scholarly journals CO2-Responsive CCT Protein interacts with 14-3-3 proteins and controls the expression of starch synthesis-related genes

2021 ◽  
Author(s):  
Hiroshi Fukayama ◽  
Naoki Shibatani ◽  
Hirofumi Miyagawa ◽  
Aiko Koudou ◽  
Yasuo Yamauchi ◽  
...  
Keyword(s):  
Oryza Sativa L ◽  
Large Subunit ◽  
Starch Synthesis ◽  
Gel Filtration ◽  
Parenchyma Cell ◽  
Mesophyll Cell ◽  
Leaf Sheath ◽  
Bimolecular Fluorescence Complementation ◽  
Promoter Regions

CO2 responsive CCT protein (CRCT) is a positive regulator of starch synthesis related genes such as ADP-glucose pyrophosphorylase large subunit 1 and starch branching enzyme I particularly in the leaf sheath of rice (Oryza sativa L.). The promoter GUS analysis revealed that CRCT expressed exclusively in the vascular bundle, whereas starch synthesis related genes were expressed in different sites such as mesophyll cell and starch storage parenchyma cell. However, the chromatin immunoprecipitation (ChIP) using a FLAG-CRCT overexpression line and subsequent qPCR analyses showed that the 5’-flanking regions of these starch synthesis-related genes tended to be enriched by ChIP, suggesting that CRCT can bind to the promoter regions of these genes. The monomer of CRCT is 34.2 kDa, however CRCT was detected at 270 kDa via gel filtration chromatography, suggesting that CRCT forms a complex in vivo. Immunoprecipitation and subsequent MS analysis pulled down several 14-3-3-like proteins. A yeast two-hybrid analysis and bimolecular fluorescence complementation assays confirmed the interaction between CRCT and 14-3-3-like proteins. Although there is an inconsistency in the place of expression, this study provide important findings regarding the molecular function of CRCT to control the expression of key starch synthesis-related genes.

scholarly journals Pullulanase from rice endosperm.

2008 ◽  
Vol 55 (3) ◽  
pp. 507-510 ◽  
Author(s):  
Yoshiki Yamasaki ◽  
Susumu Nakashima ◽  
Haruyoshi Konno
Keyword(s):  
Single Unit ◽  
Oryza Sativa L ◽  
Starch Synthesis ◽  
Gel Filtration ◽  
Soluble Starch ◽  
Starch Degradation ◽  
Rice Endosperm ◽  
Beta Cyclodextrin ◽  
Sds Page ◽  
Germinating Seeds

Pullulanase (EC 3.2.1.41) in non-germinating seeds was compared with that in germinating seeds. Moreover, pullulanase from the endosperm of rice (Oryza sativa L., cv. Hinohikari) seeds was isolated and its properties investigated. The pI value of pullulanase from seeds after 8 days of germination was almost equal to that from non-germinating seeds, which shows that these two enzymes are the same protein. Therefore, the same pullulanase may play roles in both starch synthesis during ripening and starch degradation during germination in rice seeds. The enzyme was isolated by a procedure that included ammonium sulfate fractionation, DEAE-cellulofine column chromatography, preparative isoelectric focusing, and preparative disc gel electrophoresis. The enzyme was homogeneous by SDS/PAGE. The molecular weight of the enzyme was estimated to be 100 000 based on its mobility on SDS/PAGE and 105 000 based on gel filtration with TSKgel super SW 3000, which showed that it was composed of a single unit. The isoelectric point of the enzyme was 4.7. The enzyme was strongly inhibited by beta-cyclodextrin. The enzyme was not activated by thiol reagents such as dithiothreitol, 2-mercaptoethanol or glutathione. The enzyme most preferably hydrolyzed pullulan and liberated only maltotriose. The pullulan hydrolysis was strongly inhibited by the substrate at a concentration higher than 0.1%. The degree of inhibition increased with an increase in the concentration of pullulan. However, the enzyme hydrolyzed amylopectin, soluble starch and beta-limit dextrin more rapidly as their concentrations increased. The enzyme exhibited alpha-glucosyltransfer activity and produced an alpha-1,6-linked compound of two maltotriose molecules from pullulan.

Suppression of starch synthesis in rice stems splays tiller angle due to gravitropic insensitivity but does not affect yield

2015 ◽  
Vol 42 (1) ◽  
pp. 31 ◽  
Author(s):  
Masaki Okamura ◽  
Tatsuro Hirose ◽  
Yoichi Hashida ◽  
Ryu Ohsugi ◽  
Naohiro Aoki
Keyword(s):  
Double Mutant ◽  
Oryza Sativa L ◽  
Starch Content ◽  
Large Subunit ◽  
Starch Synthesis ◽  
Starch Accumulation ◽  
Gravitropic Response ◽  
Rice Mutant ◽  
Matter Production ◽  
Tiller Angle

In rice (Oryza sativa L.), tiller angle – defined as the angle between the main culm and its side tillers – is one of the important factors involved in light use efficiency. To clarify the relationship between tiller angle, gravitropism and stem-starch accumulation, we investigated the shoot gravitropic response of a low stem-starch rice mutant which lacks a large subunit of ADP-glucose pyrophosphorylase (AGP), called OsAGPL1 and exhibits relatively spread tiller angle. The insensitive gravitropic response exhibited by the mutant led us to the conclusion that insensitivity of gravitropism caused by stem-starch reduction splayed the tiller angle. Furthermore, since another AGP gene called OsAGPL3 was expressed at considerable levels in graviresponding sites, we generated a double mutant lacking both OsAGPL1 and OsAGPL3. The double mutant exhibited still lower stem-starch content, less sensitive gravitropic response and greater tiller angle spread than the single mutants. This indicated that the expansion of the tiller angle caused by the reduction in starch level was intense according to the extent of the reduction. We found there were no significant differences between the double mutant and wild-type plants in terms of dry matter production. These results provided new insight into the importance of stem-starch accumulation and ideal plant architecture.

scholarly journals HybF, a Zinc-Containing Protein Involved in NiFe Hydrogenase Maturation

2004 ◽  
Vol 186 (9) ◽  
pp. 2603-2611 ◽  
Author(s):  
Melanie Blokesch ◽  
Michaela Rohrmoser ◽  
Sabine Rode ◽  
August Böck
Keyword(s):  
Large Subunit ◽  
Gel Filtration ◽  
Stoichiometric Ratio ◽  
Amino Acid Residues ◽  
Binding Assays ◽  
Triple Mutant ◽  
Hydrogenase Maturation ◽  
Identify Amino Acid

ABSTRACT HypA and HypB are maturation proteins required for incorporation of nickel into the hydrogenase large subunit. To examine the functions of these proteins in nickel insertion, the hybF gene, which is a homolog of hypA essential for maturation of hydrogenases 1 and 2 from Escherichia coli, was overexpressed, and the product was purified. This protein behaves like a monomer in gel filtration and contains stoichiometric amounts of zinc but insignificant or undetectable amounts of nickel and iron. In filter binding assays radioactively labeled nickel binds to HybF with a KD of 1.87 μM and in a stoichiometric ratio. To identify amino acid residues of HybF involved in nickel and/or zinc binding, variants in which conserved residues were replaced were studied. An H2Q replacement eliminated both in vivo activity and in vitro binding of nickel. The purified protein, however, contained zinc at the level characteristic of the wild-type protein. When E3 was replaced by Q, activity was retained, but an E3L exchange was detrimental. Replacement of each of the four conserved cysteine residues of a zinc finger motif reduced the cellular amount of HybF protein without a loss of in vivo activity, indicating that these residues play a purely structural role. A triple mutant deficient in the synthesis or activity of HypA, HybF, and HypB was constructed, and it exhibited the same responsiveness for phenotypic complementation by high nickel as mutants with a single lesion in one of the genes exhibited. The results are interpreted in terms of a concerted action of HypB and HybF in nickel insertion in which HybF (as well as its homolog, HypA) functions as a metallochaperone and HypB functions as a regulator that controls the interaction of HybF with the target protein.

scholarly journals Affinity Purification of Specific Chromatin Segments from Chromosomal Loci in Yeast

2003 ◽  
Vol 23 (24) ◽  
pp. 9275-9282 ◽  
Author(s):  
Joachim Griesenbeck ◽  
Hinrich Boeger ◽  
J. Seth Strattan ◽  
Roger D. Kornberg
Keyword(s):  
Affinity Purification ◽  
Gel Filtration ◽  
Single Copy ◽  
Specific Gene ◽  
Promoter Regions ◽  
Activator Proteins ◽  
Activated State ◽  
Copy Gene ◽  
Chromosomal Loci

ABSTRACT Single-copy gene and promoter regions have been excised from yeast chromosomes and have been purified as chromatin by conventional and affinity methods. Promoter regions isolated in transcriptionally repressed and activated states maintain their characteristic chromatin structures. Gel filtration analysis establishes the uniformity of the transcriptionally activated state. Activator proteins interact in the manner anticipated from previous studies in vivo. This work opens the way to the direct study of specific gene regions of eukaryotic chromosomes in diverse functional and structural states.

Starch reduction in rice stems due to a lack of OsAGPL1 or OsAPL3 decreases grain yield under low irradiance during ripening and modifies plant architecture

2013 ◽  
Vol 40 (11) ◽  
pp. 1137 ◽  
Author(s):  
Masaki Okamura ◽  
Tatsuro Hirose ◽  
Yoichi Hashida ◽  
Tohru Yamagishi ◽  
Ryu Ohsugi ◽  
...  
Keyword(s):  
Grain Yield ◽  
Plant Architecture ◽  
Oryza Sativa L ◽  
Large Subunit ◽  
Dry Weight ◽  
Leaf Sheath ◽  
Starch Accumulation ◽  
Wild Type ◽  
Gene Encoding ◽  
Rice Mutant

Starch accumulated in rice (Oryza sativa L.) stems before heading as nonstructural carbohydrates (NSCs) is reported to be important for improving and stabilising grain yield. To evaluate the importance of stem starch, we investigated a retrotransposon (Tos17) insertion rice mutant lacking a gene encoding a large subunit of ADP-glucose pyrophosphorylase (AGP) called OsAGPL1 or OsAPL3. The AGP activity and starch contents of the mutant were drastically reduced in the stem (i.e. leaf sheath and culm) but not in the leaf blade or endosperm. This starch reduction in the leaf sheaths of the mutant was complemented by the introduction of wild-type OsAGPL1. These results strongly suggest that OsAGPL1 plays a principal role in stem starch accumulation. Field experimentations spanning 2 years revealed that the mutant plants were shorter than the wild-type plants. Moreover, the tiller number and angle were larger in the mutant plants than the wild-type plants, but the dry weight at heading stage was not different. The grain yield was slightly lower in control plots without shading treatment. However, this difference increased substantially with shading. Therefore, stem starch is indispensable for normal ripening under low irradiance conditions and probably contributes to the maintenance of appropriate plant architecture.

Purification of the Antihemophilic Factor by Gel Filtration on Agarose

1969 ◽  
Vol 22 (03) ◽  
pp. 577-583 ◽  
Author(s):  
M.M.P Paulssen ◽  
A.C.M.G.B Wouterlood ◽  
H.L.M.A Scheffers
Keyword(s):  
Factor Viii ◽  
Plasma Proteins ◽  
Large Scale ◽  
Gel Filtration ◽  
Large Scale Preparation ◽  
Scale Preparation ◽  
Antihemophilic Factor

SummaryFactor VIII can be isolated from plasma proteins, including fibrinogen by chromatography on agarose. The best results were obtained with Sepharose 6B. Large scale preparation is also possible when cryoprecipitate is separated by chromatography. In most fractions containing factor VIII a turbidity is observed which may be due to the presence of chylomicrons.The purified factor VIII was active in vivo as well as in vitro.

Half-Life of Platelet Factor 4 (PF-4) in Plasma and Platelets from Macaca Mulatta

1977 ◽  
Vol 37 (01) ◽  
pp. 073-080 ◽  
Author(s):  
Knut Gjesdal ◽  
Duncan S. Pepper
Keyword(s):  
Macaca Mulatta ◽  
Half Life ◽  
Human Platelet ◽  
Gel Filtration ◽  
Platelet Factor 4 ◽  
Active Protein ◽  
Platelet Factor ◽  
Membrane Bound

SummaryHuman platelet factor 4 (PF-4) showed a reaction of complete identity with PF-4 from Macaca mulatta when tested against rabbit anti-human-PF-4. Such immunoglobulin was used for quantitative precipitation of in vivo labelled PF-4 in monkey serum. The results suggest that the active protein had an intra-platelet half-life of about 21 hours. In vitro 125I-labelled human PF-4 was injected intravenously into two monkeys and isolated by immuno-precipita-tion from platelet-poor plasma and from platelets disrupted after gel-filtration. Plasma PF-4 was found to have a half-life of 7 to 11 hours. Some of the labelled PF-4 was associated with platelets and this fraction had a rapid initial disappearance rate and a subsequent half-life close to that of plasma PF-4. The results are compatible with the hypothesis that granular PF-4 belongs to a separate compartment, whereas membrane-bound PF-4 and plasma PF-4 may interchange.

Turnover of Human Extrinsic (Tissue-Type) Plasminogen Activator in Rabbits

1981 ◽  
Vol 46 (03) ◽  
pp. 658-661 ◽  
Author(s):  
C Korninger ◽  
J M Stassen ◽  
D Collen
Keyword(s):  
Plasminogen Activator ◽  
Intravenous Injection ◽  
Active Site ◽  
Half Life ◽  
Degradation Products ◽  
Gel Filtration ◽  
Tissue Type ◽  
Organ Distribution ◽  
Small Rise

SummaryThe turnover of highly purified human extrinsic plasminogen activator (EPA) (one- and two-chain form) was studied in rabbits. Following intravenous injection, EPA-activity declined rapidly. The disappearance rate of EPA from the plasma could adequately be described by a single exponential term with a t ½ of approximately 2 min for both the one-chain and two-chain forms of EPA.The clearance and organ distribution of EPA was studied by using 125I-labeled preparations. Following intravenous injection of 125I-1abeled EPA the radioactivity disappeared rapidly from the plasma also with a t ½ of approximately 2 min down to a level of 15 to 20 percent, followed by a small rise of blood radioactivity. Gel filtration of serial samples revealed that the secondary increase of the radioactivity was due to the reappearance of radioactive breakdown products in the blood. Measurement of the organ distribution of 125I at different time intervals revealed that EPA was rapidly accumulated in the liver, followed by a release of degradation products in the blood.Experimental hepatectomy markedly prolonged the half-life of EPA in the blood. Blocking the active site histidine of EPA had no effect on the half-life of EPA in blood nor on the gel filtration patterns of 125I in serial plasma samples.It is concluded that human EPA is rapidly removed from the blood of rabbits by clearance and degradation in the liver. Recognition by the liver does not require a functional active site in the enzyme. Neutralization in plasma by protease inhibitors does not represent a significant pathway of EPA inactivation in vivo.

In Vivo Studies on the Inhibition of Coagulation by Fractionated Heparin and by a Heparin Analogue I. Effects of Heparin Fractions

1981 ◽  
Vol 46 (03) ◽  
pp. 612-616 ◽  
Author(s):  
U Schmitz-Huebner ◽  
L Balleisen ◽  
F Asbeck ◽  
J van de Loo
Keyword(s):  
Molecular Weight ◽  
Day Treatment ◽  
Gel Filtration ◽  
Weight Fraction ◽  
In Vivo Studies ◽  
Low Molecular Weight ◽  
High Molecular Weight Fraction ◽  
Platelet Factor ◽  
Heparin Fractions

SummaryHigh and low molecular weight heparin fractions obtained by gel filtration chromatography of sodium mucosal heparin were injected subcutaneously into six healthy volunteers and compared with the unfractionated substance in a cross-over trial. Equal doses of 5,000 U were administered twice daily over a period of three days and heparin activity was repeatedly controlled before and 2, 4, 8 hrs after injection by means of the APTT, the anti-Xa clotting test and a chromogenic substrate assay. In addition, the in vivo effect of subcutaneously administered fractionated heparin on platelet function was examined on three of the volunteers. The results show that s.c. injections of the low molecular weight fraction induced markedly higher anti-Xa activity than injections of the other preparations. At the same time, APTT results did not significantly differ. Unfractionated heparin and the high molecular weight fraction enhanced ADP-induced platelet aggregation and collagen-mediated MDA production, while the low molecular weight fraction hardly affected these assays, but potently inhibited thrombin-induced MDA production. All heparin preparations stimulated the release of platelet Factor 4 in plasma. During the three-day treatment periods, no side-effects and no significant changes in the response to heparin injections were detected.

scholarly journals RNF141 interacts with KRAS to promote colorectal cancer progression

Oncogene ◽  
2021 ◽  
Author(s):  
Jiuna Zhang ◽  
Xiaoyu Jiang ◽  
Jie Yin ◽  
Shiying Dou ◽  
Xiaoli Xie ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Plasma Membrane ◽  
Tumor Growth ◽  
Cancer Progression ◽  
Critical Role ◽  
Ring Finger ◽  
Immunohistochemical Analysis ◽  
Bimolecular Fluorescence Complementation

AbstractRING finger proteins (RNFs) play a critical role in cancer initiation and progression. RNF141 is a member of RNFs family; however, its clinical significance, roles, and mechanism in colorectal cancer (CRC) remain poorly understood. Here, we examined the expression of RNF141 in 64 pairs of CRC and adjacent normal tissues by real-time PCR, Western blot, and immunohistochemical analysis. We found that there was more expression of RNF141 in CRC tissue compared with its adjacent normal tissue and high RNF141 expression associated with T stage. In vivo and in vitro functional experiments were conducted and revealed the oncogenic role of RNF141 in CRC. RNF141 knockdown suppressed proliferation, arrested the cell cycle in the G1 phase, inhibited migration, invasion and HUVEC tube formation but promoted apoptosis, whereas RNF141 overexpression exerted the opposite effects in CRC cells. The subcutaneous xenograft models showed that RNF141 knockdown reduced tumor growth, but its overexpression promoted tumor growth. Mechanistically, liquid chromatography-tandem mass spectrometry indicated RNF141 interacted with KRAS, which was confirmed by Co-immunoprecipitation, Immunofluorescence assay. Further analysis with bimolecular fluorescence complementation (BiFC) and Glutathione-S-transferase (GST) pull-down assays showed that RNF141 could directly bind to KRAS. Importantly, the upregulation of RNF141 increased GTP-bound KRAS, but its knockdown resulted in a reduction accordingly. Next, we demonstrated that RNF141 induced KRAS activation via increasing its enrichment on the plasma membrane not altering total KRAS expression, which was facilitated by the interaction with LYPLA1. Moreover, KRAS silencing partially abolished the effect of RNF141 on cell proliferation and apoptosis. In addition, our findings presented that RNF141 functioned as an oncogene by upregulating KRAS activity in a manner of promoting KRAS enrichment on the plasma membrane in CRC.

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