Decreased Phosphatase Activity Research Articles

Influence of Surface Segregation of Tin on the Phosphatability of Cold‐Rolled Steel Sheets

The increasing use of iron scrap to reduce greenhouse gas emissions results in the enrichment of steel with the tramp element Sn. However, the effect of Sn on phosphatase activity remains unclear. Therefore, this study aims to investigate the effect of the Sn content on the phosphatase activity of cold‐rolled steel plates. The phosphatability of the cold‐rolled steel sheets is evaluated by adding several tens of ppm of Sn. The surface characteristics of the materials are analyzed using X‐Ray photoelectron spectroscopy and transmission electron microscopy. Additionally, the reactivities of the materials in the phosphate treatment solution are assessed using an electrochemical evaluation method. Notably, a significant decrease in phosphatase activity is observed, which is attributed to the inhibition of the phosphate reaction by the Sn oxide layers formed on the steel sheet surface, with a layer thickness of a few nanometers. These Sn surface oxides can be removed by pickling with HCl, and the initial phosphate reaction occurs rapidly in areas where the oxides are removed.

Different responses of enzyme activities to 6‐year warming after transplant of the 12 types of soils

AbstractBackgroundSoil enzymes regulate terrestrial nutrient cycles, and their activities are sensitive to environmental change. Facing climate warming, there are still contradictions over soil enzymes of different soil types in response to climate warming.AimsThe present study aims to reveal the impact of elevated temperature on soil properties. Specifically, we intended to explore the relationship between the activity of soil enzyme and soil property facing elevated temperature across soil types.MethodsA long‐term warming experiment was conducted to assess the possible responses of three enzymes (urease, phosphatase, and sucrase) to elevated temperature across 12 soil types. These soils were collected across China and transported to Nanjing in 2016. Free air temperature increase system with an infrared radiation heater was used to elevate the air temperature by 2°C in elevated treatment (eT). Wheat–soybean continuous rotation was cultivated in the long‐term experiment field since 2016. Enzyme activities, soil pH, soil organic carbon, available nitrogen (AN) and phosphorus (AP), and water content were measured after wheat harvest in 2021.ResultsWarming significantly increased the activities of urease and sucrase in most soil types but dramatically decreased phosphatase activity. The responses of the three enzyme activities to warming varied across soil types, which depended on soil properties. In contrast, soil pH was the major driver in regulating urease activity. Soil AN and water content were two key factors for driving phosphatase activity. However, no such relationship was found for sucrase activity. All these responses did not show obvious regional characteristics.ConclusionThese results suggested that soil enzyme was sensitive to air warming, which was associated with different soil properties. Thus, managing soil variables might be a potential strategy to maintain soil enzymatic activity in facing future climate warming.

Activity in post-pyrogenic soils in the Utrish State Nature Reserve (Russia) in the early succession stages

Due to the increasing frequency of fires, the study of the influence of the pyrogenic factor on the state of forest ecosystems is becoming an increasingly relevant research topic for scientists around the world. Of particular interest in the study is not only the direct wildfire impact, but also its indirect influence, which manifests itself in the early stages of succession. The study has been conducted in the Utrish State Nature Reserve, located on the Abrau Peninsula, northwestern part of the Black Sea coast of the Western Caucasus. The area of the State Nature Reserve «Utrish» is similar to the territory of the entire zone of the Mediterranean climate. The wildfire of 2020 has destroyed 4800 trees, and considerably changed biological properties of brown soils that led to a violation of their ecological functions. Changes in soil properties in the first few years after the wildfire impact are studied insufficiently compared to long-term changes. This study was aimed to investigate the activity of soil enzymes from the oxidase and hydrolase classes in the Utrish State Nature Reserve, immediately after the fire impact (2020), one year later (2021), and two years later (2022). During the field surveys, nine monitoring plots were established, i.e. three plots on each site under weak, medium, and strong degrees of the fire-induced damage. During the study, 81 soil samples were collected, i.e. three soil samples from each monitoring plot. Such enzymes as catalase, peroxidase, invertase, urease, and phosphatase were investigated. To assess the ecological condition of soils, we calculated the Integral Indicator of Biological State (IIBS), and determined the content of organic carbon. The results obtained were compared with the control values, typical for the background area of the forest. A correlation and factor analysis have been carried out. The Mann-Whitney U-test was used to assess the reliability of the data obtained. The enzyme response depends on the type of enzyme and the degree of fire-induced damage. Immediately after the fire impact (2020), a significant decrease (by 53% in average) in catalase, urease, and invertase activity was found on the sites under weak, medium, and strong degrees of damage. Phosphatase activity did not change significantly, while peroxidase activity increased on average by 47%. In 2020, the ecological condition of soils was determined as unfavourable, with the IIBS equal to 82–90%. The activity of catalase, invertase, urease, and phosphatase increased by an average of 31% for soils with a weak degree of fire-induced damage. In 2021, a decrease in activity of the studied enzymes was noted in post-pyrogenic soils under medium and strong damage degree, but to a lesser extent than in 2020. The activity of catalase and urease decreased for all investigated impacts by 47%. The activity of peroxidase and phosphatase was higher in relation to the control values by 55%. For the weak degree of fire-induced damage, a decrease in phosphatase activity by 16% was found. In 2022 (two years after the fire impact), the ecological condition of soils was still recognised as unfavourable, with the IIBS decreased to 89%. However, it was demonstrated that the activity of enzymes from the class of hydrolases two years after the fire impact was closer to the control values than the activity of oxidases. Understanding the relationships between the nature of damage and the response of ecosystem components, such as soil, can allow us to predict the response of ecosystems after forest fires.

Organic fertilization promotes crop productivity through changes in soil aggregation

Soil aggregates are the key functional units of soil ecosystems which are essential to biogeochemical cycling and plant growth. However, it remains unclear how fertilization regimes influence soil aggregation, associated resources, and microbial distribution among aggregates, as well as the potential subsequent impacts for other abiotic and biotic processes. For this, a long-term maize cropping field experiment was conducted in the subtropical region of China under four fertilization treatments: no fertilizer, chemical fertilization, organic fertilization, and chemical plus organic fertilization. Additionally, we classified soil aggregates into large macro-aggregates (>2 mm), small macro-aggregates (0.25–2 mm), and micro-aggregates (<0.25 mm) and compared soil nutrients, enzyme activities, and microbial communities of each aggregate fraction and bulk soil as well as crop productivity and plant carbon (C), nitrogen (N), and phosphorus (P). Results showed that long-term organic fertilization (1) increased soil C, N, and P contents within macro-aggregates, while increased bacterial and fungal biomass within all three aggregate size fractions, (2) increased N-acquiring enzyme activity, C:P, and N:P enzyme ratios but decreased phosphatase activity and C:N enzyme ratio regardless of aggregate fractions, and (3) promoted crop productivity but decreased plant C:N, C:P, and N:P ratios compared with chemical fertilization. Additionally, the fungi:bacteria ratio and phosphatase activity decreased, but the gram-positive to gram-negative bacterial ratio, C-acquiring enzyme activity, C:N and C:P enzyme ratios increased with decreasing aggregate size. The partial least squares models confirmed that macro-aggregates had strong effects on crop performance while micro-aggregates was the main determinant of microbial community. Taken together, long-term organic fertilization promotes soil functioning and crop productivity via increasing the proportion of soil macro-aggregates.

The YAP-Interacting Phosphatase SHP2 Can Regulate Transcriptional Coactivity and Modulate Sensitivity to Chemotherapy in Cholangiocarcinoma.

The Hippo pathway effector Yes-associated protein (YAP) is localized to the nucleus and transcriptionally active in a number of tumor types, including a majority of human cholangiocarcinomas. YAP activity has been linked to chemotherapy resistance and has been shown to rescue KRAS and BRAF inhibition in RAS/RAF-driven cancers; however, the underlying mechanisms of YAP-mediated chemoresistance have yet to be elucidated. Herein, we report that the tyrosine phosphatase SHP2 directly regulates the activity of YAP by dephosphorylating pYAPY357 even in the setting of RAS/RAF mutations, and that diminished SHP2 phosphatase activity is associated with chemoresistance in cholangiocarcinomas. A screen for YAP-interacting tyrosine phosphatases identified SHP2, and characterization of cholangiocarcinomas cell lines demonstrated an inverse relationship between SHP2 levels and pYAPY357. Human sequencing data demonstrated lower SHP2 levels in cholangiocarcinomas tumors as compared with normal liver. Cell lines with low SHP2 expression and higher levels of pYAPY357 were resistant to gemcitabine and cisplatin. In cholangiocarcinomas cells with high levels of SHP2, pharmacologic inhibition or genetic deletion of SHP2 increased YAPY357 phosphorylation and expression of YAP target genes, including the antiapoptotic regulator MCL1, imparting resistance to gemcitabine and cisplatin. In vivo evaluation of chemotherapy sensitivity demonstrated significant resistance in xenografts with genetic deletion of SHP2, which could be overcome by utilizing an MCL1 inhibitor. IMPLICATIONS: These findings demonstrate a role for SHP2 in regulating YAP activity and chemosensitivity, and suggest that decreased phosphatase activity may be a mechanism of chemoresistance in cholangiocarcinoma via a MCL1-mediated mechanism.

Ibrutinib Selects for Cells with Elevated Reactive Oxygen Species and Downregulated Phosphatases

Signals from the B-cell receptor are inappropriately activated in B-cell malignancies, including Chronic lymphocytic leukemia (CLL), Mantle cell lymphoma (MCL), and Diffuse large B-cell lymphoma (DLBCL). In CLL, 90% of patients respond to treatment with ibrutinib, an inhibitor of Bruton's Tyrosine Kinase (BTK), with a mean three-year progression free survival rate of approximately 60%. Despite these promising numbers, patients who do not initially respond to ibrutinib have extremely poor outcomes, and nearly all patients eventually become resistant to ibrutinib due to mutations in BTK and its downstream effector Phospholipase C gamma 2 (PLCG2), with a median survival time of 23 months. In order to identify salvage therapies for ibrutinib-resistant patients, we employed several quantitative proteomic methods to identify signaling pathways and proteins that are essential in ibrutinib-resistant patient cells and cell lines. Total proteomic analysis of lymphoma cell lines selected with ibrutinib in vitro revealed that multiple mitochondrial proteins are increased in B cells exposed to ibrutinib. Next, we found that ibrutinib-treated B cells also exhibited increases in reactive oxygen species (ROS), which potently inhibit several protein phosphatases, including B cell receptor signaling regulators PTPN6 and INPP5D. We next looked at tyrosine phosphorylation in patient B cells collected at several time points during ibrutinib treatment and found decreased phosphorylation of both PTPN6 (Y536) and INPP5D (Y944 and Y865) during treatment. Furthermore, total levels of PTPN6 are decreased in patients within 3 months of initiating treatment, and disruption of PTPN6 in cell lines by CRISPR provided a selective advantage in ibrutinib-treated cell populations. Finally, we found that ibrutinib-treated and ibrutinib-naïve primary CLL cells are sensitive to compounds that prevent reactive oxygen species generation (SOD and PHOX inhibitors). Combined, we propose a mechanism by which ibrutinib treatment leads to increased levels of ROS and decreased phosphatase activity, ultimately leading to a growth advantage in the presence of ibrutinib without concomitant mutations in PLCG2 and BTK. We hypothesize that targeting proteins important for reactive oxygen species generation would also be useful in patients and could result in regained disease control and/or prevention of acquired resistance. Disclosures Gopal: Seattle Genetics, Pfizer, Janssen, Gilead, Sanofi, Spectrum, Amgen, Aptevo, BRIM bio, Acerta, I-Mab-pharma, Takeda, Compliment, Asana Bio, and Incyte.: Consultancy; Teva, Bristol-Myers Squibb, Merck, Takeda, Seattle Genetics, Pfizer, Janssen, Takeda, and Effector: Research Funding; Seattle Genetics, Pfizer, Janssen, Gilead, Sanofi, Spectrum, Amgen, Aptevo, BRIM bio, Acerta, I-Mab-pharma, Takeda, Compliment, Asana Bio, and Incyte: Honoraria.

Hypoxia modulates protein phosphatase 2A through HIF-1α dependent and independent mechanisms in human aortic smooth muscle cells and ventricular cardiomyocytes.

Although protein phosphatases regulate multiple cellular functions, their modulation under hypoxia remains unclear. We investigated expression of the protein phosphatase system under normoxic/hypoxic conditions and the mechanism by which hypoxia alters protein phosphatase 2A (PP2A) activity. Human cardiovascular cells were cultured in cell type specific media under normoxic or hypoxic conditions (1% O2 ). Effects on mRNA expression, phosphatase activity, post-translational modification, and involvement of hypoxia inducible factor 1α (HIF-1α) were assessed using RT-PCR, immunoblotting, an activity assay, and siRNA silencing. All components of the protein phosphatase system studied were expressed in each cell line. Hypoxia attenuated mRNA expression of the transcripts in a cell line- and time-dependent manner. In human aortic smooth muscle cells (HASMC) and AC16 cells, hypoxia decreased PP2Ac activity and mRNA expression without altering PP2Ac abundance. Hypoxia increased demethylated PP2Ac (DPP2Ac) and phosphatase methylesterase 1 (PME-1) abundance but decreased leucine carboxyl methyltransferase 1 (LCMT-1) abundance. HIF-1α siRNA prevented the hypoxia-mediated decrease in phosphatase activity and expression of the catalytic subunit of protein phosphatase 2A (PPP2CA), independently of altering pPP2Ac, DPP2Ac, LCMT-1, or PME-1 abundance. Cardiovascular cells express multiple components of the PP2A system. In HASMC and AC16 cells, hypoxia inhibits PP2A activity through HIF-1α-dependent and -independent mechanisms, with the latter being consistent with altered PP2A holoenzyme assembly. This indicates a complex inhibitory effect of hypoxia on the PP2A system, and highlights PP2A as a therapeutic target for diseases associated with dysregulated protein phosphorylation.

Stemness Is Enhanced in Gastric Cancer by a SET/PP2A/E2F1 Axis.

Gastric cancer is the fifth most common malignancy and the third leading cause of cancer-related deaths worldwide. Chemotherapies against gastric cancer often fail, with cancer recurrence due potentially to the persistence of cancer stem cells. This unique subpopulation of cells in tumors possesses the ability to self-renew and dedifferentiate. These cancer stem cells are critical for initiation, maintenance, metastasis, and relapse of cancers; however, the molecular mechanisms supporting cancer stemness remain largely unknown. Increased kinase and decreased phosphatase activity are hallmarks of oncogenic signaling. Protein phosphatase 2A (PP2A) functions as a tumor-suppressor enzyme, and elevated levels of SET/I2PP2A, an endogenous PP2A protein inhibitor, are correlated with poor prognosis of several human cancers. Here, it was determined that SET expression was elevated in tumor tissue in a gastric cancer mouse model system, and SET expression was positively correlated with poor survival of human gastric cancer patients. Mechanistically, SET knockdown decreased E2F1 levels and suppressed the stemness of cancer cell lines. Immunoprecipitations show SET associated with the PP2A-B56 complex, and the B56 subunit interacted with the E2F1 transcription factor. Treatment of gastric cancer cells with the SET-targeting drug OP449 increased PP2A activity, decreased E2F1 protein levels, and suppressed stemness of cancer cells. These data indicate that a SET/PP2A/E2F1 axis regulates cancer cell stemness and is a potential target for gastric cancer therapy.Implications: This study highlights the oncogenic role of SET/I2PP2A in gastric cancer and suggests that SET maintains cancer cell stemness by suppressing PP2A activity and stabilizing E2F1. Mol Cancer Res; 16(3); 554-63. ©2018 AACR.

Activity of Acid Phosphatases in Ectomycorrhizal Fungi

&lt;p&gt;The colonization of plant roots by mycorrhizal fungi, generally increases P content in the host plant when in soils with low levels of P. The objective of this work was to evaluate the activity of acid phosphatases in two isolates of &lt;em&gt;Pisolithus microcarpus&lt;/em&gt;, grown in different sources and concentrations of phosphate and to characterize the phosphatase isoenzymes produced by isolates. Both isolates were grown on Melin-Norkrans modified (MNM) medium enriched with organic (Po) or inorganic (Pi) sources of phosphorus, at five different concentrations, in order to study the activity of mycelial surface acid phosphatases during incubation for up to 96 h. Activity of acid phosphatases increased when the fungi were grown without or at low concentrations of Pi. Intraspecific differences were observed between the isolates with regard to acid phosphatase production. A greater decrease in phosphatase activity was observed when incubation time was increased than when Pi concentration was increased. At an incubation time of 96 h, activity of acid phosphatases in isolate 90A increased with increasing Po concentration, while for isolate RV82 remained constant over the different incubation periods and Po concentrations tested. When grown in media without Pi, an additional band appeared in the isoenzyme pattern of RV82, while isoenzyme production was not altered in isolate 90A when grown in media without Pi or with 2 mM Pi, showing differences in their isoenzyme patterns. Isolate 90A possesses a potential competitive advantage when utilized in mycorrhizal association with &lt;em&gt;Eucalyptus &lt;/em&gt;because of its ability to utilize Po.&lt;/p&gt;

Determination of the catalytic activity of LEOPARD syndrome-associated SHP2 mutants toward parafibromin, a bona fide SHP2 substrate involved in Wnt signaling

SHP2, encoded by the PTPN11 gene, is a protein tyrosine phosphatase that plays a key role in the proliferation of cells via RAS-ERK activation. SHP2 also promotes Wnt signaling by dephosphorylating parafibromin. Germline missense mutations of PTPN11 are found in more than half of patients with Noonan syndrome (NS) and LEOPARD syndrome (LS), both of which are congenital developmental disorders with multiple common symptoms. However, whereas NS-associated PTPN11 mutations give rise to gain-of-function SHP2 mutants, LS-associated SHP2 mutants are reportedly loss-of-function mutants. To determine the phosphatase activity of LS-associated SHP2 more appropriately, we performed an in vitro phosphatase assay using tyrosine-phosphorylated parafibromin, a biologically relevant substrate of SHP2 and the positive regulator of Wnt signaling that is activated through SHP2-mediated dephosphorylation. We found that LS-associated SHP2 mutants (Y279C, T468M, Q506P, and Q510E) exhibited a substantially reduced phosphatase activity toward parafibromin when compared with wild-type SHP2. Furthermore, each of the LS-associated mutants displayed a differential degree of decrease in phosphatase activity. Deviation of the SHP2 catalytic activity from a certain range, either too strong or too weak, may therefore lead to similar clinical outcomes in NS and LS, possibly through an imbalanced Wnt signal caused by inadequate dephosphorylation of parafibromin.

Dopamine D2receptors' effects on renal inflammation are mediated by regulation of PP2A function

Lack or downregulation of the dopamine D2 receptor (D2R) results in increased renal expression of injury markers and proinflammatory factors that is independent of a blood pressure increase. This study aimed to determine the mechanisms involved in the regulation of renal inflammation by D2Rs. Silencing D2Rs in mouse renal proximal tubule cells increased the expression of the proinflammatory TNF-α, monocyte chemoattractant protein-1 (MCP-1), and IL-6. D2R downregulation also increased Akt phosphorylation and activity, and glycogen synthase kinase-3β (GSK3β) phosphorylation and cyclin D1 expression, downstream targets of Akt; however. phosphatidylinositol 3-kinase (PI3K) activity was not affected. Conversely, D2R stimulation decreased Akt and GSK3β phosphorylation and cyclin D1 expression. Increased phospho-Akt, in the absence of increased PI3K activity, may result from decreased Akt dephosphorylation. Inhibition of protein phosphatase 2A (PP2A) with okadaic acid reproduced the effects of D2R downregulation on Akt, GSK3β, and cyclin D1. The PP2A catalytic subunit and regulatory subunit PPP2R2C coimmunoprecipitated with the D2R. Basal phosphatase activity and the expression of PPP2R2C were decreased by D2R silencing that also blunted the increase in phosphatase activity induced by D2R stimulation. Similarly, silencing PPP2R2C also increased the phosphorylation of Akt and GSK3β. Moreover, downregulation of PPP2R2C resulted in increased expression of TNF-α, MCP-1, and IL-6, indicating that decreased phosphatase activity may be responsible for the D2R effect on inflammatory factors. Indeed, the increase in NF-κB reporter activity induced by D2R silencing was blunted by increasing PP2A activity with protamine. Our results show that D2R controls renal inflammation, at least in part, by modulation of the Akt pathway through effects on PP2A activity/expression.

Abstract 1251: Expression of NADPH oxidase 5 (Nox5) modulates cellular morphology, proliferation and invasiveness of human melanoma UACC-257 cells

Abstract NADPH oxidase 5 (Nox5), one of the seven members of the NADPH oxidase gene family that generate reactive oxygen species (ROS), has been implicated in physiological and pathophysiological processes. Recently, we have reported substantial overexpression of Nox5 in several human cancers including those of prostate, ovary and melanomas; expression in most non-malignant tissues was weak to modest. Despite being upregulated in many human cancers and implicated in promoting cell proliferation, the molecular mechanisms regulated by Nox5 are poorly understood. In this study, the functional significance of Nox5 was assessed in human UACC-257 melanoma cells by generating stable Nox5-overexpressing clones and knockdown clones. Overexpression of Nox5 in UACC-257 cells resulted in enhanced cell growth, increased BrdU positive cells and decreased phosphatase activity. Additionally, these cells had increased normoxic Hif-1α expression and decreased p27Kip1 expression. Conversely, knockdown of endogenous Nox5 in UACC-257 cells resulted in decreased cell growth, decreased BrdU positive cells, decreased Hif-1α expression and increased p27Kip1 expression. Cadherin switch, loss of E-cadherin expression and upregulation of N-cadherin was observed in Nox5 overexpressing cells indicative of an invasive phenotype; conversely, an upregulation of E-cadherin expression in Nox5 knockdown cells was noted. Cell invasion assay through matrigel-coated membranes also demonstrated enhanced invasion by Nox5 overexpressing cells. Additionally, 3D cultures of Nox5 overexpressing cells exhibit an amoeboid morphology compared to that of the Nox5 knockdown cells that were mesenchymal, suggestive of an amoeboid - mesenchymal (AMT) transition. Strikingly, cells that overexpress Nox5 were able to proliferate in serum-free conditions for over a month. In summary, our findings suggest that Nox5 expression in human melanoma UACC-257 cells could contribute to cell proliferation, invasion, amoeboid morphology, and the ability to grow in the absence of serum or growth factors in part due to the generation of high local concentrations of extracellular ROS that modulate multiple signaling networks that regulate Hif-1α, p27Kip1, and the cadherin switch. Citation Format: Smitha Antony, Yongzhong Wu, Guojian Jiang, Jennifer L. Meitzler, Han Liu, Agnes Juhasz, Jiamo Lu, Miriam R. Anver, Krishnendu K. Roy, James H. Doroshow. Expression of NADPH oxidase 5 (Nox5) modulates cellular morphology, proliferation and invasiveness of human melanoma UACC-257 cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1251. doi:10.1158/1538-7445.AM2015-1251

Ataxin-3, DNA damage repair, and SCA3 cerebellar degeneration: on the path to parsimony?

Ataxin-3, DNA damage repair, and SCA3 cerebellar degeneration: on the path to parsimony?

Protein phosphatase-1 is involved in the maintenance of normal homeostasis and in UVA irradiation-induced pathological alterations in HaCaT cells and in mouse skin

The number of ultraviolet (UV) radiation-induced skin diseases such as melanomas is on the rise. The altered behavior of keratinocytes is often coupled with signaling events in which Ser/Thr specific protein kinases and phosphatases regulate various cellular functions. In the present study the role of protein phosphatase-1 (PP1) was investigated in the response of human keratinocyte (HaCaT) cells and mouse skin to UV radiation. PP1 catalytic subunit (PP1c) isoforms, PP1cα/γ and PP1cδ, are all localized to the cytoskeleton and cytosol of keratinocytes, but PP1cδ was found to be dominant over PP1α/γ in the nucleus. PP1c-silencing in HaCaT cells decreased the phosphatase activity and suppressed the viability of the cells. Exposure to a 10J/cm2 UVA dose induced HaCaT cell death and resulted in a 30% decrease of phosphatase activity. PP1c-silencing and UVA irradiation altered the gene expression profile of HaCaT cells and suggested that the expression of 19 genes was regulated by the combined treatments with many of these genes being involved in malignant transformation. Microarray analysis detected altered expression levels of genes coding for melanoma-associated proteins such as keratin 1/10, calcium binding protein S100A8 and histone 1b. Treatment of Balb/c mice with the PP1-specific inhibitor tautomycin (TM) exhibited increased levels of keratin 1/10 and S100A8, and a decreased level of histone 1b proteins following UVA irradiation. Moreover, TM treatment increased pigmentation of the skin which was even more apparent when TM was followed by UVA irradiation. Our data identify PP1 as a regulator of the normal homeostasis of keratinocytes and the UV-response.

Effect of fertilization levels on soil microorganism amount and soil enzyme activities

Field experiments were conducted in Shangluo pharmaceutical base in Shaanxi province to study the effect of nitrogen, phosphorus and potassium in different fertilization levels on Platycodon grandiflorum soil microorganism and activities of soil enzyme, using three-factor D-saturation optimal design with random block design. The results showed that N0P2K2, N2P2K0, N3P1K3 and N3P3K1 increased the amount of bacteria in 0-20 cm of soil compared with N0P0K0 by 144.34%, 39.25%, 37.17%, 53.58%, respectively. The amount of bacteria in 2040 cm of soil of N3P1K3 increased by 163.77%, N0P0K3 increased the amount of soil actinomycetes significantly by 192.11%, while other treatments had no significant effect. N2P0K2 and N3P1K3 increased the amounts of fungus significantly in 0-20 cm of soil compared with N0P0K0, increased by 35.27% and 92.21%, respectively. N3P0K0 increased the amounts of fungus significantly in 20-40 cm of soil by 165.35%, while other treatments had no significant effect. All treatments decrease soil catalase activity significantly in 0-20 cm of soil except for N2P0K2, and while N2P2K0 and NPK increased catalase activity significantly in 2040 cm of soil. Fertilization regime increased invertase activity significantly in 2040 cm of soil, and decreased phosphatase activity inordinately in 0-20 cm of soil, while increased phosphatase activity in 2040 cm of soil other than N1P3K3. N3P0K0, N0P0K3, N2P0K2, N2P2K0 and NPK increased soil urease activity significantly in 0-20 cm of soil compared with N0P0K0 by 18.22%, 14.87%,17.84%, 27.88%, 24.54%, respectively. Fertilization regime increased soil urease activity significantly in 2040 cm of soil other than N0P2K2.

Carbon-Degrading Enzyme Activities Stimulated by Increased Nutrient Availability in Arctic Tundra Soils

Climate-induced warming of the Arctic tundra is expected to increase nutrient availability to soil microbes, which in turn may accelerate soil organic matter (SOM) decomposition. We increased nutrient availability via fertilization to investigate the microbial response via soil enzyme activities. Specifically, we measured potential activities of seven enzymes at four temperatures in three soil profiles (organic, organic/mineral interface, and mineral) from untreated native soils and from soils which had been fertilized with nitrogen (N) and phosphorus (P) since 1989 (23 years) and 2006 (six years). Fertilized plots within the 1989 site received annual additions of 10 g N⋅m-2⋅year-1 and 5 g P⋅m-2⋅year-1. Within the 2006 site, two fertilizer regimes were established – one in which plots received 5 g N⋅m-2⋅year-1 and 2.5 g P⋅m-2⋅year-1 and one in which plots received 10 g N⋅m-2⋅year-1 and 5 g P⋅m-2⋅year-1. The fertilization treatments increased activities of enzymes hydrolyzing carbon (C)-rich compounds but decreased phosphatase activities, especially in the organic soils. Activities of two enzymes that degrade N-rich compounds were not affected by the fertilization treatments. The fertilization treatments increased ratios of enzyme activities degrading C-rich compounds to those for N-rich compounds or phosphate, which could lead to changes in SOM chemistry over the long term and to losses of soil C. Accelerated SOM decomposition caused by increased nutrient availability could significantly offset predicted increased C fixation via stimulated net primary productivity in Arctic tundra ecosystems.

The response of microbial biomass and hydrolytic enzymes to a decade of nitrogen, phosphorus, and potassium addition in a lowland tropical rain forest

Nutrient availability is widely considered to constrain primary productivity in lowland tropical forests, yet there is little comparable information for the soil microbial biomass. We assessed microbial nutrient limitation by quantifying soil microbial biomass and hydrolytic enzyme activities in a long-term nutrient addition experiment in lowland tropical rain forest in central Panama. Multiple measurements were made over an annual cycle in plots that had received a decade of nitrogen, phosphorus, potassium, and micronutrient addition. Phosphorus addition increased soil microbial carbon (13 %), nitrogen (21 %), and phosphorus (49 %), decreased phosphatase activity by ~65 % and N-acetyl β-glucosaminidase activity by 24 %, but did not affect β-glucosidase activity. In contrast, addition of nitrogen, potassium, or micronutrients did not significantly affect microbial biomass or the activity of any enzyme. Microbial nutrients and hydrolytic enzyme activities all declined markedly in the dry season, with the change in microbial biomass equivalent to or greater than the annual nutrient flux in fine litter fall. Although multiple nutrients limit tree productivity at this site, we conclude that phosphorus limits microbial biomass in this strongly-weathered lowland tropical forest soil. This finding indicates that efforts to include enzymes in biogeochemical models must account for the disproportionate microbial investment in phosphorus acquisition in strongly-weathered soils.

Histological, cytological and biochemical alterations induced by microcystin-LR and cylindrospermopsin in white mustard (Sinapis albaL.) seedlings

This study compares the histological, cytological and biochemical effects of the cyanobacterial toxins microcystin-LR (MCY-LR) and cylindrospermopsin (CYN) in white mustard (Sinapis alba L.) seedlings, with special regard to the developing root system. Cyanotoxins induced different alterations, indicating their different specific biochemical activities. MCY-LR stimulated mitosis of root tip meristematic cells at lower concentrations (1 μg ml-1) and inhibited it at higher concentrations, while CYN had only inhibitory effects. Low CYN concentrations (0.01 μg ml-1) stimulated lateral root formation, whereas low MCY-LR concentrations increased only the number of lateral root primordia. Both inhibited lateral root development at higher concentrations. They induced lignifications, abnormal cell swelling and inhibited xylem differentiation in roots and shoots. MCY-LR and CYN induced the disruption of metaphase and anaphase spindles, causing altered cell divisions. Similar alterations could be related to decreased protein phosphatase (PP1 and PP2A) activities in shoots and roots. However, in vitro phosphatase assay with purified PP1 catalytic subunit proved that CYN in contrast to MCY-LR, decreased phosphatase activities of mustard in a non-specific way. This study intends to contribute to the understanding of the mechanisms of toxic effects of a protein phosphatase (MCY-LR) and a protein synthesis (CYN) inhibitory cyanotoxin in vascular plants.

The Structural Basis for Substrate Recognition by Mammalian Polynucleotide Kinase 3′ Phosphatase

Mammalian polynucleotide kinase 3' phosphatase (PNK) plays a key role in the repair of DNA damage, functioning as part of both the nonhomologous end-joining (NHEJ) and base excision repair (BER) pathways. Through its two catalytic activities, PNK ensures that DNA termini are compatible with extension and ligation by either removing 3'-phosphates from, or by phosphorylating 5'-hydroxyl groups on, the ribose sugar of the DNA backbone. We have now determined crystal structures of murine PNK with DNA molecules bound to both of its active sites. The structure of ssDNA engaged with the 3'-phosphatase domain suggests a mechanism of substrate interaction that assists DNA end seeking. The structure of dsDNA bound to the 5'-kinase domain reveals a mechanism of DNA bending that facilitates recognition of DNA ends in the context of single-strand and double-strand breaks and suggests a close functional cooperation in substrate recognition between the kinase and phosphatase active sites.

Identification of AKAP79 as a protein phosphatase 1 catalytic binding protein.

The ubiquitously expressed and highly promiscuous protein phosphatase 1 (PP1) regulates many cellular processes. Targeting PP1 to specific locations within the cell allows for the regulation of PP1 by conferring substrate specificity. In the present study, we identified AKAP79 as a novel PP1 regulatory subunit. Immunoprecipitaiton of the AKAP from rat brain extract found that the PP1 catalytic subunit copurified with the anchoring protein. This is a direct interaction, demonstrated by pulldown experiments using purified proteins. Interestingly, the addition of AKAP79 to purified PP1 catalytic subunit decreased phosphatase activity with an IC(50) of 811 ± 0.56 nM of the anchoring protein. Analysis of AKAP79 identified a PP1 binding site that conformed to a consensus PP1 binding motif (FxxR/KxR/K) in the first 44 amino acids of the anchoring protein. This was confirmed when a peptide mimicking this region of AKAP79 was able to bind PP1 by both pulldown assay and surface plasmon resonance. However, PP1 was still able to bind to AKAP79 upon deletion of this region, suggesting additional sites of contact between the anchoring protein and the phosphatase. Importantly, this consensus PP1 binding motif was found not to be responsible for PP1 inhibition, but rather enhanced phosphatase activity, as deletion of this domain resulted in an increased inhibition of PP1 activity. Instead, a second interaction domain localized to residues 150-250 of AKAP79 was required for the inhibition of PP1. However, the inhibitory actions of AKAP79 on PP1 are substrate dependent, as the anchoring protein did not inhibit PP1 dephosphorylation of phospho-PSD-95, a substrate found in AKAP79 complexes in the brain. These combined observations suggest that AKAP79 acts as a PP1 regulatory subunit that can direct PP1 activity toward specific targets in the AKAP79 complex.