Effects of heterodimerization and proteolytic processing on Derrière and Nodal activity: implications for mesoderm induction in Xenopus

Development ◽  
2002 ◽  
Vol 129 (13) ◽  
pp. 3089-3103 ◽  
Author(s):  
Peter M. Eimon ◽  
Richard M. Harland
Keyword(s):  
Feedback Loop ◽  
Expression System ◽  
Direct Interaction ◽  
Proteolytic Processing ◽  
Dominant Negative ◽  
Mesoderm Induction ◽  
Mesoderm Formation ◽  
Oocyte Expression System ◽  
The Right ◽  
Tgfβ Superfamily

Derrière is a recently discovered member of the TGFβ superfamily that can induce mesoderm in explant assays and is expressed at the right time and location to mediate mesoderm induction in response to VegT during Xenopus embryogenesis. We show that the ability of Derrière to induce dorsal or ventral mesoderm depends strictly on the location of expression and that a dominant-negative Derrière cleavage mutant completely blocks all mesoderm formation when ectopically expressed. This differs from the activity of similar Xnr2 cleavage mutant constructs, which are secreted and retain signaling activity. Additional analysis of mesoderm induction by Derrière and members of the Nodal family indicates that these molecules are involved in a mutual positive-feedback loop and antagonism of either one of the signals can reduce the other. Interaction between Derrière and members of the Nodal family is also shown to occur through the formation of heterodimeric ligands. Using an oocyte expression system we show direct interaction between the mature Derrière ligand and members of both the Nodal and BMP families. Taken together, these findings indicate that Derrière and Nodal proteins probably work cooperatively to induce mesoderm throughout the marginal zone during early Xenopus development.

VON WILLEBRAND FACTOR (vWF) PRO-POLYPEPTIDE IS REQUIRED FOR vWF MULTIMER FORMATION

1987 ◽  
Author(s):  
C L Verweij ◽  
M Hart ◽  
H Pannekoek
Keyword(s):  
Endothelial Cells ◽  
Von Willebrand Factor ◽  
Secretory Pathway ◽  
Vascular Endothelial Cells ◽  
Expression System ◽  
Proteolytic Processing ◽  
Information Encoding ◽  
Interchain Interactions ◽  
Von Willebrand ◽  
Willebrand Factor

The von Willebrand factor (vWF) is a multimeric plasma glycoprotein synthesized in vascular endothelial cells as a pre-pro-polypeptide with a highly repetitive domain structure, symbolized by the formula:(H)-D1-D2-D'-D3-A1-A2-A3-D4-B1-B2-B3-C1-C2-(0H).A heterologous expression system, consisting of a monkey kidney cell line (C0S-1), transfected with full-length vWF cDNA, is shown to mimic the constitutively, secretory pathway of vWF in endothelial cells. The assembly of pro-vWF into multimers and the proteolytic processing of these structures is found to oro-ceed along the following, consecutive steps. Pro-vWF subunits associate to form dimers, a process that does not involve the pro-polypeptide of pro-vWF. This observation is derived from transfection of C0S-1 cells with vWF cDNA, lacking the genetic information encoding the pro-polypeptide, composed of the domains D1 and D2. Pro-vWF dimers are linked intracellularly to form a regular series of multimeric structures that are secreted and cannot be distinguished from those released constitutively by endothelial cells. The presence of the pro-polypeptide, embedded in pro-vWF, is obligatory for multimerization since the deletion mutant lacking the D1 and D2 domains fails to assemble beyond the dimer stage. It is argued that the D domains are involved in interchain interactions.

scholarly journals An Endogenous RNA Transcript Antisense to CNGα1 Cation Channel mRNA

2002 ◽  
Vol 13 (10) ◽  
pp. 3696-3705 ◽  
Author(s):  
Chin-Hung Cheng ◽  
David Tai-Wai Yew ◽  
Hiu-Yee Kwan ◽  
Qing Zhou ◽  
Yu Huang ◽  
...  
Keyword(s):  
Expression System ◽  
Expression Patterns ◽  
Long Term Potentiation ◽  
Information Storage ◽  
Term Potentiation ◽  
Hippocampal Ca1 ◽  
Oocyte Expression System ◽  
Neuronal Functions ◽  
Rna Transcript

CNG channels are cyclic nucleotide-gated Ca2+-permeable channels that are suggested to be involved in the activity-dependent alterations of synaptic strength that are thought to underlie information storage in the CNS. In this study, we isolated an endogenous RNA transcript antisense to CNGα1 mRNA. This transcript was capable of down-regulating the expression of sense CNGα1 in theXenopus oocyte expression system. RT-PCR, Northern blot, and in situ hybridization analyses showed that the transcript was coexpressed with CNGα1 mRNA in many regions of human brain, notably in those regions that were involved in long-term potentiation and long-term depression, such as hippocampal CA1 and CA3, dentate gyrus, and cerebellar Purkinje layer. Comparison of expression patterns between adult and fetal cerebral cortex revealed that there were concurrent developmental changes in the expression levels of anti-CNG1 and CNGα1. Treatment of human glioma cell T98 with thyroid hormone T3 caused a significant increase in anti-CNG1 expression and a parallel decrease in sense CNGα1 expression. These data suggest that the suppression of CNGα1 expression by anti-CNG1 may play an important role in neuronal functions, especially in synaptic plasticity and cortical development. Endogenous antisense RNA-mediated regulation may represent a new mechanism through which the activity of ion channels can be regulated in the human CNS.

scholarly journals Specific and Nonspecific Effects of Protein Kinase C on the Epithelial Na + Channel

2000 ◽  
Vol 115 (5) ◽  
pp. 559-570 ◽  
Author(s):  
Mouhamed S. Awayda
Keyword(s):  
Membrane Trafficking ◽  
Expression System ◽  
Na Channel ◽  
Specific Effects ◽  
Nonspecific Effects ◽  
Oocyte Expression ◽  
Xenopus Oocyte Expression ◽  
Oocyte Expression System ◽  
Baseline Values ◽  
Epithelial Na Channel

The Xenopus oocyte expression system was used to explore the mechanisms of inhibition of the cloned rat epithelial Na+ channel (rENaC) by PKC (Awayda, M.S., I.I. Ismailov, B.K. Berdiev, C.M. Fuller, and D.J. Benos. 1996. J. Gen. Physiol. 108:49–65) and to determine whether human ENaC exhibits similar regulation. Effects of PKC activation on membrane and/or channel trafficking were determined using impedance analysis as an indirect measure of membrane area. hENaC-expressing oocytes exhibited an appreciable activation by hyperpolarizing voltages. This activation could be fit with a single exponential, described by a time constant (τ) and a magnitude (ΔI V). A similar but smaller magnitude of activation was also observed in oocytes expressing rENaC. This activation likely corresponds to the previously described effect of hyperpolarizing voltage on gating of the native Na+ channel (Palmer, L.G., and G. Frindt. 1996. J. Gen. Physiol. 107:35–45). Stimulation of PKC with 100 nM PMA decreased ΔIV in hENaC-expressing oocytes to a plateau at 57.1 ± 4.9% (n = 6) of baseline values at 20 min. Similar effects were observed in rENaC-expressing oocytes. PMA decreased the amiloride-sensitive hENaC slope conductance (gNa) to 21.7 ± 7.2% (n = 6) of baseline values at 30 min. This decrease was similar to that previously reported for rENaC. This decrease of g Na was attributed to a decrease of membrane capacitance (C m), as well as the specific conductance (gm/Cm ). The effects on gm/Cm reached a plateau within 15 min, at ∼60% of baseline values. This decrease is likely due to the specific ability of PKC to inhibit ENaC. On the other hand, the decrease of Cm was unrelated to ENaC and is likely an effect of PKC on membrane trafficking, as it was observed in ENaC-expressing as well as control oocytes. At lower PMA concentrations (0.5 nM), smaller changes of Cm were observed in rENaC- and hENaC-expressing oocytes, and were preceded by larger changes of gm and by changes of gm/Cm, indicating specific effects on ENaC. These findings indicate that PKC exhibits multiple and specific effects on ENaC, as well as nonspecific effects on membrane trafficking. Moreover, these findings provide the electrophysiological basis for assessing channel-specific effects of PKC in the Xenopus oocyte expression system.

PGE2 potentiates tonicity-induced COX-2 expression in renal medullary cells in a positive feedback loop involving EP2-cAMP-PKA signaling

2009 ◽  
Vol 296 (1) ◽  
pp. C75-C87 ◽  
Author(s):  
Daniela Steinert ◽  
Christoph Küper ◽  
Helmut Bartels ◽  
Franz-X. Beck ◽  
Wolfgang Neuhofer
Keyword(s):  
Osmotic Stress ◽  
Cell Survival ◽  
Positive Feedback ◽  
Feedback Loop ◽  
Mdck Cells ◽  
Dominant Negative ◽  
Dibutyryl Camp ◽  
Positive Feedback Loop ◽  
Cox 2 ◽  
Medullary Cells

Cyooxygenase-2 (COX-2)-derived PGE2 is critical for the integrity and function of renal medullary cells during antidiuresis. The present study extended our previous finding that tonicity-induced COX-2 expression is further stimulated by the major COX-2 product PGE2 and investigated the underlying signaling pathways and the functional relevance of this phenomenon. Hyperosmolality stimulated COX-2 expression and activity in Madin-Darby canine kidney (MDCK) cells, a response that was further increased by PGE2-cAMP signaling, suggesting the existence of a positive feedback loop. This effect was diminished by AH-6809, an EP2 antagonist, and by the PKA inhibitor H-89, but not by AH-23848, an EP4 antagonist. The effect of PGE2 was mimicked by forskolin and dibutyryl-cAMP, suggesting that the stimulatory effect of PGE2 on COX-2 is mediated by a cAMP-PKA-dependent mechanism. Accordingly, cAMP-responsive element (CRE)-driven reporter activity paralleled the effects of PGE2, AH-6809, AH-23848, H-89, forskolin, and dibutyryl-cAMP on COX-2 expression. In addition, the stimulatory effect of PGE2 on tonicity-induced COX-2 expression was blunted in cells transfected with dominant-negative CRE binding (CREB) protein, as was the case in a COX-2 promoter reporter construct in which a putative CRE was deleted. Furthermore, PGE2 resulted in PKA-dependent phosphorylation of the pro-apoptotic protein Bad at Ser155, a mechanism that is known to inactivate Bad, which coincided with reduced caspase-3 activity during osmotic stress. Conversely, pharmacological interruption of the PGE2-EP2-cAMP-PKA pathway abolished Ser155 phosphorylation of Bad and blunted the protective effect of PGE2 on cell survival during osmotic stress. These observations indicate the existence of a positive feedback loop of PGE2 on COX-2 expression during osmotic stress, an effect that apparently is mediated by EP2-cAMP-PKA signaling, and that contributes to cell survival under hypertonic conditions.

Expression of a novel FGF in the Xenopus embryo. A new candidate inducing factor for mesoderm formation and anteroposterior specification

Development ◽  
1992 ◽  
Vol 114 (3) ◽  
pp. 711-720 ◽  
Author(s):  
H.V. Isaacs ◽  
D. Tannahill ◽  
J.M. Slack
Keyword(s):  
Specific Activity ◽  
Biological Properties ◽  
The Body ◽  
Mesoderm Induction ◽  
Gastrula Stage ◽  
Blastula Stage ◽  
Mesoderm Formation ◽  
Low Levels

We have cloned and sequenced a new member of the fibroblast growth factor family from Xenopus laevis embryo cDNA. It is most closely related to both mammalian kFGF (FGF-4) and FGF-6 but as it is not clear whether it is a true homologue of either of these genes we provisionally refer to it as XeFGF (Xenopus embryonic FGF). Two sequences were obtained, differing by 11% in derived amino acid sequence, which probably represent pseudotetraploid variants. Both the sequence and the behaviour of in vitro translated protein indicates that, unlike bFGF (FGF-2), XeFGF is a secreted molecule. Recombinant XeFGF protein has mesoderm-inducing activity with a specific activity similar to bFGF. XeFGF mRNA is expressed maternally and zygotically with a peak during the gastrula stage. Both probe protection and in situ hybridization showed that the zygotic expression is concentrated in the posterior of the body axis and later in the tailbud. Later domains of expression were found near the midbrain/hindbrain boundary and at low levels in the myotomes. Because of its biological properties and expression pattern, XeFGF is a good candidate for an inducing factor with possible roles both in mesoderm induction at the blastula stage and in the formation of the anteroposterior axis at the gastrula stage.

scholarly journals CTRP9 Mediates Protective Effects in Cardiomyocytes via AMPK- and Adiponectin Receptor-Mediated Induction of Anti-Oxidant Response

Cells ◽  
2020 ◽  
Vol 9 (5) ◽  
pp. 1229
Author(s):  
Bernd Niemann ◽  
Ling Li ◽  
Dorothee Siegler ◽  
Benedikt H. Siegler ◽  
Fabienne Knapp ◽  
...  
Keyword(s):  
Transcriptional Activation ◽  
Direct Interaction ◽  
Left Ventricular ◽  
Cardiac Cells ◽  
Adiponectin Receptor ◽  
Protective Effects ◽  
Necrosis Factor Alpha ◽  
Anti Oxidant ◽  
Cardioprotective Effects ◽  
The Right

The C1q/tumor necrosis factor-alpha-related protein 9 (CTRP9) has been reported to exert cardioprotective effects, but its role in the right ventricle (RV) remains unclear. To investigate the role of CTRP9 in RV hypertrophy and failure, we performed pulmonary artery banding in weanling rats to induce compensatory RV hypertrophy seven weeks after surgery and RV failure 22 weeks after surgery. CTRP9 expression, signal transduction and mechanisms involved in protective CTRP9 effects were analyzed in rat and human RV tissue and cardiac cells. We demonstrate that CTRP9 was induced during compensatory RV hypertrophy but almost lost at the stage of RV failure. RV but not left ventricular (LV) cardiomyocytes or RV endothelial cells demonstrated increased intracellular reactive oxygen species (ROS) and apoptosis activation at this stage. Exogenous CTRP9 induced AMP-activated protein kinase (AMPK)-dependent transcriptional activation of the anti-oxidant thioredoxin-1 (Trx1) and superoxide dismutase-2 (SOD2) and reduced phenylephrine-induced ROS. Combined knockdown of adiponectin receptor-1 (AdipoR1) and AdipoR2 or knockdown of calreticulin attenuated CTRP9-mediated anti-oxidant effects. Immunoprecipitation showed an interaction of AdipoR1 with AdipoR2 and the co-receptor T-cadherin, but no direct interaction with calreticulin. Thus, CTRP9 mediates cardioprotective effects through inhibition of ROS production induced by pro-hypertrophic agents via AMPK-mediated activation of anti-oxidant enzymes.

scholarly journals rhEPO (recombinant human eosinophil peroxidase): expression in Pichia pastoris and biochemical characterization

2006 ◽  
Vol 395 (2) ◽  
pp. 295-301 ◽  
Author(s):  
Chiara Ciaccio ◽  
Alessandra Gambacurta ◽  
Giampiero DE Sanctis ◽  
Domenico Spagnolo ◽  
Christina Sakarikou ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Biochemical Characterization ◽  
Expression System ◽  
Gel Filtration ◽  
Proteolytic Processing ◽  
Kinetic Properties ◽  
Specific Substrate ◽  
Human Eosinophil ◽  
Eosinophil Peroxidase ◽  
Pichia Pastoris Expression System

A Pichia pastoris expression system has for the first time been successfully developed to produce rhEPO (recombinant human eosinophil peroxidase). The full-length rhEPO coding sequence was cloned into the pPIC9 vector in frame with the yeast α-Factor secretion signal under the transcriptional control of the AOX (acyl-CoA oxidase) promoter, and transformed into P. pastoris strain GS115. Evidence for the production of rhEPO by P. pastoris as a glycosylated dimer precursor of approx. 80 kDa was determined by SDS/PAGE and gel filtration chromatography. Recombinant hEPO undergoes proteolytic processing, similar to that in the native host, to generate two chains of approx. 50 and 20 kDa. A preliminary biochemical characterization of purified rhEPO demonstrated that the spectral and kinetic properties of the recombinant wild-type EPO are comparable with those of the native enzyme and are accompanied by oxidizing activity towards several physiological anionic substrates such as SCN−, Br− and Cl−. On the basis of the estimated Km and kcat values it is evident that the pseudohalide SCN− is the most specific substrate for rhEPO, consistent with the catalytic properties of other mammalian EPOs purified from blood.

scholarly journals Reversal of Bimanual Feedback Responses With Changes in Task Goal

2009 ◽  
Vol 101 (1) ◽  
pp. 283-288 ◽  
Author(s):  
Jörn Diedrichsen ◽  
Samantha Gush
Keyword(s):  
Force Field ◽  
Covariance Structure ◽  
Direct Interaction ◽  
Virtual Object ◽  
Upward Movement ◽  
Finish Line ◽  
Task Goal ◽  
The Right ◽  
Task Goals ◽  
Feedback Responses

We show that fast bimanual coordinative feedback responses can be reversed with changes in task goals. Participants moved a flexible virtual object across a finish line with an upward movement of both hands. In one condition, the middle of the object had to be aligned with a spatial goal at the end of the movement. In the second condition, the object had to be kept at a specific length. During the movement, a velocity-dependent force field was applied randomly to one of the hands to the left or to the right. Depending on the task condition, the unperturbed hand showed fast feedback corrections, either in or against the direction of the force field on the other hand. In the object-length condition we found evidence for a mixture of task goals: early in the movement the correction of the unperturbed hand was aimed at stabilizing object length; later in the movement, the correction reversed direction to reach a symmetric body posture in the end of the movement. The observed differences in feedback responses between task conditions also influenced the covariance structure of unperturbed movements and the adaptation when a specific force field was applied repeatedly to one of the hands. The results are congruent with the notion that coordination is established flexibly through a representation of the task-relevant controlled variables, rather than through a direct interaction between motor commands.

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