scholarly journals Mutagenesis of the ADAM17-phosphatidylserine–binding motif leads to embryonic lethality in mice

2019 ◽  
Vol 2 (5) ◽  
pp. e201900430 ◽  
Author(s):  
Martin Veit ◽  
Björn Ahrens ◽  
Jana Seidel ◽  
Anselm Sommer ◽  
Sucharit Bhakdi ◽  
...  
Keyword(s):  
Amino Acids ◽  
Embryonic Lethality ◽  
Binding Motif ◽  
The Novel ◽  
Primary Hepatocytes ◽  
Cellular Functions ◽  
Anionic Phospholipid ◽  
Adam Family ◽  
Novel Concept

ADAM17, prominent member of the “Disintegrin and Metalloproteinase” (ADAM) family, controls vital cellular functions through cleavage of transmembrane substrates. Several of these play central roles in oncogenesis and inflammation, yet despite its importance, the mechanism by which ADAM17 is activated is not fully understood. We recently presented evidence that surface exposure of phosphatidylserine (PS) is the penultimate event required for sheddase activation, which occurs upon binding of a membrane-proximal, cationic binding motif to the anionic phospholipid headgroup. Here, we show that mutagenesis of the 3 amino acids constituting the PS-binding motif leads to embryonic lethality in mice. Heterozygotes showed no abnormalities. Primary hepatocytes and fibroblasts were analysed and found to express the mutant protease on the cell surface. However, PMA-stimulated release of ADAM17 substrates was completely abolished. The results directly support the novel concept of transiently externalised PS as essential trigger of extracellular protease function in vivo.

In vitro hydrodynamics of the Embol-X cannula

Perfusion ◽  
2002 ◽  
Vol 17 (2) ◽  
pp. 153-156 ◽  
Author(s):  
Anja Gerdes ◽  
Thorsten Hanke ◽  
Hans-H Sievers
Keyword(s):  
In Vivo Studies ◽  
Clinical Effects ◽  
Pressure Gradients ◽  
The Novel ◽  
Flow Rates ◽  
Mock Circulation ◽  
Aortic Cannula ◽  
Novel Concept

Background: Prevention of intraoperative plaque dislodgement in patients with atherosclerotic ascending aorta by development of innovative aortic cannula designs gains growing interest in cardiac surgery. To increase knowledge about the hydrodynamics of the innovative Embol-X™ cannula, which includes an intra-aortic filter device targeting at atheromatous emboli capture, was the aim of the present study. Methods: Pressure gradients and back pressures of the Embol-X™ cannula were measured at varying flow rates in a mock circulation and compared with two commonly used single-stream cannulae. Results: At a flow rate of 5.5 l/min, pressure gradients across the Argyle™ and the RMI cannulae were 48% and 62% and back pressures 25% and 47% lower than the corresponding values across the Embol-X™ cannula. Conclusions: The novel concept of integrating a filter device may provide clinical advantages concerning neurologic outcome. Further in vivo studies seem to be desirable to obtain more information concerning the clinical effects of the Embol-X™ cannula hydrodynamics.

Clinical Studies of New Icodextrin Formulations

1994 ◽  
Vol 14 (2_suppl) ◽  
pp. 55-57 ◽  
Author(s):  
Chandra D. Mistry ◽  
B. Bhowmick ◽  
Ruth Ashman ◽  
L. Uttley
Keyword(s):  
Glucose Solution ◽  
Single Agent ◽  
The Novel ◽  
Safe Level ◽  
Dialysis Solution ◽  
Osmotic Agent ◽  
Novel Concept ◽  
Osmotic Agents ◽  
Unique Potential

The osmotic effectiveness of icodextrin as a “colloid” osmotic agent has been well established and shown to be particularly well suited for long dwell exchanges (8 12 hours). More recently, its advanced application generated the first in vivo demonstration of sustained ultrafiltration with hypo-osmolar dialysis solution (i.e., against the osmolality gradient). This led to the “novel” concept of an iso-osmolar combination of “crystalloid” and “colloid” osmotic agents to achieve a range of ultrafiltration optimized for dwell time. In accordance with our objective of developing physiological solutions for all continuous ambulatory peritoneal dialysis (CAPD) exchanges, we tested this hypothesis by comparing an iso-osmolar combination of 2.0% -2.5% of dextrin 20 (Mw 20000 dalton; Mn 5800 dalton) and 0.68% glucose with 1.36% glucose solution in 11 CAPD patients over a 6-hour dwell. An iso-osmolar icodextrin mixture produced ultrafiltration of similar magnitude to that of a 1.36% glucose solution with less than half the calorie load. This solution, therefore, has the potential to replace all the currently used hyperosmolar exchanges with physiological solutions containing half the daily calorie load without exceeding the current safe level of maltose accumulation. This also offers a unique potential for a combination of unrelated but compatible agents, each with its own metabolic advantages, which would offer a wider spectrum of metabolic advantages than a single agent alone.

scholarly journals Rapid changes in chromatin structure during dedifferentiation of primary hepatocytes in vitro

2020 ◽  
Author(s):  
Morten Seirup ◽  
Srikumar Sengupta ◽  
Scott Swanson ◽  
Brian E. McIntosh ◽  
Mike Colins ◽  
...  
Keyword(s):  
Late Phase ◽  
Chromatin Accessibility ◽  
Binding Motif ◽  
Open Chromatin ◽  
Primary Hepatocytes ◽  
Motif Analysis ◽  
Full Spectrum ◽  
Rapid Changes

AbstractPrimary hepatocytes are widely used in the pharmaceutical industry to screen drug candidates for hepatotoxicity, but isolated hepatocytes quickly dedifferentiate and lose their mature metabolic function in culture. Attempts have been made to better recapitulate the in vivo liver environment in culture, but the full spectrum of signals required to maintain hepatocyte function in vitro remains elusive. Here we studied the dedifferentiation process in detail through RNA-sequencing of hepatocytes cultured over eight days. We identified three distinct phases of dedifferentiation. An early phase, where mature hepatocyte genes are rapidly downregulated in a matter of hours. A middle phase, where fetal genes are activated, leading to hepatocytes with a fetal phenotype. A late phase, where initially rare contaminating non-parenchymal cells over-grow the culture as the hepatocytes gradually die. Using genetically tagged hepatocytes, we demonstrate that the cells reactivating fetal marker alpha-fetoprotein arise from cells previously expressing the mature hepatocyte marker albumin, and not from albumin negative precursor cells, proving that hepatocytes undergo true dedifferentiation. To better understand the signaling events that result in the rapid down-regulation of mature hepatocyte genes, we examined changes in chromatin accessibility of hepatocytes during the first 24h of culture using ATAC-seq. We find that drastic and rapid changes in chromatin accessibility occurs immediately upon start of culture. Using binding motif analysis of the areas of open chromatin sharing similar temporal profiles, we identify several candidate transcription factors potentially involved in the dedifferentiation of primary hepatocytes in culture.

scholarly journals Requirements for Septal Localization and Chromosome Segregation Activity of the DNA Translocase SftA from Bacillus subtilis

2017 ◽  
Vol 27 (1) ◽  
pp. 29-42 ◽  
Author(s):  
Nina El Najjar ◽  
Christine Kaimer ◽  
Thomas Rösch ◽  
Peter L. Graumann
Keyword(s):  
Amino Acids ◽  
Bacillus Subtilis ◽  
Chromosome Segregation ◽  
Protein Interactions ◽  
Binding Motif ◽  
Cell Fractionation ◽  
Binding Studies ◽  
Mobility Shift ◽  
Septal Localization

<i>Bacillus subtilis</i> possesses 2 DNA translocases that affect late stages of chromosome segregation: SftA separates nonsegregated DNA prior to septum closure, while SpoIIIE rescues septum-entrapped DNA. We provide evidence that SftA is associated with the division machinery via a stretch of 47 amino acids within its N-terminus, suggesting that SftA is recruited by protein-protein interactions with a component of the division machinery. SftA was also recruited to mid-cell in the absence of its first 20 amino acids, which are proposed to contain a membrane-binding motif. Cell fractionation experiments showed that SftA can be found in the cytosolic fraction, and to a minor degree in the membrane fraction, showing that it is a soluble protein in vivo. The expression of truncated SftA constructs led to a dominant <i>sftA</i> deletion phenotype, even at very low induction rates of the truncated proteins, indicating that the incorporation of nonfunctional monomers into SftA hexamers abolishes functionality. Mobility shift experiments and surface plasmon binding studies showed that SftA binds to DNA in a cooperative manner, and demonstrated low ATPase activity when binding to short nucleotides rather than to long stretches of DNA.

scholarly journals Rational Design of Improved Pharmabiotics

2009 ◽  
Vol 2009 ◽  
pp. 1-7 ◽  
Author(s):  
Roy D. Sleator ◽  
Colin Hill
Keyword(s):  
Rational Design ◽  
Ex Vivo ◽  
Clinical Applications ◽  
Survival Strategies ◽  
The Novel ◽  
Recent Advances ◽  
Stress Survival ◽  
Novel Concept

Herein we review the most recent advances in probiotic research and applications with particular emphasis on the novel concept of patho-biotechnology: the application of pathogen-derived (ex vivo and in vivo) stress survival strategies for the design of more technologically robust and effective probiotic cultures with improved biotechnological and clinical applications.

scholarly journals Antagonism of STAT1 by Nipah virus P gene products modulates disease course but not lethal outcome in the ferret model

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Benjamin A. Satterfield ◽  
Viktoriya Borisevich ◽  
Stephanie L. Foster ◽  
Sergio E. Rodriguez ◽  
Robert W. Cross ◽  
...  
Keyword(s):  
Amino Acids ◽  
Nipah Virus ◽  
Minor Role ◽  
Published Data ◽  
Binding Motif ◽  
Type I ◽  
Disease Course ◽  
Similar Reduction ◽  
P Gene

Abstract Nipah virus (NiV) is a pathogenic paramyxovirus and zoononis with very high human fatality rates. Previous protein over-expression studies have shown that various mutations to the common N-terminal STAT1-binding motif of the NiV P, V, and W proteins affected the STAT1-binding ability of these proteins thus interfering with he JAK/STAT pathway and reducing their ability to inhibit type-I IFN signaling, but due to differing techniques it was unclear which amino acids were most important in this interaction or what impact this had on pathogenesis in vivo. We compared all previously described mutations in parallel and found the amino acid mutation Y116E demonstrated the greatest reduction in binding to STAT1 and the greatest reduction in interferon antagonism. A similar reduction in binding and activity was seen for a deletion of twenty amino acids constituting the described STAT1-binding domain. To investigate the contribution of this STAT1-binding motif in NiV-mediated disease, we produced rNiVs with complete deletion of the STAT1-binding motif or the Y116E mutation for ferret challenge studies (rNiVM-STAT1blind). Despite the reduced IFN inhibitory function, ferrets challenged with these rNiVM-STAT1blind mutants had a lethal, albeit altered, NiV-mediated disease course. These data, together with our previously published data, suggest that the major role of NiV P, V, and W in NiV-mediated disease in the ferret model are likely to be in the inhibition of viral recognition/innate immune signaling induction with a minor role for inhibition of IFN signaling.

scholarly journals Regulation of glucose-6-phosphate dehydrogenase synthesis and mRNA abundance in cultured rat hepatocytes

1991 ◽  
Vol 276 (1) ◽  
pp. 245-250 ◽  
Author(s):  
P Manos ◽  
R Nakayama ◽  
D Holten
Keyword(s):  
Energy Source ◽  
Amino Acids ◽  
Rat Hepatocytes ◽  
Fold Increase ◽  
G6pd Activity ◽  
Primary Hepatocytes ◽  
Cultured Rat Hepatocytes ◽  
Free Media ◽  
Glucose 6 Phosphate Dehydrogenase

Conditions were identified which, for the first time, demonstrate that primary hepatocytes can express the same range of glucose-6-phosphate dehydrogenase (G6PD) synthesis and mRNA as in live rats. Primary hepatocytes were cultured without prior exposure to serum, hormones or carbohydrates. Five modulators implicated in G6PD induction in vivo were examined: insulin, dexamethasone, tri-iodothyronine (T3), glucose and fructose, T3 did not affect G6PD activity, and did not interact with carbohydrate to affect the activity of G6PD. Neither glucose nor fructose alone affected G6PD activity, and they did not interact with insulin to increase G6PD activity. Hepatocytes isolated from fasted rats and cultured in serum-free media with amino acids ad the only energy source how a 12-fold increase in G6PD synthesis and mRNA (measured by a solution-hybridization assay). This induction does not require added hormones or carbohydrate. The addition of insulin alone caused another increase in G6PD synthesis and mRNA. There are at least three distinct phases to G6PD induction under these conditions. The largest increase in G6PD synthesis (12-fold) occurs in the absence of any hormones and with amino acids as the only energy source. This phase is due to increased G6PD mRNA. Insulin causes an additional 2-3-fold increase in G6PD synthesis and mRNA. However, dexamethasone and insulin are both required before G6PD synthesis is equal to that in rats which are fasted and refed on a high-carbohydrate diet.

SAC3B is a target of CML19, the centrin 2 of Arabidopsis thaliana

2020 ◽  
Vol 477 (1) ◽  
pp. 173-189 ◽  
Author(s):  
Marco Pedretti ◽  
Carolina Conter ◽  
Paola Dominici ◽  
Alessandra Astegno
Keyword(s):  
Excision Repair ◽  
Complex Structure ◽  
Binding Motif ◽  
C Terminus ◽  
Repair Protein ◽  
Nucleotide Excision ◽  
Ef Hand ◽  
Molecular Determinants ◽  
Structure In Solution

Arabidopsis centrin 2, also known as calmodulin-like protein 19 (CML19), is a member of the EF-hand superfamily of calcium (Ca2+)-binding proteins. In addition to the notion that CML19 interacts with the nucleotide excision repair protein RAD4, CML19 was suggested to be a component of the transcription export complex 2 (TREX-2) by interacting with SAC3B. However, the molecular determinants of this interaction have remained largely unknown. Herein, we identified a CML19-binding site within the C-terminus of SAC3B and characterized the binding properties of the corresponding 26-residue peptide (SAC3Bp), which exhibits the hydrophobic triad centrin-binding motif in a reversed orientation (I8W4W1). Using a combination of spectroscopic and calorimetric experiments, we shed light on the SAC3Bp–CML19 complex structure in solution. We demonstrated that the peptide interacts not only with Ca2+-saturated CML19, but also with apo-CML19 to form a protein–peptide complex with a 1 : 1 stoichiometry. Both interactions involve hydrophobic and electrostatic contributions and include the burial of Trp residues of SAC3Bp. However, the peptide likely assumes different conformations upon binding to apo-CML19 or Ca2+-CML19. Importantly, the peptide dramatically increases the affinity for Ca2+ of CML19, especially of the C-lobe, suggesting that in vivo the protein would be Ca2+-saturated and bound to SAC3B even at resting Ca2+-levels. Our results, providing direct evidence that Arabidopsis SAC3B is a CML19 target and proposing that CML19 can bind to SAC3B through its C-lobe independent of a Ca2+ stimulus, support a functional role for these proteins in TREX-2 complex and mRNA export.

Uptake, Internalization and Degradation of the Novel Plasminogen Activator Reteplase (BM 06.022) in the Rat

1995 ◽  
Vol 74 (06) ◽  
pp. 1501-1510 ◽  
Author(s):  
J Kuiper ◽  
H van de Bilt ◽  
U Martin ◽  
Th J C van Berkel
Keyword(s):  
Plasminogen Activator ◽  
Liver Cells ◽  
The Novel ◽  
Uptake Mechanism ◽  
Liver Uptake ◽  
Lysosomal Compartment ◽  
Β Phase ◽  
Α Phase ◽  
Parenchymal Liver

SummaryThe catabolism of the novel plasminogen activator reteplase (BM 06.022) was described. For this purpose BM 06.022 was radiolabelled with l25I or with the accumulating label l25I-tyramine cellobiose (l25I-TC).BM 06.022 was injected at a pharmacological dose of 380 μg/kg b.w. and it was cleared from the plasma in a biphasic manner with a half-life of about 1 min in the α-phase and t1/2of 20-28 min in the β-phase. 28% and 72% of the injected dose was cleared in the α-phase and β-phase, respectively. Initially liver, kidneys, skin, bones, lungs, spleen, and muscles contributed mainly to the plasma clearance. Only liver and the kidneys, however, were responsible for the uptake and subsequent degradation of BM 06.022 and contributed for 75% to the catabolism of BM 06.022. BM 06.022 was degraded in the lysosomal compartment of both organs. Parenchymal liver cells were responsible for 70% of the liver uptake of BM 06.022. BM 06.022 associated rapidly to isolated rat parenchymal liver cells and was subsequently degraded in the lysosomal compartment of these cells. BM 06.022 bound with low-affinity to the parenchymal liver cells (550 nM) and the binding of BM 06.022 could be displaced by t-PA (IC50 5.6 nM), indicating that the low-density lipoprotein receptor-related protein (LRP) could be involved in the binding of BM 06.022. GST-RAP, which is an inhibitor of LRP, could in vivo significantly inhibit the uptake of BM 06.022 in the liver.It is concluded that BM 06.022 is metabolized primarily in the liver and the kidneys. These organs take up and degrade BM 06.022 in the lysosomes. The uptake mechanism of BM 06.022 in the kidneys is unknown, while LRP is responsible for a low-affinity binding and uptake of BM 06.022 in parenchymal liver cells.

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