scholarly journals Method for identifying the phosphorylation site of Maize Starch Synthase I

SURG Journal ◽  
2009 ◽  
Vol 3 (1) ◽  
pp. 49-58
Author(s):  
Clare Barker ◽  
Nadya Romanova ◽  
Michael J. Emes ◽  
Ian J. Tetlow
Keyword(s):  
Higher Plants ◽  
Phosphorylation Site ◽  
Reversed Phase ◽  
Starch Synthase ◽  
Maize Starch ◽  
Nucleotide Sugar ◽  
Ionization Time ◽  
Metal Affinity ◽  
Phosphoamino Acid Analysis

Multiple forms of starch synthases (EC 2.4.1.21) are critical for the synthesis of starch in higher plants. These enzymes catalyze the extension of linear glucan chains by the transfer of glucose from the nucleotide sugar ADPglucose. Recombinant maize starch synthase I (rSSI) was purified from Escherichia coli in order to study the regulation of this enzyme by protein phosphorylation. The rSSI was phosphorylated in vitro by incubation with [γ 32P]-ATP and maize amyloplast lysates, which were used as a source of protein kinase. Maximal phosphorylation of rSSI was achieved within 20 minutes, and there was no noticeable change in the amount of phosphorylation beyond this time. Phosphoamino acid analysis of rSSI indicated phosphorylation of one or more serine residues. In order to purify and identify the phosphopeptides, phosphorylated rSSI was digested with trypsin to yield smaller peptides, which were then concentrated using Immobilized Metal Affinity Chromatography (IMAC). Potential phosphopeptides eluting from the IMAC column were purified and putatively identified using reversed phase (C18 column) High Pressure Liquid Chromatography (HPLC). They were then analyzed by mass spectrometry using Matrix-Assisted Laser Desorption/Ionization-Time of Flight (MALDI-TOF) which yielded the mass of potential phosphopeptides.

scholarly journals Phosphorylation of septin 3 on Ser-91 by cGMP-dependent protein kinase-I in nerve terminals

2004 ◽  
Vol 381 (3) ◽  
pp. 753-760 ◽  
Author(s):  
Jing XUE ◽  
Peter J. MILBURN ◽  
Bernadette T. HANNA ◽  
Mark E. GRAHAM ◽  
John A. P. ROSTAS ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Phosphorylation Site ◽  
Nerve Terminals ◽  
Dependent Protein Kinase ◽  
Cgmp Dependent Protein Kinase ◽  
Dependent Protein ◽  
Phosphoamino Acid Analysis ◽  
The Brain ◽  
Major Phosphorylation Site

The septins are a family of GTPase enzymes required for cytokinesis and play a role in exocytosis. Among the ten vertebrate septins, Sept5 (CDCrel-1) and Sept3 (G-septin) are primarily concentrated in the brain, wherein Sept3 is a substrate for PKG-I (cGMP-dependent protein kinase-I) in nerve terminals. There are two motifs for potential PKG-I phosphorylation in Sept3, Thr-55 and Ser-91, but phosphoamino acid analysis revealed that the primary site is a serine. Derivatization of phosphoserine to S-propylcysteine followed by N-terminal sequence analysis revealed Ser-91 as a major phosphorylation site. Tandem MS revealed a single phosphorylation site at Ser-91. Substitution of Ser-91 with Ala in a synthetic peptide abolished phosphorylation. Mutation of Ser-91 to Ala in recombinant Sept3 also abolished PKG phosphorylation, confirming that Ser-91 is the major site in vitro. Antibodies raised against a peptide containing phospho-Ser-91 detected phospho-Sept3 only in the cytosol of nerve terminals, whereas Sept3 was located in a peripheral membrane extract. Therefore Sept3 is phosphorylated on Ser-91 in nerve terminals and its phosphorylation may contribute to the regulation of its subcellular localization in neurons.

In vitro and in vivo polysaccharides assembly: ultrastructural and cytochemical study of growing plant cell wall components.

1978 ◽  
Vol 36 (2) ◽  
pp. 434-435
Author(s):  
D. Reis ◽  
B. Vian ◽  
J. C. Roland
Keyword(s):  
Cell Wall ◽  
Self Assembly ◽  
Plant Cell Wall ◽  
Higher Plants ◽  
Three Dimensional ◽  
Cytochemical Study ◽  
Wall Components

Wall morphogenesis in higher plants is a problem still open to controversy. Until now the possibility of a transmembrane control and the involvement of microtubules were mostly envisaged. Self-assembly processes have been observed in the case of walls of Chlamydomonas and bacteria. Spontaneous gelling interactions between xanthan and galactomannan from Ceratonia have been analyzed very recently. The present work provides indications that some processes of spontaneous aggregation could occur in higher plants during the formation and expansion of cell wall.Observations were performed on hypocotyl of mung bean (Phaseolus aureus) for which growth characteristics and wall composition have been previously defined.In situ, the walls of actively growing cells (primary walls) show an ordered three-dimensional organization (fig. 1). The wall is typically polylamellate with multifibrillar layers alternately transverse and longitudinal. Between these layers intermediate strata exist in which the orientation of microfibrils progressively rotates. Thus a progressive change in the morphogenetic activity occurs.

Influence of New Synthetic Xanthones on the Proliferation and Migration Potential of Cancer Cell Lines In Vitro

2020 ◽  
Vol 19 (16) ◽  
pp. 1949-1965 ◽  
Author(s):  
Natalia Szkaradek ◽  
Daniel Sypniewski ◽  
Dorota Żelaszczyk ◽  
Sabina Gałka ◽  
Paulina Borzdziłowska ◽  
...  
Keyword(s):  
Cancer Cells ◽  
Cell Lines ◽  
Cancer Cell ◽  
Higher Plants ◽  
Biological Activities ◽  
Human Tumor ◽  
Plant Metabolites ◽  
Xtt Assay ◽  
Treatment Protocols

Background: Natural plant metabolites and their semisynthetic derivatives have been used for years in cancer therapy. Xanthones are oxygenated heterocyclic compounds produced as secondary metabolites by higher plants, fungi or lichens. Xanthone core may serve as a template in the synthesis of many derivatives that have broad biological activities. Objective: This study synthesized a series of 17 new xanthones, and their anticancer potential was also evaluated. Methods: The anticancer potential was evaluated in vitro using a highly invasive T24 cancer cell line. Direct cytotoxic effects of the xanthones were established by IC50 estimation based on XTT assay. Results: 5 compounds of the total 17 showed significant cytotoxicity toward the studied cancer cultures and were submitted to further detailed analysis, including studies examining their influence on gelatinase A and B expression, as well as on the cancer cells migration and adhesion to an extracellular matrix. These analyses were carried out on five human tumor cell lines: A2780 (ovarian cancer), A549 (lung cancer), HeLa (cervical cancer), Hep G2 (liver cancer), and T24 (urinary bladder cancer). All the compounds, especially 4, showed promising anticancer activity: they exhibited significant cytotoxicity towards all the evaluated cell lines, including MCF-7 breast cancer, and hindered migration-motility activity of cancer cells demonstrating more potent activity than α-mangostin which served as a reference xanthone. Conclusion: These results suggest that our xanthone derivatives may be further analyzed in order to include them in cancer treatment protocols.

Spastin Interacts with CRMP2 to Regulate Neurite Outgrowth by Controlling Microtubule Dynamics through Phosphorylation Modifications

2020 ◽  
Vol 19 ◽  
Author(s):  
Sumei Li ◽  
Jifeng Zhang ◽  
Jiaqi Zhang ◽  
Jiong Li ◽  
Longfei Cheng ◽  
...  
Keyword(s):  
Neurite Outgrowth ◽  
Hippocampal Neurons ◽  
Neuronal Development ◽  
Phosphorylation Site ◽  
Microtubule Dynamics ◽  
Microtubule Assembly ◽  
C Terminus ◽  
Mediator Protein ◽  
Branch Formation

Aims: Our work aims to revealing the underlying microtubule mechanism of neurites outgrowth during neuronal development, and also proposes a feasible intervention pathway for reconstructing neural network connections after nerve injury. Background: Microtubule polymerization and severing are the basis for the neurite outgrowth and branch formation. Collapsin response mediator protein 2 (CRMP2) regulates axonal growth and branching as a binding partner of the tubulin heterodimer to promote microtubule assembly. And spastin participates in the growth and regeneration of neurites by severing microtubules into small segments. However, how CRMP2 and spastin cooperate to regulate neurite outgrowth by controlling the microtubule dynamics needs to be elucidated. Objective: To explore whether neurite outgrowth was mediated by coordination of CRMP2 and spastin. Method: Hippocampal neurons were cultured in vitro in 24-well culture plates for 4 days before being used to perform the transfection. Calcium phosphate was used to transfect the CRMP2 and spastin constructs and their control into the neurons. An interaction between CRMP2 and spastin was examined by using pull down, CoIP and immunofluorescence colocalization assays. And immunostaining was also performed to determine the morphology of neurites. Result: We first demonstrated that CRMP2 interacted with spastin to promote the neurite outgrowth and branch formation. Furthermore, our results identified that phosphorylation modification failed to alter the binding affinities of CRMP2 for spastin, but inhibited their binding to microtubules. CRMP2 interacted with the MTBD domain of spastin via its C-terminus, and blocking the binding sites of them inhibited the outgrowth and branch formation of neurites. In addition, we confirmed one phosphorylation site S210 at spastin in hippocampal neurons and phosphorylation spastin at site S210 promoted the neurite outgrowth but not branch formation by remodeling microtubules. Conclusion: Taken together, our data demonstrated that the interaction of CRMP2 and spastin is required for neurite outgrowth and branch formation and their interaction is not regulated by their phosphorylation.

scholarly journals In-vitro and in-vivo metabolism of different aspirin formulations studied by a validated liquid chromatography tandem mass spectrometry method

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Michele Dei Cas ◽  
Jessica Rizzo ◽  
Mariangela Scavone ◽  
Eti Femia ◽  
Gian Marco Podda ◽  
...  
Keyword(s):  
Oral Administration ◽  
Whole Blood ◽  
Serum Levels ◽  
Reversed Phase ◽  
Weekly Treatment ◽  
Low Dose Aspirin ◽  
Liquid Chromatography Tandem Mass ◽  
Enteric Coated

AbstractLow-dose aspirin (ASA) is used to prevent cardiovascular events. The most commonly used formulation is enteric-coated ASA (EC-ASA) that may be absorbed more slowly and less efficiently in some patients. To uncover these “non-responders” patients, the availability of proper analytical methods is pivotal in order to study the pharmacodynamics, the pharmacokinetics and the metabolic fate of ASA. We validated a high-throughput, isocratic reversed-phase, negative MRM, LC–MS/MS method useful for measuring circulating ASA and salicylic acid (SA) in blood and plasma. ASA-d4 and SA-d4 were used as internal standards. The method was applied to evaluate: (a) the "in vitro" ASA degradation by esterases in whole blood and plasma, as a function of time and concentration; (b) the "in vivo" kinetics of ASA and SA after 7 days of oral administration of EC-ASA or plain-ASA (100 mg) in healthy volunteers (three men and three women, 37–63 years). Parameters of esterases activity were Vmax 6.5 ± 1.9 and Km 147.5 ± 64.4 in plasma, and Vmax 108.1 ± 20.8 and Km 803.2 ± 170.7 in whole blood. After oral administration of the two formulations, tmax varied between 3 and 6 h for EC-ASA and between 0.5 and 1.0 h for plain-ASA. Higher between-subjects variability was seen after EC-ASA, and one subject had a delayed absorption over eight hours. Plasma AUC was 725.5 (89.8–1222) for EC-ASA, and 823.1(624–1196) ng h/mL (median, 25–75% CI) for plain ASA. After the weekly treatment, serum levels of TxB2 were very low (< 10 ng/mL at 24 h from the drug intake) in all the studied subjects, regardless of the formulation or the tmax. This method proved to be suitable for studies on aspirin responsiveness.

scholarly journals The Potential of Rhizoctonia-Like Fungi for the Biological Protection of Cereals against Fungal Pathogens

Plants ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 349
Author(s):  
Dominik Bleša ◽  
Pavel Matušinský ◽  
Romana Sedmíková ◽  
Milan Baláž
Keyword(s):  
Biological Control ◽  
Fungal Pathogens ◽  
Plant Pathogens ◽  
Higher Plants ◽  
Fusarium Culmorum ◽  
Suppressive Effect ◽  
Plant Production ◽  
Shoot Biomass ◽  
Organic Substrates

The use of biological control is becoming a common practice in plant production. One overlooked group of organisms potentially suitable for biological control are Rhizoctonia-like (Rh-like) fungi. Some of them are capable of forming endophytic associations with a large group of higher plants as well as mycorrhizal symbioses. Various benefits of endophytic associations were proved, including amelioration of devastating effects of pathogens such as Fusarium culmorum. The advantage of Rh-like endophytes over strictly biotrophic mycorrhizal organisms is the possibility of their cultivation on organic substrates, which makes their use more suitable for production. We focused on abilities of five Rh-like fungi isolated from orchid mycorrhizas, endophytic fungi Serendipita indica, Microdochium bolleyi and pathogenic Ceratobasidium cereale to inhibit the growth of pathogenic F. culmorum or Pyrenophora teres in vitro. We also analysed their suppressive effect on wheat infection by F. culmorum in a growth chamber, as well as an effect on barley under field conditions. Some of the Rh-like fungi affected the growth of plant pathogens in vitro, then the interaction with plants was tested. Beneficial effect was especially noted in the pot experiments, where wheat plants were negatively influenced by F. culmorum. Inoculation with S. indica caused higher dry shoot biomass in comparison to plants treated with fungicide. Prospective for future work are the effects of these endophytes on plant signalling pathways, factors affecting the level of colonization and surviving of infectious particles.

scholarly journals Infective Endocarditis Due to Corynebacterium Species: Clinical Features and Antibiotic Resistance

2021 ◽  
Vol 8 (3) ◽  
Author(s):  
Anna Bläckberg ◽  
Linn Falk ◽  
Karl Oldberg ◽  
Lars Olaison ◽  
Magnus Rasmussen
Keyword(s):  
Antibiotic Resistance ◽  
Infective Endocarditis ◽  
Prosthetic Valve Endocarditis ◽  
Penicillin G ◽  
Severe Condition ◽  
Ionization Time ◽  
Prosthetic Valves ◽  
Corynebacterium Species ◽  
Corynebacterium Striatum

Abstract Background Corynebacterium species are often dismissed as contaminants in blood cultures, but they can also cause infective endocarditis (IE), which is a severe condition. Antibiotic resistance of corynebacteria is increasing making treatment challenging. Reports on IE caused by Corynebacterium species are scarce and more knowledge is needed. Methods Cases of IE caused by Corynebacterium species were identified through the Swedish Registry of Infective Endocarditis. Isolates were collected for species redetermination by matrix-assisted laser desorption ionization-time of flight and for antibiotic susceptibility testing using Etests. Results Thirty episodes of IE due to Corynebacterium species were identified between 2008 and 2017. The median age of patients was 71 years (interquartile range, 60–76) and 77% were male. Corynebacterium striatum (n = 11) was the most common IE causing pathogen followed by Corynebacterium jeikeium (n = 5). Surgery was performed in 50% and in-hospital mortality rate was 13%. Patients with IE caused by Corynebacterium species were significantly more likely to have prosthetic valve endocarditis (70%), compared with patients with IE due to Staphylococcus aureus or non-beta-hemolytic streptococci (14% and 26%, respectively) (P &lt; .0001). Vancomycin was active towards all Corynebacterium isolates, whereas resistance towards penicillin G was common. Conclusions Corynebacterium species cause IE, where prosthetic valves are mainly affected and surgery is often performed. Corynebacterium striatum is an important causative agent of IE within the genus. Antibiotic resistance of corynebacteria is relatively common but resistance towards vancomycin could not be detected in vitro.

scholarly journals Molecular rearrangement of waxy and normal maize starch granules during in vitro digestion

2016 ◽  
Vol 139 ◽  
pp. 10-19 ◽  
Author(s):  
Anju Teng ◽  
Torsten Witt ◽  
Kai Wang ◽  
Ming Li ◽  
Jovin Hasjim
Keyword(s):  
In Vitro Digestion ◽  
Starch Granules ◽  
Maize Starch ◽  
Molecular Rearrangement ◽  
Normal Maize

scholarly journals Phosphorylation of Serine 303 Is a Prerequisite for the Stress-Inducible SUMO Modification of Heat Shock Factor 1

2003 ◽  
Vol 23 (8) ◽  
pp. 2953-2968 ◽  
Author(s):  
Ville Hietakangas ◽  
Johanna K. Ahlskog ◽  
Annika M. Jakobsson ◽  
Maria Hellesuo ◽  
Niko M. Sahlberg ◽  
...  
Keyword(s):  
Heat Shock ◽  
Phosphorylation Site ◽  
Stress Granules ◽  
Heat Shock Factor ◽  
Transcriptional Level ◽  
Heat Shock Factor 1 ◽  
Protection Mechanism ◽  
Sumo Modification

ABSTRACT The heat shock response, which is accompanied by a rapid and robust upregulation of heat shock proteins (Hsps), is a highly conserved protection mechanism against protein-damaging stress. Hsp induction is mainly regulated at transcriptional level by stress-inducible heat shock factor 1 (HSF1). Upon activation, HSF1 trimerizes, binds to DNA, concentrates in the nuclear stress granules, and undergoes a marked multisite phosphorylation, which correlates with its transcriptional activity. In this study, we show that HSF1 is modified by SUMO-1 and SUMO-2 in a stress-inducible manner. Sumoylation is rapidly and transiently enhanced on lysine 298, located in the regulatory domain of HSF1, adjacent to several critical phosphorylation sites. Sumoylation analyses of HSF1 phosphorylation site mutants reveal that specifically the phosphorylation-deficient S303 mutant remains devoid of SUMO modification in vivo and the mutant mimicking phosphorylation of S303 promotes HSF1 sumoylation in vitro, indicating that S303 phosphorylation is required for K298 sumoylation. This finding is further supported by phosphopeptide mapping and analysis with S303/7 phosphospecific antibodies, which demonstrate that serine 303 is a target for strong heat-inducible phosphorylation, corresponding to the inducible HSF1 sumoylation. A transient phosphorylation-dependent colocalization of HSF1 and SUMO-1 in nuclear stress granules provides evidence for a strictly regulated subnuclear interplay between HSF1 and SUMO.

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