◽  
Ectopic Expression ◽  
Expression Cloning ◽  
Functional Assay ◽  
T Box ◽  
Zygotic Transcription ◽  
Vegetal Cortex ◽  
Dorsal Mesoderm ◽  
Partial Dependence

We have used a functional assay to identify a putative T-box transcription factor (Xombi) that has the ability to induce sites of invagination in the ectoderm of Xenopus embryos that resemble the blastopore lip. Maternal Xombi transcript is first localized to the oocyte's vegetal cortex and cytoplasm, early sources of mesoderm and endoderm-inducing signals. Soon after zygotic transcription begins, there is a wave of Xombi expression, beginning in dorsal mesoderm and then extending to lateral and ventral mesoderm, that precedes and parallels blastopore lip formation at the border between the mesoderm and endoderm. Transcripts encoding brachyury, Xwnt8 and goosecoid colocalize with Xombi transcripts within the marginal zone; ectopic expression of Xombi induces expression of all three mesodermal genes. In ectodermal explants, Xombi expression is induced by the secreted mesoderm inducers activinA, activinB, Xnrl and eFGF, and by brachyury, another Xenopus T-box containing gene. The time course and location of Xombi expression, its biological activities and the partial dependence of Xombi expression and blastopore lip formation on fibroblast growth factor (FGF) signaling suggest that Xombi contributes to a traveling wave of morphogenesis and differentiation during Xenopus gastrulation.

Xenopus VegT RNA is localized to the vegetal cortex during oogenesis and encodes a novel T-box transcription factor involved in mesodermal patterning

Development ◽  
1996 ◽  
Vol 122 (12) ◽  
pp. 4119-4129 ◽  
Author(s):  
J. Zhang ◽  
M.L. King
Keyword(s):  
Transcription Factor ◽  
Xenopus Oocytes ◽  
Nuclear Protein ◽  
Marginal Zone ◽  
Ectopic Expression ◽  
Developmental Pathways ◽  
T Box ◽  
Vegetal Cortex ◽  
Head Formation ◽  
Lateral Mesoderm

An RNA localized to the vegetal cortex of Xenopus oocytes encodes a novel T-box protein (VegT) capable of inducing either dorsal or posterior ventral mesoderm at different times in development. VegT is a nuclear protein and its C-terminal domain can activate transcription in a yeast reporter assay, observations consistent with VegT functioning as a transcription factor. Zygotic expression is dynamic along the dorsoventral axis, with transcripts first expressed in the dorsal marginal zone. By the end of gastrulation, VegT is expressed exclusively in posterior ventral and lateral mesoderm and is excluded from the notochord. Later expression is confined to a subset of Rohon-Beard cells, a type of primary sensory neuron. In animal cap assays, VegT is capable of converting prospective ectoderm into ventral lateral mesoderm. Such ectopic expression of VegT induces its own expression as well as that of Xwnt-8 in caps, suggesting that a Wnt pathway may be involved. Mis-expression of VegT in dorsal animal blastomeres fated to contribute to brain suppresses head formation. Our results suggest that VegT is a localized transcription factor, which operates sequentially in several developmental pathways during embryogenesis, including dorsoventral and posterior patterning of mesoderm.

scholarly journals Subcellular Targeting of p33ING1b by Phosphorylation-Dependent 14-3-3 Binding Regulates p21WAF1 Expression

2006 ◽  
Vol 26 (8) ◽  
pp. 2947-2954 ◽  
Author(s):  
Wei Gong ◽  
Michael Russell ◽  
Keiko Suzuki ◽  
Karl Riabowol
Keyword(s):  
Kinase Inhibitor ◽  
Biological Activities ◽  
Ectopic Expression ◽  
Growth Stress ◽  
Nuclear Localization Sequence ◽  
Binding Motif ◽  
Subcellular Targeting ◽  
Cyclin Dependent Kinase Inhibitor ◽  
Cargo Proteins ◽  
Localization Sequence

ABSTRACT ING1 is a type II tumor suppressor that affects cell growth, stress signaling, apoptosis, and DNA repair by altering chromatin structure and regulating transcription. Decreased ING1 expression is seen in several human cancers, and mislocalization has been noted in diverse types of cancer cells. Aberrant targeting may, therefore, functionally inactivate ING1. Bioinformatics analysis identified a sequence between the nuclear localization sequence and plant homeodomain domains of ING1 that closely matched the binding motif of 14-3-3 proteins that target cargo proteins to specific subcellular locales. We find that the widely expressed p33ING1b splicing isoform of ING1 interacts with members of the 14-3-3 family of proteins and that this interaction is regulated by the phosphorylation status of ING1. 14-3-3 binding resulted in significant amounts of p33ING1b protein being tethered in the cytoplasm. As shown previously, ectopic expression of p33ING1b increased levels of the p21Waf1 cyclin-dependent kinase inhibitor upon UV-induced DNA damage. Overexpression of 14-3-3 inhibited the up-regulation of p21Waf1 by p33ING1b, consistent with the idea that mislocalization blocks at least one of ING1's biological activities. These data support the idea that the 14-3-3 proteins play a crucial role in regulating the activity of p33ING1b by directing its subcellular localization.

XBF-2 is a transcriptional repressor that converts ectoderm into neural tissue

Development ◽  
1998 ◽  
Vol 125 (24) ◽  
pp. 5019-5031 ◽  
Author(s):  
F.V. Mariani ◽  
R.M. Harland
Keyword(s):  
Dna Binding ◽  
Neural Development ◽  
Ectopic Expression ◽  
Transcriptional Repressor ◽  
Neural Plate ◽  
Binding Domain ◽  
Expression Cloning ◽  
Dna Binding Domain ◽  
Neural Fate ◽  
Striking Contrast

We have identified Xenopus Brain Factor 2 (XBF-2) as a potent neuralizing activity in an expression cloning screen. In ectodermal explants, XBF-2 converts cells from an epidermal to a neural fate. Such explants contain neurons with distinct axonal profiles and express both anterior and posterior central nervous system (CNS) markers. In striking contrast to X-ngnR-1a or X-NeuroD, ectopic expression of XBF-2 in Xenopus embryos results in an expansion of the neural plate to the ventral midline. The enlarged neural plate consists predominantly of undifferentiated neurons. XBF-2 lies downstream of the BMP antagonists noggin, cerberus, and gremlin since ectodermal explants expressing these molecules exhibit strong expression of XBF-2. While XBF-2 does not upregulate the expression of secreted neural inducers, it downregulates the transcription of BMP-4, an epidermal inducer. We show that XBF-2 acts as a transcriptional repressor and that its effects can be phenocopied with either the engrailed or hairy repressor domain fused to the XBF-2 DNA-binding domain. A fusion of the DNA-binding domain to the activator domain of VP16 blocks the effects of XBF-2 and prevents neural plate development in the embryo. This provides evidence that a transcriptional repressor can affect both regional neural development and neurogenesis in vertebrates.

Endogenous patterns of TGFbeta superfamily signaling during early Xenopus development

Development ◽  
2000 ◽  
Vol 127 (13) ◽  
pp. 2917-2931 ◽  
Author(s):  
S. Faure ◽  
M.A. Lee ◽  
T. Keller ◽  
P. ten Dijke ◽  
M. Whitman
Keyword(s):  
Transforming Growth Factor Beta ◽  
Transforming Growth Factor ◽  
Ectopic Expression ◽  
Xenopus Embryo ◽  
Bmp Signaling ◽  
Type I ◽  
Embryonic Cells ◽  
Xenopus Development ◽  
Zygotic Transcription ◽  
Carboxy Terminal

Transforming growth factor beta (TGFbeta) superfamily signaling has been implicated in patterning of the early Xenopus embryo. Upon ligand stimulation, TGFbeta receptors phosphorylate Smad proteins at carboxy-terminal SS(V/M)S consensus motifs. Smads 1/5/8, activated by bone morphogenetic protein (BMP) signaling, induce ventral mesoderm whereas Smad2, activated by activin-like ligands, induces dorsal mesoderm. Although ectopic expression studies are consistent with roles for TGFbeta signals in early Xenopus embryogenesis, when and where BMP and activin-like signaling pathways are active endogenously has not been directly examined. In this study, we investigate the temporal and spatial activation of TGFbeta superfamily signaling in early Xenopus development by using antibodies specific for the type I receptor-phosphorylated forms of Smad1/5/8 and Smad2. We find that Smad1/5/8 and two distinct isoforms of Smad2, full-length Smad2 and Smad2(delta)exon3, are phosphorylated in early embryos. Both Smad1/5/8 and Smad2/Smad2(delta)exon3 are activated after, but not before, the mid-blastula transition (MBT). Endogenous activation of Smad2/Smad2(delta)exon3 requires zygotic transcription, while Smad1/5/8 activation at MBT appears to involve transcription-independent regulation. We also find that the competence of embryonic cells to respond to TGF(delta) superfamily ligands is temporally regulated and may be a determinant of early patterning. Levels of phospho-Smad1/5/8 and of phospho-Smad2/Smad2(delta)exon3 are asymmetrically distributed across both the animal-vegetal and dorsoventral axes. The timing of the development of these asymmetries differs for phospho-Smad1/5/8 and for phospho-Smad2/Smad2(delta)exon3, and the spatial distribution of phosphorylation of each Smad changes dramatically as gastrulation begins. We discuss the implications of our results for endogenous functions of BMP and activin-like signals as candidate morphogens regulating primary germ layer formation and dorsoventral patterning of the early Xenopus embryo.

scholarly journals Parallelism of Chemicostructural Properties between Filgrastim Originator and Three of Its Biosimilar Drugs

2019 ◽  
Vol 2019 ◽  
pp. 1-15 ◽  
Author(s):  
Alessandra Gianoncelli ◽  
Michela Bertuzzi ◽  
Michela Guarienti ◽  
Sara Vezzoli ◽  
Sara Anna Bonini ◽  
...  
Keyword(s):  
Biological Activity ◽  
Reference Product ◽  
Biological Activities ◽  
Chemical Characterization ◽  
Structural Similarity ◽  
Functional Assay ◽  
Biological Drugs ◽  
Reference Drug ◽  
Rp Hplc

The approval and granting of marketing authorization for a putative biosimilar is based on strong comparability studies with its biological reference product. This is due to the complexity of the structure and nature of the manufacturing process of biological drugs. Hence, a rigorous analytical workflow for chemical characterization and clinical trials to evaluate the efficacy and safety is required to demonstrate their high similarities to the reference drug. This work is focused on the comparison of the originator of filgrastim with three of its biosimilars by evaluating their structural similarity and biological activity. Qualiquantitative analyses were performed by MALDI-TOF/TOF-MS and RP-HPLC-UV. An innovative functional assay using zebrafish as the animal model was developed to evaluate the biological activities of the drugs. The different analyses performed in this study highlighted the structural similarity of biosimilar drugs and their originator. This result was further confirmed by a similar in vivo biological activity.

scholarly journals Simultaneous Optimal Production of Flavonol Aglycones and Degalloylated Catechins from Green Tea Using a Multi-Function Food-Grade Enzyme

Catalysts ◽  
2019 ◽  
Vol 9 (10) ◽  
pp. 861
Author(s):  
Chan-Su Rha ◽  
Shin-Woo Kim ◽  
Kyoung Hee Byoun ◽  
Yong Deog Hong ◽  
Dae-Ok Kim
Keyword(s):  
Green Tea ◽  
Time Course ◽  
Biological Activities ◽  
Hydrolase Activity ◽  
Enzyme Specificity ◽  
Optimum Conditions ◽  
Enzymatic Process ◽  
Food Grade ◽  
Phytochemical Compounds ◽  
Kaempferol Glycosides

(1) Background: Green tea (GT) contains well-known phytochemical compounds; namely, it is rich in flavan-3-ols (catechins) and flavonols comprising all glycoside forms. These compounds in GT might show better biological activities after a feasible enzymatic process, and the process on an industrial scale should consider enzyme specificity and cost-effectiveness. (2) Methods: In this study, we evaluated the most effective method for the enzymatic conversion of flavonoids from GT extract. One enzyme derived from Aspergillus niger (molecular weight 80–90 kDa) was ultimately selected, showing two distinct but simultaneous activities: intense glycoside hydrolase activity via deglycosylation and weak tannin acyl hydrolase activity via degalloylation. (3) Results: The optimum conditions for producing flavonol aglycones were pH 4.0 and 50 °C. Myricetin glycosides were cleaved 3.7–7.0 times faster than kaempferol glycosides. Flavonol aglycones were produced effectively by both enzymatic and hydrochloride treatment in a time-course reaction. Enzymatic treatment retained 80% (w/w) catechins, whereas 70% (w/w) of catechins disappeared by hydrochloride treatment. (4) Conclusions: This enzymatic process offers an effective method of conditionally producing flavonol aglycones and de-galloylated catechins from conversion of food-grade enzyme.

Hypoxia-activated ligand HAL-1/13 is lupus autoantigen Ku80 and mediates lymphoid cell adhesion in vitro

2001 ◽  
Vol 280 (4) ◽  
pp. C897-C911 ◽  
Author(s):  
Eileen M. Lynch ◽  
Robert B. Moreland ◽  
Irene Ginis ◽  
Susan P. Perrine ◽  
Douglas V. Faller
Keyword(s):  
Tumor Cell ◽  
Tumor Cells ◽  
Cell Lines ◽  
Lymphoid Cell ◽  
Ectopic Expression ◽  
Lymphoid Cells ◽  
Expression Cloning ◽  
Hypoxic Exposure ◽  
Tumor Cell Lines ◽  
Human Lymphoid Cell

Hypoxia is known to induce extravasation of lymphocytes and leukocytes during ischemic injury and increase the metastatic potential of malignant lymphoid cells. We have recently identified a new adhesion molecule, hypoxia-activated ligand-1/13 (HAL-1/13), that mediates the hypoxia-induced increases in lymphocyte and neutrophil adhesion to endothelium and hypoxia-mediated invasion of endothelial cell monolayers by tumor cells. In this report, we used expression cloning to identify this molecule as the lupus antigen and DNA-dependent protein kinase-associated nuclear protein, Ku80. The HAL-1/13-Ku80 antigen is present on the surface of leukemic and solid tumor cell lines, including T and B lymphomas, myeloid leukemias, neuroblastoma, rhabdomyosarcoma, and breast carcinoma cells. Transfection and ectopic expression of HAL-1/13-Ku80 on (murine) NIH/3T3 fibroblasts confers the ability of these normally nonadhesive cells to bind to a variety of human lymphoid cell lines. This adhesion can be specifically blocked by HAL-1/13 or Ku80-neutralizing antibodies. Loss of expression variants of these transfectants simultaneously lost their adhesive properties toward human lymphoid cells. Hypoxic exposure of tumor cell lines resulted in upregulation of HAL-1/13-Ku80 expression at the cell surface, mediated by redistribution of the antigen from the nucleus. These studies indicate that the HAL-1/13-Ku80 molecule may mediate, in part, the hypoxia-induced adhesion of lymphocytes, leukocytes, and tumor cells.

Effect of Prostaglandin on the Composition of Chinchilla Middle Ear Effusion

1980 ◽  
Vol 89 (3_suppl) ◽  
pp. 153-160 ◽  
Author(s):  
Timothy T. K. Jung ◽  
S. K. Juhn ◽  
Douglas M. Smith ◽  
Jonathan M. Gerrard
Keyword(s):  
Alkaline Phosphatase ◽  
Bone Resorption ◽  
Otitis Media ◽  
Middle Ear ◽  
Time Course ◽  
Unsaturated Fatty Acids ◽  
Biological Activities ◽  
Middle Ear Mucosa ◽  
Acid And Alkaline Phosphatase ◽  
Wide Range

Prostaglandins (PGs) are naturally occurring, cyclic, unsaturated fatty acids which possess a wide range of potent biological activities. PGs have been found in human middle ear effusions and might have implications for understanding the inflammation and possibly the bone resorption seen in chronic otitis media. We have measured PGs by radioimmunoassay in middle ear effusions (MEE) from experimentally induced serous otitis media (SOM) and purulent otitis media (POM) in chinchillas. PGE2 levels were significantly higher in the POM group compared to the SOM group. We have also demonstrated that chinchilla middle ear mucosa can convert arachidonic acid (AA), a precursor of PGs, to PG by injecting 14C-AA into bullae and assaying using radiochromatography. This conversion was completely blocked by both indomethacin and aspirin given orally or by direct injection into the middle ear. We then injected 50 μg of PGE2 into chinchilla bullae to assess its effect on the composition of MEE. First, the time course of PGE2 metabolism after its injection into the middle ear (ME) was determined by thin-layer chromatography (TLC) of labelled and unlabelled PGE2. Following this, serial daily injections of PGE2 and normal saline as control were made for one, three, and seven days. MEE and serum were collected and assayed for lactate dehydrogenase (LDH), acid and alkaline phosphatase, calcium, protein and hexosamine. Compared to the control, the levels of LDH, acid and alkaline phosphatase, calcium and protein were significantly elevated. Hexosamine levels were higher than the control at one and three days but did not differ significantly at seven days from the control. We have therefore demonstrated that chinchilla middle ear mucosa has the ability to synthesize PG from AA and suggest an active role for PGs in the inflammation and in the bone resorption seen in otitis media.

Measurement of Blood-Borne Tissue Factor Procoagulant Activity in Healthy Individuals and Cancer Patients Using a Novel Assay.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1938-1938
Author(s):  
Rachel E. Tilley ◽  
Todd Holscher ◽  
Rajesh Belani ◽  
Jorge Nieva ◽  
Nigel Mackman
Keyword(s):  
Cancer Patients ◽  
Tissue Factor ◽  
Whole Blood ◽  
Time Course ◽  
Mononuclear Cells ◽  
Ex Vivo ◽  
Procoagulant Activity ◽  
Functional Assay ◽  
Healthy Individuals ◽  
Stimulation Of

Abstract Tissue factor (TF) is the cellular initiator of the extrinsic pathway of coagulation. Recent studies have reported circulating blood-borne TF (BBTF) antigen by methods including ELISA and flow cytometry. Whether this BBTF is active has been rarely addressed due to the lack of a simple functional assay. We have developed a new assay that measures BBTF procoagulant activity. Whole blood was collected into tubes containing citrate and corn trypsin inhibitor (FXIIa inhibitor). First, we compared the procoagulant activity obtained in detergent lyzed platelets and microparticles with peripheral blood mononuclear cells (PBMCs) following a 5 hour ex vivo stimulation of whole blood with LPS. Platelets and microparticles had 3% (23 ± 10 mU/mL) and 0.1% (1.0 ± 0.4 mU/mL), respectively, of the procoagulant activity of PBMCs (1048 ± 200 mU/mL). The procoagulant activity of PBMCs and platelets was inhibited by >90% in the presence of an anti-TF polyclonal antibody. Next, we determined the time course of TF-dependent procoagulant activity of microparticles and platelets after ex vivo LPS stimulation of whole blood. In both cases, a dramatic increase in microparticle and platelet procoagulant activity was observed after 12 hours of LPS stimulation, with a maximum activity observed at 48 hours (48 mU/mL for microparticles and 1028 mU/mL for platelets at 48 hours). The majority (>90%) of this procoagulant activity was TF-dependent. Therefore, we subsequently analyzed combined platelet and microparticle (P+MP) fractions for BBTF activity. We determined functional BBTF activity in P+MP fractions from the blood of healthy individuals with and without LPS ex vivo stimulation. In healthy individuals, very low levels of BBTF procoagulant activity was detected in the absence of LPS stimulation (0.22 ± 0.09 mU/mL) and this activity was decreased on average by 28% with an anti-TF antibody (0.16 ± 0.1 mU/mL; p = 0.01). In contrast, P+MP from LPS stimulated blood demonstrated on average 30 fold higher procoagulant activity, of which >90% was TF-dependent. Cancer is associated with an increased susceptibility to develop pathological thrombosis. Using our new assay, we demonstrate a significant elevation of the procoagulant activity of P+MP fractions from advanced (stage IV) solid tumor patients of varying histology’s (n=14; 0.88 ± 0.55 mU/mL) compared with normal subjects (p <0.001). Furthermore, we showed that on average 50% of the procoagulant activity was TF-dependent (procoagulant activity was reduced to 0.57 ± 0.35 mU/mL in the presence of an anti-TF antibody; p < 0.01), suggesting that circulating BBTF in cancer patients has the potential to contribute to thrombosis in vivo. In summary, we have developed a novel assay that measures BBTF activity. This assay may be useful in the detection of a pre-thrombotic state in cancer patients.

scholarly journals Porcine Haemagglutinating Encephalomyelitis Virus Triggers Neural Autophagy Independent of ULK1

2021 ◽  
Author(s):  
Zi Li ◽  
Feng Gao ◽  
Yungang Lan ◽  
Jiyu Guan ◽  
Jing Zhang ◽  
...  
Keyword(s):  
Time Course ◽  
Ectopic Expression ◽  
Neuroblastoma Cells ◽  
Neurological Dysfunction ◽  
Global Public Health ◽  
Complex Interactions ◽  
Encephalomyelitis Virus ◽  
Bona Fide

Uncoordinated 51-like kinase 1 (ULK1) is a well-characterized initiator of canonical autophagy under basal or pathological conditions. Porcine haemagglutinating encephalomyelitis virus (PHEV), a neurotropic betacoronavirus (β-CoV), impairs ULK1 kinase but hijacks autophagy to facilitate viral proliferation. However, the machinery of PHEV-induced autophagy initiation upon ULK1 kinase deficiency remains unclear. Here, the time course of PHEV infection showed a significant accumulation of autophagosomes (APs) in nerve cells in vivo and in vitro. Utilizing the ULK1-knockout neuroblastoma cells, we have identified that ULK1 was not essential for productive AP formation induced by PHEV. In vitro phosphorylation studies discovered that mTORC1-regulated ULK1 activation stalls during PHEV infection, whereas the AP biogenesis was controlled by AMPK-driven BECN1 phosphorylation. A lack of BECN1 is sufficient to block LC3 lipidation and disrupt recruitment of the LC3-ATG14 complex. Moreover, BECN1 acts as a bona fide substrate for ULK1-independent neural autophagy, and ectopic expression of BECN1 somewhat enhances PHEV replication. These findings highlight a novel machinery of non-canonical autophagy independent of ULK1 that bypasses the conserved initiation circuit of AMPK-mTORC1-ULK1, providing new insights into the interplay between neurotropic β-CoV and the host. IMPORTANCE The ongoing COVID-19 pandemic alongside the outbreaks of SARS and MERS pose betacoronavirus (β-CoV) as a global public health challenge. Coronaviruses subvert, haijack, or utilize autophagy to promote proliferation, thus exploring the cross-talk between β-CoV and autophagy of great significance in confronting future β-CoV outbreaks. Porcine haemagglutinating encephalomyelitis virus (PHEV) is a highly neurotropic β-CoV and invades the central nervous system (CNS) in pigs, but understanding of the pathogenesis for PHEV-induced neurological dysfunction yet limited. Here, we discovered a novel regulatory principle of neural autophagy initiation during PHEV infection, where productive autophagosome (AP) biogenesis bypassing the multifaceted regulation of ULK1 kinase. The PHEV-triggered non-canonical autophagy underscores the complex interactions of virus-host, and will help in the development of therapeutic strategies targeting non-canonical autophagy to treat β-CoV disease.

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