Two native types of phytochrome A, phyAʹ and phyAʺ, differ by the state of phosphorylation at the N-terminus as revealed by fluorescence investigations of the Ser/Ala mutant of rice phyA expressed in transgenic Arabidopsis

2018 ◽  
Vol 45 (2) ◽  
pp. 150 ◽  
Author(s):  
Vitaly A. Sineshchekov ◽  
Larissa A. Koppel ◽  
Cordelia Bolle
Keyword(s):  
Transgenic Arabidopsis ◽  
Emission Spectra ◽  
Total Content ◽  
The State ◽  
Serine Phosphorylation ◽  
Phytochrome A ◽  
Wild Type ◽  
Etiolated Seedlings ◽  
Pre Treatment

Phytochrome A (phyA) mediates different photoresponses what may be connected with the existence of its two types, phyAʹ and phyAʹʹ, differing by spectroscopic, photochemical and functional properties. We investigated a role of phyA phosphorylation in their formation turning to transgenic Arabidopsis thaliana (L. Heynh.) phyA or phyAphyB mutants overexpressing rice wild-type phyA (phyA WT) or mutant phyA (phyA SA) with the first 10 serines substituted by alanines. This prevents phyA phosphorylation at these sites and modifies photoresponses. Etiolated seedlings were employed and phyA parameters were evaluated with the use of low temperature fluorescence spectroscopy and photochemistry. Germination of seeds was induced by white light (WL) pre-treatment for 15 min or 3 h. Emission spectra of rice phyA WT and phyA SA were similar and their total content was comparable. However, the phyAʹ/phyAʹʹ proportion in phyA WT was high and varied with the duration of the WL pre-treatment, whereas in phyA SA it was substantially shifted towards phyAʹʹ and did not depend on the pre-illumination. This suggests that phyA SA comprises primarily or exclusively the phyAʹʹ pool and supports the notion that the two phyA types differ by the state of serine phosphorylation. phyAʹʹ was also found to be much more effective in the germination induction than phyAʹ.

Role of O-GlcNAcylation and endoplasmic reticulum stress on obesity and insulin resistance

2019 ◽  
Vol 44 (5) ◽  
pp. 599-610 ◽  
Author(s):  
Benan Pelin Sermikli ◽  
Gulizar Aydogdu ◽  
Afsar Abbasi Taghidizaj ◽  
Erkan Yilmaz
Keyword(s):  
Insulin Resistance ◽  
Endoplasmic Reticulum ◽  
Western Blot ◽  
Er Stress ◽  
Insulin Receptor ◽  
Serine Phosphorylation ◽  
Wild Type ◽  
Adipose Tissues ◽  
Insulin Resistant

Abstract Background Obesity is a global public health problem. Obesity closely associated with various metabolic diseases such as; insulin resistance, hypertension, dyslipidemia and cardiovascular diseases. Endoplasmic reticulum (ER) stress is a critical factor for insulin resistance. O-linked N-acetyl-glucosamine (O-GlcNAc); is the post-translational modification which is has a vital role in biological processes; including cell signaling, in response to nutrients, stress and other extracellular stimuli. Materials and methods In this study, we aimed to investigate the role of O-GlcNAc modification in the context of obesity and obesity-associated insulin resistance in adipose tissue. For this purpose, first, the visceral and epididymal adipose tissues of obese and insulin resistant C57BL/6 Lepob/Lepob and wild-type mice were used to determine the O-GlcNAc modification pattern by western blot. Secondly, the external stimulation of O-GlcNAc modification in wild-type mice achieved by intraperitoneal 5 mg/kg/day glucosamine injection every 24 h for 5 days. The effect of increased O-GlcNAc modification on insulin resistance and ER stress investigated in adipose tissues of glucosamine challenged wild-type mice through regulation of the insulin signaling pathway and unfolded protein response (UPR) elements by western blot. In addition to that, the O-GlcNAc status of the insulin receptor substrate-1 (IRS1) investigated in epididymal and visceral adipose tissues of ob/ob, wild-type and glucosamine challenged mice by immunoprecipitation. Results We found that reduced O-GlcNAc levels in visceral and epididymal adipose tissues of obese and insulin-resistant ob/ob mice, although interestingly we observed that increased O-GlcNAc modification in glucosamine challenged wild-type mice resulted in insulin resistance and ER stress. Furthermore, we demonstrated that the IRS1 was modified with O-GlcNAc in visceral and epididymal adipose tissues in both ob/ob mice and glucosamine-injected mice, and was compatible with the serine phosphorylation of this modification. Conclusion Our results suggest that O-GlcNAcylation of proteins is a crucial factor for intracellular trafficking regulates insulin receptor signaling and UPR depending on the cellular state of insulin resistance.

The Regulation of STAT3 and Its Role in the Adhesion and Migration of Chronic Lymphocytic Leukaemia Cells

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4347-4347
Author(s):  
Sarah Brophy ◽  
Fiona M Quinn ◽  
David O'Brien ◽  
Paul Browne ◽  
Elisabeth A. Vandenberghe ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cell Adhesion ◽  
Cell Survival ◽  
Lymphocytic Leukaemia ◽  
Serine Phosphorylation ◽  
Pre Treatment ◽  
And Migration ◽  
Induced Apoptosis ◽  
Stat3 Inhibition

Abstract The bone marrow and lymph node microenvironments are important in promoting cell proliferation, survival and protection from drug induced apoptosis in chronic lymphocytic leukaemia (CLL). Chemokine networks, such as the CXCR4/CXCL12 axis, in combination with selectins, such as CD62L and integrins allow the migration of CLL cells to these protective niches. The B-cell receptor (BCR) signalling pathway is the most important pathway involved in micro-environmental crosstalk and CLL cell survival. Further, it has been shown to interact with the signal transducer and activator of transcription 3 (STAT3) signalling pathway. The role of the STAT3 in CLL pathogenesis is unclear; however, it is constitutively phosphorylated on serine residue 727 (serine pSTAT3) in CLL cells. Here, we investigate the role of STAT3 in CLL cell survival and migration, using pharmacological inhibition and siRNA knockdown. Phospho-tyrosine and phospho-serine STAT3 were assessed by flow cytometry and western blotting. Apoptosis was assessed by Annexin V/Propidium Iodide staining by flow cytometry. The expression of cell surface markers involved in cell adhesion and homing was determined by multicolour flow cytometry. Stimulation of the BCR using immunoglobulin F(ab´)2 fragments induced tyrosine phosphorylation of STAT3 in CLL cells with unmutated immunoglobulin (IgVH) genes (n=7) but not mutated IgVH genes (n=5). This induced tyrosine phosphorylation was abrogated by pre-treatment with the Janus kinase (JAK) inhibitor Ruxolitinib and the BCR inhibitors Ibrutinib and Idelalisib (p<0.05, n=5). Gene expression studies using Taqman Assays showed BCR stimulation resulted in an upregulation of STAT3 regulated genes in CLL cells with unmutated IgVH genes. Interestingly, stimulation of BCR resulted in a significant increase in CD62L expression, which was inhibited by pre-treatment with Ibrutinib and Ruxolitinib (p<0.05, n=5). STAT3 inhibition was shown to have a divergent effect on CLL cell survival: In patient samples with >70% positive serine pSTAT3 cells, the STAT3 inhibitor cucurbitacin I induced apoptosis with a concurrent downregulation in serine phosphorylation (n=3); while in patient samples with <70% positive serine pSTAT3 cells, treatment with cucurbitacin I resulted in a decrease in apoptosis and a concurrent increase in serine phosphorylation (n=3). The STAT3 inhibitor S3I-201 had a similar effect but the upstream JAK inhibitor Ruxolitinib had no effect on serine phosphorylation and no effect on the apoptosis of CLL cells. In addition, siRNA mediated STAT3 knockdown and treatment with cucurbitacin I and S31-201, resulted in a significant decrease in CD62L positive CLL cells (p<0.0001, n=29). The role of STAT3 in CLL cell adhesion under shear flow conditions was investigated using a microfluidics system including a neMESYS Low Pressure syringe pump system and Human Umbilical Vein Endothelial Cells (HUVEC) coated biochips. Treatment of CLL cells with cucurbitacin I resulted in a significant decrease in adhesion to endothelial cells (p<0.001, n=4). The effect of STAT3 inhibition on the chemotaxis of CLL cells was investigated using Neuroprobe 96-well ChemoTx plates. Treatment with cucurbitacin I resulted in a significant decrease in CLL cells migrated in response to the chemokine CXCL12 compared to control (p=0.0001, n=8). In addition, treatment of CLL cells with CXCL12 resulted in an increase in serine pSTAT3 that was downregulated by pretreatment with cucurbitacin I. This study has shown: 1. Activation of STAT3 by BCR stimulation occurs in poor prognostic unmutated IgVH genes 2. Serine pSTAT3 has a role in cell survival in response to STAT3 inhibition 3. A role for STAT3 in CLL cell adhesion and migration, in particular in the regulation of the expression of CD62L. In conclusion, this study shows a role for the STAT3 pathway in cell survival and CLL cell-microenvironment crosstalk, suggesting therapeutic potential by interfering with the migration and homing of CLL cells to the lymph node and bone marrow microenvironments. Disclosures No relevant conflicts of interest to declare.

Innate inflammatory and phagocytic responses to Plasmodium falciparum: linked processes or molecularly discrete pathways?s

2007 ◽  
Vol 30 (4) ◽  
pp. 81
Author(s):  
Laura Erdman ◽  
Gabriela Cosio ◽  
Samir N. Patel ◽  
Sergio Grinstein ◽  
Kevin C. Kain
Keyword(s):  
Severe Malaria ◽  
Scavenger Receptor ◽  
Innate Immune ◽  
Innate Immune Responses ◽  
Wild Type ◽  
Blood Stage Infection ◽  
Representative Model ◽  
Pre Treatment ◽  
Stage Infection

Background: Effective innate immune responses are important for control of malaria blood-stage infection and in preventing progression to severe malaria in non-immune individuals. Key innate defenses include a tightly regulated inflammatory response and host clearance of parasitized erythrocytes (PE). Pattern recognition receptors on macrophages mediate these processes: parasite glycosylphosphatidylinositol (GPI) activates TLR2 to induce inflammation – an excess of which is associated with severe malaria – while scavenger receptor CD36 mediates non-opsonic uptake of PEs. Both pathways are potential therapeutic targets, but it is unclear whether they are interdependent. Findings in other systems implicate CD36 in inflammation and TLR2 in phagocytosis, and recent evidence indicates that CD36 and TLR2 can directly cooperate. Methods: We investigated whether the innate inflammatory and phagocytic responses of macrophages to P. falciparum are separable: does CD36-mediated PE internalization have inflammatory outcomes, and does TLR2 regulate PE uptake? CD36-mediated endocytosis failed to induce TNFα production. As a more representative model of innate PE uptake, α-CD36 EBABs (Erythrocyte-Biotin-Avidin-Biotinylated antibody) were generated; macrophages internalized EBABs in a CD36-specific manner via a signaling pathway similar to that of PE uptake. Results: Compared to controls, neither PE nor EBAB internalization induced TNFα release, indicating that the inflammatory consequences of CD36 engagement are ligand dependent. Regarding TLR2 regulation of PE uptake, wild type and Tlr2-/- macrophages showed no differences in EBAB or PE uptake. Pre-treatment of macrophages with P. falciparum GPI enhanced EBAB internalization, but this effect was CD36-independent and generalizable to other TLR ligands. Conclusions: These results suggest that innate inflammatory and phagocytic responses of macrophages to malaria are discrete. Thus, therapeutic augmentation of CD36-mediated PE uptake should not exacerbate inflammation, nor should inhibition of the TLR2 pathway compromise CD36-mediated PE clearance. The role of TLR-enhanced internalization in malaria will be further examined.

scholarly journals MCA1 and MCA2 Are Involved in the Response to Hypergravity in Arabidopsis Hypocotyls

Plants ◽  
2020 ◽  
Vol 9 (5) ◽  
pp. 590 ◽  
Author(s):  
Takayuki Hattori ◽  
Yasuhiro Otomi ◽  
Yohei Nakajima ◽  
Kouichi Soga ◽  
Kazuyuki Wakabayashi ◽  
...  
Keyword(s):  
Normal Growth ◽  
Elongation Growth ◽  
Gravitational Acceleration ◽  
Gravity Level ◽  
Wild Type ◽  
Gravity Perception ◽  
Signal Perception ◽  
Etiolated Seedlings ◽  
In The Wild

Plants respond to and resist gravitational acceleration, but the mechanism of signal perception in the response is unknown. We studied the role of MCA (mid1-complementing activity) proteins in gravity perception by analyzing the expression of the MCA1 and MCA2 genes, and the growth of hypocotyls of mca mutants, under hypergravity conditions in the dark. An MCA1 promoter::GUS fusion reporter gene construct (MCA1p::GUS) and MCA2p::GUS were expressed almost universally in etiolated seedlings. Under hypergravity conditions, the expression levels of both genes increased compared with that under the 1 g condition, and remained higher, especially in the basal supporting region. On the other hand, mca-null and MCA-overexpressing seedlings showed normal growth under the 1 g condition. Hypergravity suppressed elongation growth of hypocotyls, but this effect was reduced in hypocotyls of mca-null mutants compared with the wild type. In contrast, MCA-overexpressing seedlings were hypersensitive to increased gravity; suppression of elongation growth was detected at a lower gravity level than that in the wild type. These results suggest that MCAs are involved in the perception of gravity signals in plants, and may be responsible for resistance to hypergravity.

scholarly journals Essential Role of STAT3 in Postnatal Survival and Growth Revealed by Mice Lacking STAT3 Serine 727 Phosphorylation

2004 ◽  
Vol 24 (1) ◽  
pp. 407-419 ◽  
Author(s):  
Yuhong Shen ◽  
Karni Schlessinger ◽  
Xuejun Zhu ◽  
Eric Meffre ◽  
Fred Quimby ◽  
...  
Keyword(s):  
Acute Phase Response ◽  
Transcriptional Response ◽  
Serine Phosphorylation ◽  
Wild Type ◽  
Embryonic Fibroblasts ◽  
Survival And Growth ◽  
Supplemental Activation ◽  
Phosphorylated Stat3

ABSTRACT A large number of extracellular polypeptides bound to their cognate receptors activate the transcription factor STAT3 by phosphorylation of tyrosine 705. Supplemental activation occurs when serine 727 is also phosphorylated. STAT3 deletion in mice leads to embryonic lethality. We have produced mice with alanine substituted for serine 727 in STAT3 (the SA allele) to examine the function of serine 727 phosphorylation in vivo. Embryonic fibroblasts from SA/SA mice had ∼50% of the transcriptional response of wild-type cells. However, SA/SA mice were viable and grossly normal. STAT3 wild-type/null (+/−) animals were also normal and were interbred with SA/SA mice to study SA/− mice. The SA/− mice progressed through gestation, showing 10 to 15% reduced birth weight, three-fourths died soon after birth, and the SA/− survivors reached only 50 to 60% of normal size at 1 week of age. The lethality and decreased growth were accompanied by altered insulin-like growth factor 1 (IGF-1) levels in serum, establishing a role for the STAT3 serine phosphorylation acting through IGF-1 in embryonic and perinatal growth. The SA/− survivors have decreased thymocyte number associated with increased apoptosis, but unexpectedly normal STAT3-dependent liver acute phase response. These animals offer the opportunity to study defined reductions in the transcriptional capacity of a widely used signaling pathway.

scholarly journals Role of Human Immunodeficiency Virus Type 1 Matrix Phosphorylation in an Early Postentry Step of Virus Replication

2004 ◽  
Vol 78 (5) ◽  
pp. 2319-2326 ◽  
Author(s):  
Rajnish Kaushik ◽  
Lee Ratner
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Serine Phosphorylation ◽  
Wild Type Virus ◽  
Wild Type ◽  
Particle Assembly ◽  
Immunodeficiency Virus

ABSTRACT The matrix domain (MA) is important for targeting of human immunodeficiency virus type 1 Gag assembly to the plasma membrane, envelope incorporation into virions, preintegration complex import into the nucleus, and nuclear export of viral RNA. Myristylation and phosphorylation are key regulatory events for MA function. Previous studies have indicated that MA phosphorylation at serine (Ser) residues is important for viral replication. This study defines the molecular mechanisms of virus particle assembly and infectivity through a detailed study of the role of MA serine phosphorylation. We show that the combined mutation of Ser residues at positions 9, 67, 72, and 77 impairs viral infectivity in dividing and nondividing cells, although the assembly of these Ser mutant viruses is comparable to that of wild-type virus. This defect can be rescued by pseudotyping these mutant viruses with vesicular stomatitis virus G protein, suggesting that these serine residues are critical in an early postentry step of viral infection. The phosphorylation level of MA in defective mutant viruses was severely reduced compared to that of the wild type, suggesting that phosphorylation of Ser-9, -67, -72, and -77 is important for an early postentry step during virus infection.

Differential Role of Components of the Fibrinolytic System in the Formation and Removal of Thrombus Induced by Endothelial Injury

1999 ◽  
Vol 81 (04) ◽  
pp. 601-604 ◽  
Author(s):  
Hiroyuki Matsuno ◽  
Osamu Kozawa ◽  
Masayuki Niwa ◽  
Shigeru Ueshima ◽  
Osamu Matsuo ◽  
...  
Keyword(s):  
Plasminogen Activator ◽  
Thrombus Formation ◽  
Endothelial Injury ◽  
Fibrinolytic System ◽  
Tissue Type ◽  
Clot Lysis ◽  
Wild Type ◽  
Type Plasminogen Activator ◽  
Pai 1

SummaryThe role of fibrinolytic system components in thrombus formation and removal in vivo was investigated in groups of six mice deficient in urokinase-type plasminogen activator (u-PA), tissue-type plasminogen activator (t-PA), or plasminogen activator inhibitor-1 (PAI-1) (u-PA-/-, t-PA-/- or PAI-1-/-, respectively) or of their wild type controls (u-PA+/+, t-PA+/+ or PAI-1+/+). Thrombus was induced in the murine carotid artery by endothelial injury using the photochemical reaction between rose bengal and green light (540 nm). Blood flow was continuously monitored for 90 min on day 0 and for 20 min on days 1, 2 and 3. The times to occlusion after the initiation of endothelial injury in u-PA+/+, t-PA+/+ or PAI-1+/+ mice were 9.4 ± 1.3, 9.8 ± 1.1 or 9.7 ± 1.6 min, respectively. u-PA-/- and t-PA-/- mice were indistinguishable from controls, whereas that of PAI-1-/- mice were significantly prolonged (18.4 ± 3.7 min). Occlusion persisted for the initial 90 min observation period in 10 of 18 wild type mice and was followed by cyclic reflow and reocclusion in the remaining 8 mice. At day 1, persistent occlusion was observed in 1 wild type mouse, 8 mice had cyclic reflow and reocclusion and 9 mice had persistent reflow. At day 2, all injured arteries had persistent reflow. Persistent occlusion for 90 min on day 0 was observed in 3 u-PA-/-, in all t-PA-/- mice at day 1 and in 2 of the t-PA-/-mice at day 2 (p <0.01 versus wild type mice). Persistent patency was observed in all PAI-1-/- mice at day 1 and in 5 of the 6 u-PA-/- mice at day 2 (both p <0.05 versus wild type mice). In conclusion, t-PA increases the rate of clot lysis after endothelial injury, PAI-1 reduces the time to occlusion and delays clot lysis, whereas u-PA has little effect on thrombus formation and spontaneous lysis.

A Re-Appraisal of the Role of Blood Coagulation and Platelets in the Generalized Shwartzman Phenomenon

1966 ◽  
Vol 15 (03/04) ◽  
pp. 519-538 ◽  
Author(s):  
J Levin ◽  
E Beck
Keyword(s):  
Blood Coagulation ◽  
The State ◽  
Coagulation System ◽  
Renal Lesions ◽  
Intravascular Coagulation ◽  
Renal Glomeruli ◽  
Coagulation Mechanism ◽  
Shwartzman Phenomenon ◽  
Do So

SummaryThe role of intravascular coagulation in the production of the generalized Shwartzman phenomenon has been evaluated. The administration of endotoxin to animals prepared with Thorotrast results in activation of the coagulation mechanism with the resultant deposition of fibrinoid material in the renal glomeruli. Anticoagulation prevents alterations in the state of the coagulation system and inhibits development of the renal lesions. Platelets are not primarily involved. Platelet antiserum produces similar lesions in animals prepared with Thorotrast, but appears to do so in a manner which does not significantly involve intravascular coagulation.The production of adrenal cortical hemorrhage, comparable to that seen in the Waterhouse-Friderichsen syndrome, following the administration of endotoxin to animals that had previously received ACTH does not require intravascular coagulation and may not be a manifestation of the generalized Shwartzman phenomenon.

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