Myosin Phosphatase Is Implicated in the Control of THP-1 Monocyte to Macrophage Differentiation

Monocyte to macrophage differentiation is characterized by the activation of various signal transduction pathways, which may be modulated by protein phosphorylation; however, the impact of protein kinases and phosphatases is not well understood yet. It has been demonstrated that actomyosin rearrangement during macrophage differentiation is dependent on Rho-associated protein kinase (ROCK). Myosin phosphatase (MP) target subunit-1 (MYPT1) is one of the major cellular substrates of ROCK, and MP is often a counter enzyme of ROCK; therefore, MP may also control macrophage differentiation. Changes in MP activity and the effects of MP activation were studied on PMA or l,25(OH)₂D₃-induced differentiation of monocytic THP-1 cells. During macrophage differentiation, phosphorylation of MYPT1 at Thr696 and Thr853 increased significantly, resulting in inhibition of MP. The ROCK inhibitor H1152 and the MP activator epigallocatechin-3-gallate (EGCG) attenuated MYPT1 phosphorylation and concomitantly decreased the extent of phosphorylation of 20 kDa myosin light chain. H1152 and EGCG pretreatment also suppressed the expression of CD11b and weakened the PMA-induced adherence of the cells. Our results indicate that MP activation/inhibition contributes to the efficacy of monocyte to macrophage differentiation, and this enzyme may be a target for pharmacological interventions in the control of disease states that are affected by excessive macrophage differentiation.     

The Protein Phosphatase PP2A Plays Multiple Roles in Plant Development by Regulation of Vesicle Traffic—Facts and Questions

The protein phosphatase PP2A is essential for the control of integrated eukaryotic cell functioning. Several cellular and developmental events, e.g., plant growth regulator (PGR) mediated signaling pathways are regulated by reversible phosphorylation of vesicle traffic proteins. Reviewing present knowledge on the relevant role of PP2A is timely. We discuss three aspects: (1) PP2A regulates microtubule-mediated vesicle delivery during cell plate assembly. PP2A dephosphorylates members of the microtubule associated protein family MAP65, promoting their binding to microtubules. Regulation of phosphatase activity leads to changes in microtubule organization, which affects vesicle traffic towards cell plate and vesicle fusion to build the new cell wall between dividing cells. (2) PP2A-mediated inhibition of target of rapamycin complex (TORC) dependent signaling pathways contributes to autophagy and this has possible connections to the brassinosteroid signaling pathway. (3) Transcytosis of vesicles transporting PIN auxin efflux carriers. PP2A regulates vesicle localization and recycling of PINs related to GNOM (a GTP–GDP exchange factor) mediated pathways. The proper intracellular traffic of PINs is essential for auxin distribution in the plant body, thus in whole plant development. Overall, PP2A has essential roles in membrane interactions of plant cell and it is crucial for plant development and stress responses.     

An inhibition type alkaline phosphatase biosensor for amperometric determination of caffeine

A biosensor based on the inhibition of alkaline phosphatase (ALP) enzyme was developed for caffeine determination. The biosensor was prepared by a chemical covalent immobilization of alkaline phosphatase with a cross-linking agent, glutaraldehyde on a ceramic based gold screen printed electrode that was modified with cysteamine by forming a self-assembled monolayer. Caffeine competitively inhibits ALP enzyme and the determination method of caffeine by the biosensor was based on this inhibition effect of caffeine. The principle of the measurement was based on the determination of the differentiation of biosensor responses in the enzymatic reaction catalyzed by ALP in the absence and the presence of caffeine. Differences between the biosensor responses were related to caffeine concentration which was added in to the reaction medium. Caffeine concentration can be determined accurately between 0.1 and 10 μM using the biosensor. Detection limit of the biosensor is 0.08 μM. In the optimization studies of the biosensor, glycine buffer (pH 10.5; 50 mM) and 30 °C were obtained as the optimum working conditions.     

Acid phosphatase in the tubers of Dioscorea species and its purification from the white yam (D. rotundata poir)

Acid phosphatase activity was determined in 15 cultivars from four species of yam. A 12-fold purification of the enzyme from Dioscorea rotundata (cv. chikakwondo) gave a homogeneous preparation as demonstrated by polyacrylamide gel electrophoresis. This enzyme preparation has an apparent molecular weight of 115 000 ±2000 and an optimum activity at a pH of 5·20 and a temperature of 50°C. The K_m of the enzyme is 3·81 mM with disodium p-nitrophenylphosphate (p-NNP) as a substrate. The energy of activation, heat of activation, energy of inactivation and heat of inactivation are 7·0, 6·4, 4·41 and 4·34 kcal M^?1, respectively. Although it has very little activity with most organic phosphoric acid esters, it is significantly inhibited by Ca²⁺, Hg²⁺ and EDTA and activated by Mg²⁺. The enzyme has a half-life of 50,17 or 13 days, respectively, when stored at 6-8°C, 0°C or room temperature (29±2°C).     

A comparative study of two advanced spraying techniques for the deposition of biologically active enzyme coatings onto bone-substituting implants

Surface modification of implant materials with biomolecule coatings is of high importance to improve implant fixation in bone tissue. In the current study, we present two techniques for the deposition of biologically active enzyme coatings onto implant materials. The well-established thin film ElectroSpray Deposition (ESD) technique was compared with the SAW-ED technique that combines high-frequency Surface Acoustic Wave atomization with Electrostatic Deposition. By immobilizing the enzyme alkaline phosphatase (ALP) onto implant surfaces, the influence of both SAW-ED and ESD deposition parameters on ALP deposition efficiency and ALP biological activity was investigated. ALP coatings with preserved enzyme activity were deposited by means of both the SAW-ED and ESD technique. The advantages of SAW-ED over ESD include the possibility to spray highly conductive protein solutions, and the 60-times faster deposition rate. Furthermore, significantly higher deposition efficiencies were observed for the SAW-ED technique compared to ESD. Generally, it was shown that protein inactivation is highly dependent on both droplet dehydration and the applied electrical field strength. The current study shows that SAW-ED is a versatile and flexible technique for the fabrication of functionally active biomolecule coatings.     

新型染料配基对碱性磷酸酶的亲和纯化

设计合成了一种含有碱性磷酸酶抑制剂——对氨基苯磷酸的新型偶氮染料配基并将其偶联到凝胶Sepharose CL-6B 上,制备出新型的亲和层析介质,分离纯化小牛肠中的碱性磷酸酶。通过考察不同配基密度的层析介质对碱性磷酸酶的选择性,得出配基密度为4.55mg 配基(g 湿胶)-1的层析介质选择性高。用NaCl和Na2HPO4进行阶段洗脱,可使酶的纯化倍数一步达到65倍,活力回收率达到89%。通过测定不同配基浓度下酶的动力学,发现新合成的配基是碱性磷酸酶的竞争性抑制剂,其KI为3.0×10-3mol L-1。     

A regulated MET3-GLC7 gene fusion provides evidence of a mitotic role for Saccharomyces cerevisiae protein phosphatase 1

Saccharomyces cerevisiae possesses a single essential gene (GLC7) encoding protein phosphatase 1 (PP1). Elevated expression of this gene from the GAL1 promoter is highly detrimental to the cell, causing a growth defect and aberrant bud morphology, which leads to cells exhibiting long, extended buds. By comparison, expression of GLC7 from the weaker MET3 promoter was without significant effect on either growth or morphology. However, repression of GLC7 expression from the MET3 promoter in cells where the MET3-GLC7 fusion was the sole source of PP1 resulted in a mitotic delay. Such cultures showed a massive decrease in the rate of proliferation in conjunction with a significant increase in the proportion of large, budded cells. 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) staining and anti-tubulin immunofluorescence analysis of these cells revealed that many were blocked in mitosis, with a short spindle and DAPI-stained material stretched between the mother and daughter cell within the bud neck. These results support a role for PP1 in the completion of mitosis in S. cerevisiae.     

Beneficial effects of fucoidan on osteoblastic MG-63 cell differentiation

Fucoidan extracted from the marine brown alga Undaria pinnatifida, significantly induced osteoblastic cell differentiation. Our results indicated that a non-toxic sulphated polysaccharide, fucoidan, can increase activity of alkaline phosphatase (ALP) and level of osteocalcin (OC) as phenotypic markers for early-stage osteoblastic differentiation and terminally osteoblastic differentiation, respectively. Furthermore, the results showed positive effects of fucoidan on bone morphogenic protein-2 (BMP-2) as an important factor for bone formation, remodeling and mineralization. The present study may provide new insights in the osteoblastic differentiation of fucoidan and possibility for its application in bone health supplement.     

High pressure processing extend the shelf life of fresh salmon,cod and mackerel

High pressure processing (HPP) is used on several types of seafood, though its potential for extending the shelf life of fresh fish has not been fully exploited. This study compared the effect of HPP (200 and 500 MPa, 120 s) on salmon, cod and mackerel. Immediately after processing, and during storage up to 26 days different analysis were carried out to evaluate the microbiological shelf life, oxidation, acid phosphatase level and pH of the different fish species during the storage period. For cod and mackerel, HPP at 500 MPa restrained the bacterial flora and it did not reach spoilage level, opposite to the salmon samples exposed to 500 MPa. Analysis of TBARS is often used as a measure of lipid oxidation. The TBARS-level was differently affected by pressure in salmon and cod. HPP at 200 MPa did not have any effect on the oxidation level in salmon during the storage period while this was observed for cod. The TBARS level in mackerel was high, independent of pressure treatment or not. Phosphatase activity in fish has been suggested to be related to freshness. The acid phosphatase (ACP) levels in control samples showed significant differences between salmon and cod. For salmon both control and samples treated at 200 MPa showed a decrease in ACP level at day 11 compared with day 0, whereas the HPP500 values were even throughout the study. This latter observation was also the case for cod treated at 500 MPa. This study have shown that HPP induce different levels of changes in various fish species.