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The Discodermia calyx Toxin Calyculin A Enhances Cyclin D1 Phosphorylation and Degradation, and Arrests Cell Cycle Progression in Human Breast Cancer Cells  [PDF]
Jessica R. Edelson,David L. Brautigan
Toxins , 2011, DOI: 10.3390/toxins3010105
Abstract: Cyclin D1 is a key regulator of the cell cycle that is over expressed in more than half of breast cancer patients. The levels of cyclin D1 are controlled primarily through post-translational mechanisms and phosphorylation of cyclin D1 at T286 induces its proteasomal degradation. To date, no studies have explored the involvement of phosphatases in this process. Here we treated human breast cancer cells with the structurally distinct toxins calyculin A, okadaic acid, and cantharidin, which are known to inhibit Ser/Thr phosphatases of the PPP family. At low nanomolar concentrations calyculin A induced T286 phosphorylation and degradation of cyclin D1 via the proteosome in MDA-MB-468 and MDA-MB-231 cells. Cyclin D1 degradation also was dose-dependently induced by okadaic acid and catharidin, implicating a negative regulatory role for type-2A phosphatases. These effects occurred without increasing phosphorylation of p70S6K, cyclin D3, or myosin light chain that were used as endogenous reporters of cellular PP2A and PP1 activity. A reverse phase phosphoprotein array analysis revealed increased phosphorylation of only 6 out of 33 Ser/Thr phosphosites, indicating selective inhibition of phosphatases by calyculin A. Calyculin A treatment induced cell cycle arrest in MDA-MB-468 and MCF-7 breast cancer cells. These findings suggest that a specific pool of type-2A phosphatase is inhibited by calyculin A leading to the degradation of cyclin D1 in human breast cancer cells. The results highlight the utility of toxins as pharmacological probes and points to the T286 cyclin D1 phosphatase inhibited by calyculin A as a possible target for chemotherapy to treat triple negative breast cancer.
Cloning, expression, purification of spinach carboxyl-terminal processing protease of D1 protein with hydrolysis activity and preparation of polyclonal antibody
D1蛋白酶的克隆表达、纯化、生物活性测定及多克隆抗体的制备

Hui Li,Wei Zhang,Mingxia Sheng,Weiguo Li,Yanli Liu,Sufang Liu,Chao Qi,
李慧
,张巍,申明霞,李伟国,刘艳丽,刘素芳,祁超

生物工程学报 , 2010,
Abstract: Carboxyl-terminal processing protease of D1 protein (CtpA) catalyzes carboxyl terminal processing of the D1 protein of photosystem II, which is essential for the assembly of a manganese cluster and consequent light-mediated water oxidation. It is a target for the discovery of wide-spectrum herbicide. We amplified the CtpA gene from spinach cDNA with standard PCR method and constructed it into pET-28a vector to generate a recombinant expression plasmid. Recombinant CtpA fusion protein with His-tag was expressed as soluble protein in Escherichia coli BL21(DE3) after induction with 0.1 mmol/L IPTG at 8°C for 72 h. We purified the CtpA protein with the Ni-NTA affinity chromatography and Superdex 75 gel filtration chromatography respectively, and verified the protein by SDS-PAGE and Western blotting with anti-his antibody. Hydrolysis activity of CtpA was assayed by HPLC method with a synthetic 24-mer oligopeptide corresponding to carboxyl terminal of precursor D1 protein, and gave a total activity of 1.10 nmol/(mg?min). We used the purified CtpA protein as antigen to immune rabbit for the production of polyclonal antibody, and prepared antibody with high specificity and sensitivity. The results obtained in this paper provided the feasibility of high-throughput screening of lead compounds for the protease as inhibitors and mechanism analysis of CtpA enzyme.
Cross-Linked Enzyme Aggregates of Naringinase: Novel Biocatalysts for Naringin Hydrolysis  [PDF]
Maria H. L. Ribeiro,Marco Raba?a
Enzyme Research , 2011, DOI: 10.4061/2011/851272
Abstract: Cross-linked enzyme aggregates (CLEAs) have emerged as interesting biocatalyst design for immobilization. These new generation enzyme biocatalysts, CLEAs, in addition to exhibiting good mechanical stability, can be highly active, since they do not include large amounts of foreign particulate nonenzymatic material and may have increased stability. Naringinase (NGase) is an enzyme complex with high potential in pharmaceutical and food industries. In fact, NGase can be used in the biotransformation of steroids, of antibiotics and mainly on glycosides hydrolysis. In this paper, the formation of CLEAs was tried using ammonium sulphate, polyethylene glycol 6000 and tert-butyl alcohol as precipitant agents and glutaraldehyde as cross-linking agent, at different pH, time, and temperature conditions. However, among the precipitant agents tested, only tert-butyl alcohol cross-linked with glutaraldehyde allowed the formation of CLEAs, at pH 4.0 and at temperature between 7 and . Different enzyme loadings were tested. The NGase-CLEAs were highly effective in naringin hydrolysis. The operational stability of the NGase-CLEAs aggregates was studied through six successive reutilizations. 1. Introduction Cross-linked enzyme aggregates (CLEAs) have emerged as interesting biocatalysts design for immobilization. Additionally, since precipitation can be used on the purification of enzymes, and on the preparation of CLEAs, this technique may integrate in a single/or reduced operation: enzyme purification and immobilization. This new generation of biocatalysts, CLEAs, in addition to exhibiting good mechanical stability, can be highly active, since they do not include large amounts of foreign particulate nonenzymatic material and may have increased stability. The CLEAs technology present many advantages on different applications, as it is simple and amenable to rapid optimization, leading to low costs and short time-to-market processes. The self-immobilization techniques of CLEAs are referred to as providing higher volumetric and specific activity. The cross-linking agents such as glutaraldehyde, ethylene polymers and aldehyde dextran, bind to the enzymes without the need for support when they are close [1–3]. Immobilization by cross-linked enzyme aggregates (CLEAs) involves the precipitation mechanism of enzymes by salting out, addition of nonionic polymers or mixture of solvents. The most common used precipitating agents are ammonium sulphate, polyethylene glycol (PEG 6000, PEG 8000), or ter-butyl alcohol among others [3, 4]. Therefore, the formation of CLEAS comprises two
Effects of salicylic acid on protein kinase activity and chloroplast D1 protein degradation in wheat leaves subjected to heat and high light stress

Hui-Jie Zhao,Xue-Juan Zhao,Pei-Fang M,Yue-Xia Wang,Wei-Wei Hu,Li-Hong Li,Yi-Dan Zhao,

生态学报 , 2011,
Abstract: In the north of China, wheat plants are often stressed by heat and high light during grain-filling stage, which leads to injury in photosynthetic apparatus and decline in photosynthetic rate. In order to develop a method to protect photosynthetic apparatus in wheat leaves subjected to heat and high light stress, the effects of SA (salicylic acid) and FSBA (5′-p-fluorosulfonylbenzoyl adenosine) on PK (protein kinase) activity, D1 protein degradation and the performance of PSII were investigated in present work. Our results showed that PK activity enhanced under heat and high light stress and declined when stress was removed. FSBA pretreatment resulted in marked decreases in PK activity and D1 protein level, suggesting a correlationship between degradation of D1 protein and phosphorylation. After 2 h of stress, D1 protein level in water-pretreated leaves decreased to 79% of control and then recovered to 81% after 3 h of recovery. This clearly indicated that the damage of D1 protein induced by heat and high light stress was reversible. Compared to the control, SA pretreatment could not only increase PK activity, retard the degradation of D1 protein during heat and high light stress, but also accelerate the recovery of D1 protein level when the stress was removed. Correspondingly, Fv/Fm (maximum photochemical efficiency of PSII), ΦPSII (actual photochemical efficiency of PSII), ETR (electron transfer rate) and Pn (net photosynthetic rate) in SA-treated leaves were higher than that in leaves of control under both stress and non-stress conditions. Taken together, our results revealed that SA pretreatment could significantly alleviate damages of heat and high light stress on D1 protein and PSII of wheat leaves, and accelerate restoration of photosynthetic function.
SKF83959, an Agonist of Phosphatidylinositol-Linked D1-Like Receptors, Promotes ERK1/2 Activation and Cell Migration in Cultured Rat Astrocytes  [PDF]
Chao Huang, Jingjing Wu, Rujia Liao, Wei Zhang
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0049954
Abstract: Extracellular signal-regulated kinase 1/2 (ERK1/2) is a member of the mitogen-activated protein kinase family. It can mediate cell migration. Classical dopamine receptor-mediated ERK1/2 phosphorylation is widely studied in neurons. Here, we report that ERK1/2 phosphorylation is also modulated by putative phosphatidylinositol-linked D1-like receptors in cultured rat astrocytes. 6-chloro-7,8-dihydroxy-3-methyl-1-(3-met?hylphenyl)-2,3,4,5-tetrahydro-1H-3-benza?zepine(SKF83959), an agonist of the putative phosphatidylinositol-linked D1-like receptors, was found to enhance ERK1/2 phosphorylation, which then promoted the migration of cultured astrocytes. The SKF83959-induced ERK1/2 phosphorylation was found to be Ca2+-independent based on the following observations: i. chelating intracellular Ca2+ did not inhibit ERK1/2 phosphorylation and astrocyte migration; ii. blockage of the release of intracellular Ca2+ from the endoplasmic reticulum by an inhibitor of inositol 1,4,5-trisphosphate (IP3) receptor did not attenuate ERK1/2 phosphorylation. However, inhibition of phospholipase C (PLC), the upstream molecule of internal Ca2+ release, disabled SKF83959’s ability to elevate the level of ERK1/2 phosphorylation. Both non-selective protein kinase C (PKC) inhibitor and PKCδ selective inhibitor prevented ERK1/2 phosphorylation increase and astrocyte migration, but PKCα inhibitor did not. This suggests that Ca2+-independent and diacylglycerol-dependent PKCδ acts downstream of putative phosphatidylinositol-linked D1-like receptor activation and mediates SKF83959-induced elevation of ERK1/2 phosphorylation in order to modulate astrocyte migration. In conclusion, our results demonstrate that SKF83959-induced increases in ERK1/2 phosphorylation and astrocyte migration are dependent on PLC-PKCδ signals. This might help us to further understand the functions of the putative phosphatidylinositol-linked D1-like receptors in the nervous system.
Isolation of chlorophyll a from spinach leaves
E.D. Dikio, D.A. Isabirye
Bulletin of the Chemical Society of Ethiopia , 2008,
Abstract: An efficient method for separating chlorophyll a from spinach leaves by column chromatography and solvent extraction techniques has been developed. The purity and identity of the chlorophyll a have been confirmed by UV-Vis, IR and mass spectrometry. Yields from 100 g of freeze-dried spinach were 23 – 24 mg of chlorophyll a. KEY WORDS: Chlorophyll a, Spinach leaves, Separation, Column chromatography, Solvent extraction, Pheophytin a Bull. Chem. Soc. Ethiop. 2008, 22(2), 301-304.
Resolvin D1 reverses chronic pancreatitis-induced mechanical allodynia, phosphorylation of NMDA receptors, and cytokines expression in the thoracic spinal dorsal horn  [cached]
Quan-Xin Feng,Fan Feng,Xiang-Ying Feng,Shu-Jun Li
BMC Gastroenterology , 2012, DOI: 10.1186/1471-230x-12-148
Abstract: Background We previously reported that immune activation in the spinal dorsal horn contributes to pain induced by chronic pancreatitis (CP). Targeting immune response in the CNS may provide effective treatments for CP-induced pain. Recent findings demonstrate that resolvin D1 (RvD1) can potently dampen inflammatory pain. We hypothesized that intrathecal injection of RvD1 may inhibit pain of CP. Methods Rat CP model was built through intrapancreatic infusion of trinitrobenzene sulfonic acid (TNBS). All the rats were divided into three groups: TNBS, sham, and na ve controls and were further divided for intrathecal RvD1 administration. Pain behavior of rats was tested with von Frey filaments. Anxiety-like behavior and free locomotor and exploration of rats were evaluated by open field test and elevated plus maze. Pancreatic histology was evaluated with hematoxylin and eosin staining. Phosphorylation of NMDA receptor and expression of inflammatory cytokines were examined with Western blot, real-time RT-PCR and ELISA. Results Behavioral study indicated that compared to the vehicle control, RvD1 (100 ng/kg) significantly decreased TNBS-induced mechanical allodynia at 2 h after administration (response frequencies: 49.2 ± 3.7% vs 71.3 ± 6.1%), and this effect was dose-dependent. Neither CP nor RvD1 treatment could affect anxiety-like behavior. CP or RvD1 treatment could not affect free locomotor and exploration of rats. Western blot analysis showed that compared with that of na ve group, phosphorylated NR1 (pNR1) and pNR2B in TNBS rats were significantly increased in the spinal cord (pNR1: 3.87±0.31 folds of na ve control, pNR2B: 4.17 ± 0.24 folds of na ve control). Compared to vehicle control, 10 ng/kg of RvD1 could significantly block expressions of pNR1 (2.21 ± 0.26 folds of na ve) and pNR2B (3.31 ± 0.34 folds of na ve). Real-time RT-PCR and ELISA data showed that RvD1 (10 ng/kg) but not vehicle could significantly block expressions of TNF-alpha, IL-1beta and IL-6. In addition, RvD1 did not influence pain behavior, NMDA receptor phosphorylation or cytokines production in sham-operated rats. Conclusions These data highly suggest that RvD1 could be a novel and effective treatment for CP-induced chronic pain.
The Impact of Pesticide Application on Heavy Metal (Cd, Pb and Cu) Levels in Spinach  [cached]
Timothy Musa CHIROMA,Bala Isah ABDULKARIM,Haruna Mavakumba KEFAS
Leonardo Electronic Journal of Practices and Technologies , 2007,
Abstract: The impact of Pesticides (DELVAP 1000EC) application on the levels of Cd, Pb and Cu in two species of Spinach (maroon and green) was investigated. The results shows highest accumulation in leaves compared to stem and roots for both species. The levels of Cd, Pb and Cu in leaves of maroon Spinach treated with pesticides are 6.8, 1.4 and 18.6 times respectively higher than the maximum tolerable levels of 30μg/g, 300μg/g and100μg/g for Cd, Pb and Cu respectively in plants, while in green Spinach treated with pesticides the levels of Cd and Cu in leaves are 4.9 and 14.7 times respectively higher than the maximum tolerable levels. The concentration of Cd, Pb and Cu in leaves, stem and roots of maroon Spinach treated with pesticides are 163%, 222% and 178%; 364%, 325% and 449%; and 254%, 363%and 224% respectively higher than the untreated Spinach while their corresponding concentration in green Spinach treated with pesticides are 1! 56%, 238% and 150%; 163%, 454% and 462%; and 156%, 407% and 346% respectively higher than the untreated green Spinach.
Isolation of chlorophyll a from spinach leaves  [cached]
E.D. Dikio,D.A. Isabirye
Bulletin of the Chemical Society of Ethiopia , 2008,
Abstract: An efficient method for separating chlorophyll a from spinach leaves by column chromatography and solvent extraction techniques has been developed. The purity and identity of the chlorophyll a have been confirmed by UV-Vis, IR and mass spectrometry. Yields from 100 g of freeze-dried spinach were 23 – 24 mg of chlorophyll a.
Optimization of Enzymatic Synthesis of Ampicillin Using Cross-Linked Aggregates of Penicillin G Acylase
Daryoush Abedi,Mohammad Reza Fazeli,Abbas Jafarian
Iranian Journal of Pharmaceutical Research , 2004,
Abstract: Penicillin G acylase from E. coli TA1 was immobilized by Cross-Linked Enzyme Aggregates (CLEA), a new method for immobilization. This biocatalyst and commercial immobilized penicillin G acylase (PGA-450) were used to study the effect of pH, temperatureand substrate concentration on the synthesis of ampicillin from phenyl glycine methyl ester (PGME) and 6-aminopenicillanic acid (6-APA). Compared with PGA-450, this immobilized enzyme showed a high synthesis activity. The optimum conditions for synthetic activity was at pH 6, 25°C and 2:6 (6-APA:PGME) substrate ratio.
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