Stage-specific tyrosine phosphorylation of actin in Dictyostelium discoideum cells

1992 ◽  
Vol 102 (3) ◽  
pp. 601-609 ◽  
Author(s):  
A. Schweiger ◽  
O. Mihalache ◽  
M. Ecke ◽  
G. Gerisch
Keyword(s):  
Tyrosine Phosphorylation ◽  
Dictyostelium Discoideum ◽  
Binding Activity ◽  
Amino Acid Residues ◽  
Physiological Conditions ◽  
Cytoskeleton Organization ◽  
Electrophoretic Behavior ◽  
In Vivo Labelling ◽  
Phosphotyrosine Phosphatases

A 45 kDa protein in Dictyostelium discoideum cells that was recognized by a phosphotyrosine-specific antibody was identified by its binding activity to DNase I and its 2D-electrophoretic behavior as actin. The reactivity of actin with the antibody was transiently enhanced for about 30 minutes shortly after starving cells were reintroduced into nutrient medium. This effect indicates a modification of actin that is regulated under physiological conditions. A similar effect was obtained when growing cells were treated with phenylarsine oxide (PAO), an inhibitor of phosphotyrosine phosphatases. This effect was reversed and the cells fully recovered upon addition of the PAO antagonist 2,3-dimercaptopropanol. Starved cells did not show this enhancement of antibody labelling, which indicates that the response to PAO depends on the developmental stage. Phosphorylated amino acid residues were identified after in vivo labelling with [32P]phosphate in the presence of PAO. Part of the radioactivity in the actin band was recovered as phosphotyrosine, another part as phosphoserine. PAO caused the cells to form elongated blebs, to round up and finally to become immobilized. Fluorescence labelling with phalloidin of cells that were fixed at different times of PAO treatment revealed a progressive decrease in the staining for actin filaments and showed that these alterations in cytoskeleton organization were readily reversible, in accordance with the reversal of tyrosine phosphorylation at actin.

Investigation of a potential role for aldose reductase AlrA in tetrahydropteridine synthesis in Dictyostelium discoideum Ax2

Pteridines ◽  
2017 ◽  
Vol 28 (2) ◽  
pp. 97-103
Author(s):  
Hye-Lim Kim ◽  
Hyun-Chul Ryu ◽  
Young Shik Park
Keyword(s):  
Dictyostelium Discoideum ◽  
Aldose Reductase ◽  
Potential Role ◽  
Amino Acid Residues ◽  
Wild Type ◽  
High Sequence Identity ◽  
Physiological Conditions ◽  
Antioxidant Function ◽  
Cellular Extract ◽  
Sr Gene

AbstractDictyostelium discoideum Ax2 is well-known for the synthesis of d-threo-tetrahydrobiopterin (DH4) with a smaller amount of l-erythro-tetrahydrobiopterin (BH4). DH4 synthesis from 6-pyruvoyltetrahydropterin (PPH4) is catalyzed by aldose reductase (AR)-like protein and sepiapterin reductase (SR) via an intermediate 1′-oxo-2′-d-hydroxypropyl tetrahydropterin, which is non-enzymatically oxidized to d-sepiapterin in the absence of SR. However, l-sepiapterin was a dominant product in the reaction of a cellular extract of spr− disrupted in the SR gene. In order to investigate its potential role in tetrahydropteridine synthesis, the enzyme catalyzing l-sepiapterin synthesis from PPH4 was purified from spr−. Via mass spectrometry, the protein was identified to be encoded by alrA. AlrA consists of 297 amino acid residues sharing a high sequence identity with human AR. However, in the co-incubation assay, DH4 synthesis was not detected and, furthermore, the recombinant AlrA was observed to suppress BH4 synthesis by SR, which was known to prefer 1′-oxo-2′-d-hydroxypropyl tetrahydropterin to PPH4. Although intracellular DH4 level in alrA− was decreased to 60% of the wild type, it is presumed to result from the antioxidant function of DH4. Therefore, despite the structural and catalytic identities with human AR, AlrA seems to be involved in neither BH4, nor DH4 synthesis under normal physiological conditions.

Anti-Thrombotic Effects of Targeting Outside-in αIIbβ3 Signaling in Platelets

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 411-411
Author(s):  
Ararat J. Ablooglu ◽  
Jian Kang ◽  
Brian G. Petrich ◽  
Mark H. Ginsberg ◽  
Sanford J. Shattil
Keyword(s):  
Carotid Artery ◽  
Tyrosine Phosphorylation ◽  
Amino Acid Residues ◽  
Spontaneous Bleeding ◽  
Src Signaling ◽  
Fibrinogen Binding ◽  
Vessel Injury ◽  
Alternative Approach ◽  
Platelet Spreading

Abstract Platelet activation by agonists stimulates high affinity binding of adhesive ligands, such as fibrinogen, to integrin αIIbβ3. In turn, ligand binding initiates Src kinase-dependent outside-in signaling, platelet spreading on sub-endothelial matrices, and platelet aggregation. Short-term integrin blockade by parenteral αIIbβ3 antagonists is effectively anti-thrombotic in certain clinical situations, but long-term blockade by oral αIIbβ3 antagonists has been problematic. As an alternative approach, we evaluated the feasibility of disrupting outside-in αIIbβ3 signaling in vivo. A “β3(Δ760-762)” knock-in mouse was generated that lacked the three C-terminal β3 amino acid residues (RGT) necessary for αIIbβ3-dependent c-Src signaling, but retained β3 residues necessary for agonist- and talin-dependent fibrinogen binding. β3(Δ760-762) mice were compared with wild-type β3+/+ littermates, β3+/− heterozygotes, and “β3/β1(EGK)” knock-in mice where β3 RGT was replaced by three C-terminal residues in β1 (EGK) to potentially enable outside-in integrin signaling by certain Src kinases other than c-Src. Whereas β3+/+, β3+/− and β3/β1(EGK) platelets spread and underwent tyrosine phosphorylation normally on fibrinogen, β3(Δ760-762) platelets spread poorly and exhibited reduced tyrosine phosphorylation of c-Src substrates, including the β3 cytoplasmic domain itself (Tyr-747). Furthermore, unlike the three groups of control mice, all of the β3(Δ760-762) mice tested were protected from carotid artery occlusive thrombosis following vessel injury with FeCl3. The median tail bleeding time for the β3(Δ760-762) mice was approximately two-fold longer than that of the control mice. However, β3(Δ760-762) mice did not exhibit spontaneous bleeding, excess bleeding after surgical exposure of the carotid artery, fecal blood loss or anemia. Fibrinogen binding to β3(Δ760-762) platelets was normal in response to saturating concentrations of agonists to the PAR4 thrombin receptor or the GP VI collagen receptor; however, responses to ADP were impaired. These studies establish that deletion of β3 RGT disrupts outside-in αIIbβ3 signaling and confers protection from arterial thrombosis in vivo. Currently available anti-platelet drugs either inhibit agonist-induced activation of αIIbβ3 or block fibrinogen binding to the integrin. The studies reported here suggest that targeting outside-in αIIbβ3 signaling may represent an alternative anti-thrombotic strategy.

scholarly journals Differential sorting of tyrosine kinases and phosphotyrosine phosphatases acting on band 3 during vesiculation of human erythrocytes

2004 ◽  
Vol 377 (2) ◽  
pp. 489-497 ◽  
Author(s):  
Giampaolo MINETTI ◽  
Annarita CIANA ◽  
Cesare BALDUINI
Keyword(s):  
Tyrosine Phosphorylation ◽  
Tyrosine Kinases ◽  
Membrane Skeleton ◽  
Band 3 ◽  
Human Erythrocytes ◽  
Parent Cell ◽  
Ionophore A23187 ◽  
Tight Association ◽  
Phosphotyrosine Phosphatases

One of the most intensively studied post-translational modifications of erythrocyte proteins is the phosphorylation of tyrosine residues of band 3, which is strictly regulated in vivo by PTKs (protein-tyrosine kinases) and PTPs (protein-phosphotyrosine phosphatases). Two PTKs (p72syk and p56/53lyn) and two PTP activities (PTP1B and SHPTP-2) have been immunologically identified so far in mature human erythrocytes. We have shown previously that band 3 undergoes tyrosine phosphorylation upon a decrease in cell volume, as occurs when erythrocytes treated with Ca2+/Ca2+ ionophore (A23187) lose KCl and release microvesicles. Similar levels of band 3 tyrosine phosphorylation in vesicles and in the parent cells are induced by this treatment. However, we have found that tyrosine phosphorylation of band 3 in vesicles is more stable than in whole erythrocytes. Examination of how the identified PTPs and PTKs are partitioned between the vesicles and the remnant cells during vesiculation reveals that PTP1B, unlike the PTKs, is retained entirely in the parent cell compartment. Since a tight association between PTP1B and band 3 has been documented previously, we have investigated the partitioning of PTP1B and band 3 between the membrane and the membrane-skeletal fractions prepared from resting or Ca2+/A23187-treated cells. Our results rule out the possibility that the preferential retention of PTP1B within the cell was due to an increase in the amount of membrane-skeleton-associated band 3 (and of PTP1B) during the release of spectrin-free vesicles, suggesting a more complex modality of interaction of PTP1B with band 3 in the erythrocyte membrane. Analysis of erythrocytes of different cell ages revealed that PTP1B, unlike the other enzymes examined, was quantitatively conserved during erythrocyte aging. This suggests important roles for the down-regulation of tyrosine phosphorylation of band 3 in erythrocyte physiology, and for vesiculation as a mechanism of human erythrocyte senescence.

scholarly journals Genetic analysis of β1 integrin “activation motifs” in mice

2006 ◽  
Vol 174 (6) ◽  
pp. 889-899 ◽  
Author(s):  
Aleksandra Czuchra ◽  
Hannelore Meyer ◽  
Kyle R. Legate ◽  
Cord Brakebusch ◽  
Reinhard Fässler
Keyword(s):  
Genetic Analysis ◽  
Tyrosine Phosphorylation ◽  
Aspartic Acid ◽  
Salt Bridge ◽  
Β1 Integrin ◽  
Cytoplasmic Domains ◽  
Integrin Activation ◽  
Physiological Conditions ◽  
Npxy Motif

Akey feature of integrins is their ability to regulate the affinity for ligands, a process termed integrin activation. The final step in integrin activation is talin binding to the NPXY motif of the integrin β cytoplasmic domains. Talin binding disrupts the salt bridge between the α/β tails, leading to tail separation and integrin activation. We analyzed mice in which we mutated the tyrosines of the β1 tail and the membrane-proximal aspartic acid required for the salt bridge. Tyrosine-to-alanine substitutions abolished β1 integrin functions and led to a β1 integrin–null phenotype in vivo. Surprisingly, neither the substitution of the tyrosines with phenylalanine nor the aspartic acid with alanine resulted in an obvious defect. These data suggest that the NPXY motifs of the β1 integrin tail are essential for β1 integrin function, whereas tyrosine phosphorylation and the membrane-proximal salt bridge between α and β1 tails have no apparent function under physiological conditions in vivo.

scholarly journals STAT3 serine phosphorylation by ERK-dependent and -independent pathways negatively modulates its tyrosine phosphorylation.

1997 ◽  
Vol 17 (11) ◽  
pp. 6508-6516 ◽  
Author(s):  
J Chung ◽  
E Uchida ◽  
T C Grammer ◽  
J Blenis
Keyword(s):  
Growth Factors ◽  
Tyrosine Phosphorylation ◽  
Map Kinases ◽  
Nuclear Translocation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Binding Activity ◽  
Serine Phosphorylation

Recent studies have indicated that serine phosphorylation regulates the activities of STAT1 and STAT3. However, the kinase(s) responsible and the role of serine phosphorylation in STAT function remain unresolved. In the present studies, we examined the growth factor-dependent serine phosphorylation of STAT1 and STAT3. We provide in vitro and in vivo evidence that the ERK family of mitogen-activated protein (MAP) kinases, but not JNK or p38, specifically phosphorylate STAT3 at serine 727 in response to growth factors. Evidence for additional mitogen-regulated serine phosphorylation is also provided. STAT1 is a relatively poor substrate for all MAP kinases tested both in vitro and in vivo. STAT3 serine phosphorylation, not its tyrosine phosphorylation, results in retarded mobility of the STAT3 protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Importantly, serine 727 phosphorylation negatively modulates STAT3 tyrosine phosphorylation, which is required for dimer formation, nuclear translocation, and the DNA binding activity of this transcriptional regulator. Interestingly, the cytokine interleukin-6 also stimulates STAT3 serine phosphorylation, but in contrast to growth factors, this occurs by an ERK-independent process.

Supramolecular Enhancement of Aminoxyl ORCA Contrast Agents

2019 ◽  
Author(s):  
Hamilton Lee ◽  
Jenica Lumata ◽  
Michael A. Luzuriaga ◽  
Candace Benjamin ◽  
Olivia Brohlin ◽  
...  
Keyword(s):  
Magnetic Resonance Imaging ◽  
Side Effects ◽  
Magnetic Resonance ◽  
Tobacco Mosaic Virus ◽  
Contrast Agents ◽  
Single Molecule ◽  
Organic Radical ◽  
Resonance Imaging ◽  
Physiological Conditions

<div><div><div><p>Many contrast agents for magnetic resonance imaging are based on gadolinium, however side effects limit their use in some patients. Organic radical contrast agents (ORCAs) are potential alternatives, but are reduced rapidly in physiological conditions and have low relaxivities as single molecule contrast agents. Herein, we use a supramolecular strategy where cucurbit[8]uril binds with nanomolar affinities to ORCAs and protects them against biological reductants to create a stable radical in vivo. We further over came the weak contrast by conjugating this complex on the surface of a self-assembled biomacromolecule derived from the tobacco mosaic virus.</p></div></div></div>

In-vivo anti-oxidant screening of Tectona grandis extract

2020 ◽  
Vol 9 (1) ◽  
pp. 1-4
Author(s):  
Tamilarasi G P ◽  
Sabarees G
Keyword(s):  
Biochemical Parameters ◽  
Tectona Grandis ◽  
The Body ◽  
Altered Expression ◽  
Physiological Conditions ◽  
Synthetic Drugs ◽  
Significant Difference ◽  
Anti Oxidant ◽  
Radical Generation

Oxidation is an essential reaction in the human body, which determines the expression of proteins in the body. This results in the altered expression like rapid growth resulting in cancers and other disorders. Many synthetic drugs are available in the market that is effective in limiting the free radical generation and the reaction of radicals with cells. Unfortunately, all those synthetic drugs were found to cause side effects and adverse effects in the body. But given the accuracy of the predictability of the results and administration, this research focuses on testing the anti-oxidant efficiency in rat models testing the biochemical parameters. Investigations have also been done on the anti-oxidant activity of Tectona, but every research was concentrated to prove the anti-oxidant activity only. extract had been tested for anti-oxidant activity by estimating various tissue parameters and it showed better activity. As predicted, there is a significant difference in the and results which can be explained are due to the physiological conditions that exist inside the body.

Oxytocin analogues with non-coded amino acid residues in position 8: [8-Neopentylglycine]oxytocin and [8-cycloleucine]oxytocin

1987 ◽  
Vol 52 (9) ◽  
pp. 2317-2325 ◽  
Author(s):  
Jan Hlaváček ◽  
Jan Pospíšek ◽  
Jiřina Slaninová ◽  
Walter Y. Chan ◽  
Victor J. Hruby
Keyword(s):  
Amino Acid ◽  
Solid Phase Synthesis ◽  
Solid Phase ◽  
The Other ◽  
Amino Acid Residues ◽  
Phase Synthesis ◽  
Other Hand ◽  
Fragment Condensation

[8-Neopentylglycine]oxytocin (II) and [8-cycloleucine]oxytocin (III) were prepared by a combination of solid-phase synthesis and fragment condensation. Both analogues exhibited decreased uterotonic potency in vitro, each being about 15-30% that of oxytocin. Analogue II also displayed similarly decreased uterotonic potency in vivo and galactogogic potency. On the other hand, analogue III exhibited almost the same potency as oxytocin in the uterotonic assay in vivo and in the galactogogic assay.

Synthesis of Fragments of the Vasoactive Intestinal Peptide (VIP) and Analysis of Their Residual Biological Activities

1995 ◽  
Vol 60 (7) ◽  
pp. 1229-1235 ◽  
Author(s):  
Ivana Zoulíková ◽  
Ivan Svoboda ◽  
Jiří Velek ◽  
Václav Kašička ◽  
Jiřina Slaninová ◽  
...  
Keyword(s):  
Amino Acid ◽  
Biological Activities ◽  
Residual Activity ◽  
Detailed Examination ◽  
Amino Acid Residues ◽  
Linear Peptide ◽  
Zone Electrophoresis ◽  
Capillary Zone

The vasoactive intestinal (poly)peptide (VIP) is a linear peptide containing 28 amino acid residues, whose primary structure indicates a low metabolic stability. The following VIP fragments, as potential metabolites, and their analogues were prepared by synthesis on a solid: [His(Dnp)1]VIP(1-10), VIP(11-14), [D-Arg12]VIP(11-14), [Lys(Pac)15,21,Arg20]VIP(15-22), and VIP(23-28). After purification, the peptides were characterized by amino acid analysis, mass spectrometry, RP HPLC, and capillary zone electrophoresis. In some tests, detailed examination of the biological activity of the substances in vivo and in vitro gave evidence of a low, residual activity of some fragments, viz. a depressoric activity in vivo for [His(Dnp)1]VIP(1-10) and a stimulating activity for the release of α-amylase in vitro and in vivo for [Lys(Pac)15,21,Arg20]VIP(15-22) and VIP(23-28).

scholarly journals Novel DNA Motif Binding Activity Observed In Vivo With an Estrogen Receptor α Mutant Mouse

2014 ◽  
Vol 28 (6) ◽  
pp. 899-911 ◽  
Author(s):  
Sylvia C. Hewitt ◽  
Leping Li ◽  
Sara A. Grimm ◽  
Wipawee Winuthayanon ◽  
Katherine J. Hamilton ◽  
...  
Keyword(s):  
Dna Binding ◽  
Target Gene ◽  
Mutant Mouse ◽  
Estrogen Receptor Α ◽  
Binding Activity ◽  
Estrogen Response Element ◽  
Dna Binding Domain ◽  
Estrogen Response ◽  
Uterine Growth

Abstract Estrogen receptor α (ERα) interacts with DNA directly or indirectly via other transcription factors, referred to as “tethering.” Evidence for tethering is based on in vitro studies and a widely used “KIKO” mouse model containing mutations that prevent direct estrogen response element DNA- binding. KIKO mice are infertile, due in part to the inability of estradiol (E2) to induce uterine epithelial proliferation. To elucidate the molecular events that prevent KIKO uterine growth, regulation of the pro-proliferative E2 target gene Klf4 and of Klf15, a progesterone (P4) target gene that opposes the pro-proliferative activity of KLF4, was evaluated. Klf4 induction was impaired in KIKO uteri; however, Klf15 was induced by E2 rather than by P4. Whole uterine chromatin immunoprecipitation-sequencing revealed enrichment of KIKO ERα binding to hormone response elements (HREs) motifs. KIKO binding to HRE motifs was verified using reporter gene and DNA-binding assays. Because the KIKO ERα has HRE DNA-binding activity, we evaluated the “EAAE” ERα, which has more severe DNA-binding domain mutations, and demonstrated a lack of estrogen response element or HRE reporter gene induction or DNA-binding. The EAAE mouse has an ERα null–like phenotype, with impaired uterine growth and transcriptional activity. Our findings demonstrate that the KIKO mouse model, which has been used by numerous investigators, cannot be used to establish biological functions for ERα tethering, because KIKO ERα effectively stimulates transcription using HRE motifs. The EAAE-ERα DNA-binding domain mutant mouse demonstrates that ERα DNA-binding is crucial for biological and transcriptional processes in reproductive tissues and that ERα tethering may not contribute to estrogen responsiveness in vivo.

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