抗原变异可使得多种致病微生物易于逃避宿主免疫应答。表达在感染红细胞表面的恶性疟原虫红细胞表面蛋白1（PfPMP1）与感染红细胞、内皮细胞、树突状细胞以及胎盘的单个或多个受体作用，在黏附及免疫逃避中起关键的作用。每个单倍体基因组var基因家族编码约60种成员，通过启动转录不同的var基因变异体为抗原变异提供了分子基础。

疟原虫var基因转换速率变化导致抗原变异[英]／Paul H, Robert P, Christodoulou Z, et al//Proc Natl Acad Sci U S A

The discovery that mammalian cells have the ability to synthesize the free radical nitric oxide (NO) has stimulated an extraordinary impetus for scientific research in all the fields of biology and medicine. Since its early description as an endothelial-derived relaxing factor, NO has emerged as a fundamental signaling device regulating virtually every critical cellular function, as well as a potent mediator of cellular damage in a wide range of conditions. Recent evidence indicates that most of the cytotoxicity attributed to NO is rather due to peroxynitrite, produced from the diffusion-controlled reaction between NO and another free radical, the superoxide anion. Peroxynitrite interacts with lipids, DNA, and proteins via direct oxidative reactions or via indirect, radical-mediated mechanisms. These reactions trigger cellular responses ranging from subtle modulations of cell signaling to overwhelming oxidative injury, committing cells to necrosis or apoptosis. In vivo, peroxynitrite generation represents a crucial pathogenic mechanism in conditions such as stroke, myocardial infarction, chronic heart failure, diabetes, circulatory shock, chronic inflammatory diseases, cancer, and neurodegenerative disorders. Hence, novel pharmacological strategies aimed at removing peroxynitrite might represent powerful therapeutic tools in the future. Evidence supporting these novel roles of NO and peroxynitrite is presented in detail in this review.

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Nitric Oxide and Peroxynitrite in Health and Disease

Interleukin-10 (IL-10), a cytokine with anti-inflammatory properties, has a central role in infection by limiting the immune response to pathogens and thereby preventing damage to the host. Recently, an increasing interest in how IL10 expression is regulated in different immune cells has revealed some of the molecular mechanisms involved at the levels of signal transduction, epigenetics, transcription factor binding and gene activation. Understanding the specific molecular events that regulate the production of IL-10 will help to answer the remaining questions that are important for the design of new strategies of immune intervention.

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The regulation of IL-10 production by immune cells

Somlyo, Andrew P., and Avril V. Somlyo. Ca2+ Sensitivity of Smooth Muscle and Nonmuscle Myosin II: Modulated by G Proteins, Kinases, and Myosin Phosphatase. Physiol Rev 83: 1325-1358, 2003; 10.1152...

https://www.physiology.org/doi/pdf/10.1152/physrev.00023.2003

Ca2+ Sensitivity of Smooth Muscle and Nonmuscle Myosin II: Modulated by G Proteins, Kinases, and Myosin Phosphatase

Peroxynitrite--the product of the diffusion-controlled reaction of nitric oxide with superoxide radical--is a short-lived oxidant species that is a potent inducer of cell death Conditions in which the reaction products of peroxynitrite have been detected and in which pharmacological inhibition of its formation or its decomposition have been shown to be of benefit include vascular diseases, ischaemia-reperfusion injury, circulatory shock, inflammation, pain and neurodegeneration In this Review, we first discuss the biochemistry and pathophysiology of peroxynitrite and then focus on pharmacological strategies to attenuate the toxic effects of peroxynitrite These include its catalytic reduction to nitrite and its isomerization to nitrate by metalloporphyrins, which have led to potential candidates for drug development for cardiovascular, inflammatory and neurodegenerative diseases

Peroxynitrite: biochemistry, pathophysiology and development of therapeutics

Protein phosphorylation is a frequent posttranslational modification regulating cellular processes in eukaryotes. The phosphate content of a protein is determined by the conflicting activities of protein kinases and phosphatases. Protein phosphatases were divided into Ser/Thr and Tyr specific groups, depending on the phosphorylated residue in the substrate molecules. The former group was further classified based on enzymatic criteria (reviewed in Cohen 1989 Ann. Rev. Biochem. 58:453-508). Protein phosphatase 1 (PP1) is inhibited by two heat stable proteins termed inhibitor-1 and -2. Protein phosphatase 2A is inhibited by nanomolar concentration of the tumor promoter okadaic acid. Protein phosphatase 2B (PP2B) also called calcineurin is stimulated by Ca-calmodulin, and protein phosphatase 2C (PP2C) is a Mg2+ dependent enzyme. Molecular cloning of the catalytic subunits revealed that PP1-PP2A-PP2B consist of a highly conserved superfamily of proteins. Creative Commons License This work is licensed under a Creative Commons Attribution-Share Alike 4.0 License. This regular paper is available in Fungal Genetics Reports: http://newprairiepress.org/fgr/vol41/iss1/26 Detection of Ser/Thr protein phosphatases in Neurospora crassa B. Szoor,1 Z. Feher,2 G. Szabo,2 P. Gergely,1 and V. Dombradi,1 Departments of Medical Chemistry1 and Biology2, University Medical School, Debrecen, Hungary Protein phosphorylation is a frequent posttranslational modification regulating cellular processes in eukaryotes. The phosphate content of a protein is determined by the conflicting activities of protein kinases and phosphatases. Protein phosphatases were divided into Ser/Thr and Tyr specific groups, depending on the phosphorylated residue in the substrate molecules. The former group was further classified based on enzymatic criteria (reviewed in Cohen 1989 Ann. Rev. Biochem. 58:453-508). Protein phosphatase 1 (PP1) is inhibited by two heat stable proteins termed inhibitor-1 and -2. Protein phosphatase 2A is inhibited by nanomolar concentration of the tumor promoter okadaic acid. Protein phosphatase 2B (PP2B) also called calcineurin is stimulated by Ca-calmodulin, and protein phosphatase 2C (PP2C) is a Mg2+ dependent enzyme. Molecular cloning of the catalytic subunits revealed that PP1-PP2A-PP2B consist of a highly conserved superfamily of proteins. The catalytic subunit of PP2B from N. crassa was cloned and expressed (Higuchi et al. 1991 J. Biol. Chem. 266:18104-18112). We assumed that PP1 and PP2A were also present in this organisms. In order to test this possibility we assayed phosphatase activities in N. crassa. For the detection of PP1 and PP2A we adapted a method originally described for S. cerevisiae (Cohen et al. 1989 FEBS Lett. 250:601-606). PP2B activity was measured according to Higuchi et al. (1991 J. Biol. Chem. 266:18104-18112). Neurospora crassa strain RL3-8 (FGSC stock number 2218) was cultured in Vogel's minimal medium for 48 h at 27oC. Two agar slants (one week old) were used to inoculate 400 ml of medium. Mycelia were harvested by filtering through cheesecloth. The mycelial mat was weighed, frozen in liquid nitrogen and stored at -70oC. Homogenization was carried out by disintegrating the mat at the temperature of the liquid nitrogen in mortar with pestle, then by mixing the powder with 4 volumes/weight extraction buffer (50 mM Tris-HCl pH 7.4, 1 mM EDTA, 0.1% vol/vol. beta-mercaptoethanol) containing a mixture of freshly diluted protease inhibitors (5 mM benzamidine, 0.2 mM phenylmethyl sulfonyl fluoride, 1 mM ophenanthroline). Extraction was completed by mixing the ice cold homogenate at full speed for 1 min in an MSE blender. The extract was filtered through glass wool and was centrifuged at 4oC and 15,000 g for 15 min. The supernatant was used as crude extract in the phosphatase assays. We found that small aliquots (0.1-0.5 ml) of extract can be stored, after being frozen in liquid nitrogen, at -70oC for 7 days without significant loss of phosphatase activity. PP1 and PP2A were assayed with rabbit muscle phosphorylase a substrate. Phosphorylase a was prepared from phosphorylase b with [gamma-32P]ATP and phosphorylase kinase. The assay mixture contained 5 uM 32P-phosphorylase a dimer and 5 mM caffeine in the extraction buffer in a total volume of 30 ul. The reaction was initiated by the addition of the substrate and was terminated after 10 min incubation at 30oC by the addition of 100 ul 10% TCA. TCA soluble radioactivity was counted by Cherenkov radiation. Maximal specific activity was obtained if the Published by New Prairie Press, 2017 extract was diluted with extraction buffer, so that less than 20% of the total 32P was released from the substrate. To test if the liberated radioactivity was in Pi or small phosphopeptide(s) we converted Pi into phosphomolybdic acid and extracted it with organic solvents (Antoniw and Cohen 1976 Eur. J. Biochem. 68:45-54). More than 95% of the radioactivity was recovered in the organic phase. In addition, 20 mM NaF (a well known phosphatase inhibitor) reduced the activity to 5-8%. These facts demonstrate that the assay measures phosphatase activity and the effect of proteases can be neglected. After establishing the assay system we investigated the effect of specific phosphatase inhibitors. One micromolar okadaic acid caused a nearly complete inhibition with the half maximal effect at 10 nM (Fig.1.). Since the IC50 of okadaic acid is 0.1 nM for PP2A and 1520 nM for PP1 in mammalian tissue extracts (Cohen et al. 1989 FEBS Lett. 250:596-600) we assumed that most of the phosphorylase phosphatase activity in N. crassa extract was due to PP1. To test this hypothesis we measured the effect of okadaic acid in the presence of an excess of inhibitor-2. As shown in Fig. 1, under these conditions the activity was half maximally inhibited by 0.3 nM okadaic acid and 90% inhibition was observed at 1 nM concentration. The sensitivity of N. crassa PP1 to the rabbit muscle inhibitor-2 was demonstrated in Fig.2. When PP2A was inactivated with 1 nM okadaic acid, 10,000 U/ml inhibitor-2 caused complete inhibition. 50% inhibition was observed at 30 U/ml concentration i.e. around 1 U inhibitor-2 in the 30 æl assay mixture elicited half maximal inhibition. http://newprairiepress.org/fgr/vol41/iss1/26 DOI: 10.4148/1941-4765.1388 Figure 1. Inhibition of phosphorylase phosphatase activity by okadaic acid in the presence and absence of inhibitor-2 (I-2) Published by New Prairie Press, 2017 Figure 2. Inhibition of phosphorylase phosphatase activity by inhibitor-2 in the presence of okadaic acid (OA) Our results indicate that the phosphorylase phosphatase activity inhibited by 1 nM okadaic acid can be attributed to PP2A and the one inhibited by 10,000 U/ml inhibitor-2 belongs to PP1. Protein concentration was measured according to Read and Northcote (1981 Anal. Biochem. 116:53-64). Using these methods we found 4.95 +/0.9 mU/mg protein (n=10) of total phosphorylase phosphatase activity in N. crassa mycelial extract, 20% of which was PP2A and 65% PP1. By definition one unit of the enzyme liberated 1 umol Pi in 1 min under the above conditions. PP2B was assayed with rabbit muscle inhibitor-1. The substrate was phosphorylated with [gamma-32P]ATP and bovine cAMP-dependent protein kinase. The reaction mixture contained 3 uM 32P-inhibitor-1, 40 mM Tris/HCl pH 7.0, 0.4 mM DTT, 0.2 mM CaCl2, 1 mM MnCl2 and 12 uM calmodulin in 30 ul total volume. The reaction was started by the addition of substrate and after 10 min incubation at 30oC it was terminated by the addition of 100 ul 20% TCA and 100 ul 6% BSA. The released radioactivity was counted as above. Using the extraction method, we proved that more than 95% of the TCA soluble radioactivity was due to the presence of 32Pi. About 40% of the total activity was inhibited by 0.5 mM EDTA. The activity stimulated by http://newprairiepress.org/fgr/vol41/iss1/26 DOI: 10.4148/1941-4765.1388 Ca2+-calmodulin in the presence of Mn2+ was attributed to PP2B, thus the specific activity of PP2B was estimated to be 0.044 +/0.01 U/mg protein (n=6) in the extracts. One unit of PP2B liberated 1 nmol Pi in 1 min in the above assay. The assays described in the present communication are suitable to test protein phosphatase mutants by measuring crude N. crassa extracts, and provide a handle for the purification and characterization of the respective enzymes. The results show considerable similarity between the N. crassa and animal protein phosphatases suggesting that cloning of PP1 and PP2A catalytic subunits could be possible using heterologous cDNA probes. Acknowledgements. This work was supported by the grants OTKA 6005 and 1501. Sz.B. is a recipient of a fellowship from the Hungarian Academy of Sciences, and the Foundation for the Hungarian Science. Published by New Prairie Press, 2017

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Detection of Ser/Thr protein phosphatases in Neurospora crassa

The simultaneos presence of platelet-activating factor, leukotriene B4, prostaglandin F1α and F2α are detectable in the supernatant of human neutrophil granulocytes treated with phospholipase A2 of human monocytes. This enzyme is suspected to play an important role in the pathomechanism of inflammation.

Simultaneous presence of platelet activating factor, leukotriene B4, prostaglandin F1α and F2α in the supernatant of human neutrophils treated with phospholipase A2 of human monocytes

Hyaluronan (HA) is the major glycosaminoglycan component of the extracellular matrix in either normal or malignant tissues and it may affect proliferation, motility and differentiation of various cell types. Three isoforms of plasma membrane-bound hyaluronan synthases (HAS 1, 2 and 3) secrete and simultaneously bind pericellular HA. HAS enzymes are subjects of post-translational protein phosphorylation which is believed to regulate their enzymatic activity. In this study, we investigated the HA homeostasis of normal human epidermal melanocytes, HT168 and WM35 human melanoma cell lines and melanoma metastases. HAS2 and HAS3 were detected in all the samples, while the expression of HAS1 was not detectable in any case. Malignant tissue samples and melanoma cell lines contained extra- and intracellular HA abundantly but not normal melanocytes. Applying HA as a chemoattractant facilitated the migration of melanoma cells in Boyden chamber. The amount of HA was reduced upon the inhibition of calcineurin with cyclosporine A (CsA), while the inhibition of ERK1/2 with PD098059 elevated it in both cell lines. The signals of Ser/Thr phosphoproteins at 57 kD were stronger after CsA treatment, while a markedly weaker signal was detected upon inhibition of the MAPK pathway. Our results suggest opposing effects of the two investigated enzymes on the HA homeostasis of melanoma cells. We propose that the dephosphorylation of HAS enzymes targeted by PP2B augments HA production, while their phosphorylation by the activity of MAPK pathway reduces HA synthesis. As the expression of the HA receptor RHAMM was also significantly enhanced by PD098059, the MAPK pathway exerted a complex attenuating effect on HA signalling in the investigated melanoma cells. This observation suggests that the application of MAPK-ERK pathway inhibitors requires a careful therapeutic design in melanoma treatment.

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PP2B and ERK1/2 regulate hyaluronan synthesis of HT168 and WM35 human melanoma cell lines

Ca2+-dependent stimulation of muscle and liver phosphorylase kinase by heparin

Anti-phosphorylase produced in cocks against phosphorylase from rabbit skeletal muscle inhibits phosphorylases isolated from mammalian heart and skeletal muscle to different degrees. On the basis of the differential inhibition observed an immunotitration method was developed to determine isophosphorylases in crude heart extracts. It was found that the ratio of isophosphorylases is different for every mammalian species investigated and characteristic for the given species.

Pál Gergely

Papers

Detection of Ser/Thr protein phosphatases in Neurospora crassa

Simultaneous presence of platelet activating factor, leukotriene B4, prostaglandin F1α and F2α in the supernatant of human neutrophils treated with phospholipase A2 of human monocytes

PP2B and ERK1/2 regulate hyaluronan synthesis of HT168 and WM35 human melanoma cell lines

Ca2+-dependent stimulation of muscle and liver phosphorylase kinase by heparin

Studies on heart isophosphorylases by means of immunotitration