Endogenous Phosphatase Research Articles

Abstract 13262: Improved Rabbit Model of Calcific Aortic Valve Disease Induces Severe Medial Calcification and Stenosis Equivalent to Human Disease

Introduction: Accurate animal models of calcific aortic valve stenosis (CAVS) are needed to get insights into the early stages. We had previously modified a rabbit model to achieve CAVS. Here, we sought to assess its ability to produce similar lesion to humans. Methods: New Zealand rabbits were randomized to normal diet or +1% cholesterol + 3500 I.U.s/kg Vitamin D2 daily. Aortic valve area was assessed with echocardiography. At 7 weeks, blood serum and valve cusps were retrieved from sacrificed animals. Valve cusps were ex-vivo imaged with 18F-NaF microPET/CT, snap-frozen or formalin-fixed. Cusps were homogenized with an optimized protocol. Pyrophosphate (PPi) was measured in serum and cusps, metabolites analyzed with LC-MS/MS. Phosphatase activity was detected with BCIP/NBT assay. Protein content was analyzed with immunohistochemistry, quantitative ELISA and proteomics analysis (LC-MS/MS), and compared to human databases. Fixed cusps were analyzed using FT-IR and imaged with atomic force, scanning and transmission electron microscope. EDX chemical maps were obtained from the regions of interest Results: Vitamin D and metabolites were validated in serum and tissue. Echocardiography confirmed significant valve stenosis. Sodium fluoride activity was quadrupled in experimental valves. Histology revealed severe medial calcification, positive for osteopontin, negative for TNAP, BSPII and osteocalcin. Endogenous phosphatase activity colocalized with calcification in the medium. Serum PPi levels were increased, and tissue PPi levels were decreased. Proteomics analysis revealed 96 differentially expressed proteins validating important proteins, including apolipoproteins, complement, osteonectin, matrix Gl(a), galectin-3, fetuin-A, sortiltin, and TGF-b axis proteins. Ultrastructural analysis revealed the pathognomonic calcified spherical particles composed predominantly of phosphorus in the experimental valves. Conclusions: The improved high-fat - vitamin D toxicity rabbit model produced severe phosphate-induced CAVS, with similar protein and ultrastructural content to human disease, related to TGF-b pathway. Further investigations are needed in the underlying molecular networks driving the lesion.

Affinity-based profiling of endogenous phosphoprotein phosphatases by mass spectrometry.

Phosphoprotein phosphatases (PPPs) execute >90% of serine/threonine dephosphorylation in cells and tissues. While the role of PPPs in cell biology and diseases such as cancer, cardiac hypertrophy and Alzheimer's disease is well established, the molecular mechanisms governing and governed by PPPs still await discovery. Here we describe a chemical proteomic strategy, phosphatase inhibitor beads and mass spectrometry (PIB-MS), that enables the identification and quantification of PPPs and their posttranslational modifications in as little as 12 h. Using a specific but nonselective PPP inhibitor immobilized on beads, PIB-MS enables the efficient affinity-capture, identification and quantification of endogenous PPPs and associated proteins ('PPPome') from cells and tissues. PIB-MS captures functional, endogenous PPP subunit interactions and enables discovery of new binding partners. It performs PPP enrichment without exogenous expression of tagged proteins or specific antibodies. Because PPPs are among the most conserved proteins across evolution, PIB-MS can be employed in any cell line, tissue or organism.

A Dominant Negative SHP-2 Which Abrogates PD-1 Signalling Pathways and Restores Function of Cytotoxic CAR T Cells

IntroductionRecent successes in the clinic with monoclonal antibodies against cytotoxic T lymphocyte antigen 4 (CTLA-4) and programmed-death 1 (PD-1) have highlighted the importance of developing strategies to overcome T cell inhibition when investigating anti-tumour immunotherapies. Strategies to avoid inhibitory signals by the tumour microenvironment have recently been incorporated into CAR T cells with the most exploited strategy being the conversion of the inhibitory signal from one receptor into an activating one. Many of these inhibitory receptors signal through either the SHP-1/SHP-2 phosphatases. Here we present a method to block these phosphatases using a dominant negative protein. Unlike previous methods that focus on blocking one inhibitory receptor at a time, this approach enables the disarmament of multiple inhibitory receptors at once.Methods and resultsWe generated dominant negative forms of SHP-2 (dnSHP2) that block the function of this endogenous inhibitory phosphatase. This was transduced into T cells coupled with an anti-CD19 CAR and CAR T function was assessed in the presence of PD-1/PD-L1 ligation. In the absence of dnSHP-2, CAR T cell killing, proliferation and cytokine release was significantly impaired when co-cultured with target cells that ligated both the CAR and PD-1. In contrast, CAR T cells that expressed dnSHP-2 observed no defect in killing response in the presence of PD1 ligation. Furthermore, dnSHP2 was shown to impair PD-1 mediated inhibition of cytokine release and proliferation in CAR T cells. This ability to block PD-1 mediated inhibition in CAR T cells was independent of the CAR generation, with similar results in first and second-generation CARs with either a 41BB or CD28 co-stimulatory domain.ConclusionsWe have successfully engineered a protein (dnSHP2) that is capable of abrogating the function of the inhibitory phosphatase SHP-2. The expression of dnSHP2 impaired PD-1 mediated inhibition of CAR T cells and could theoretically impair any inhibitory receptor that signals through SHP2, making this a universal approach to nullify inhibitory receptors on CAR T cells. DisclosuresBaldan:Autolus Ltd.: Employment, Equity Ownership. Ghongane:Autolus Ltd.: Employment, Equity Ownership. Kokalaki:Autolus Ltd.: Employment, Equity Ownership. Lim:Autolus Ltd.: Employment, Equity Ownership. Onuoha:Autolus Ltd.: Employment, Equity Ownership. Cordoba:Autolus Ltd.: Employment, Equity Ownership. Thomas:Autolus Ltd.: Employment, Equity Ownership. Pule:Autolus Ltd.: Employment, Equity Ownership.

Involvement of Leishmania Phosphatases in Parasite Biology and Pathogeny.

In the Leishmania lifecycle, the motile promastigote form is transmitted from the sand fly vector to a mammalian host during a blood meal. Inside vertebrate host macrophages, the parasites can differentiate into the amastigote form and multiply, causing leishmaniasis, one of the most significant neglected tropical diseases. Leishmania parasites face different conditions throughout their development inside sand flies. Once in the mammalian host, the parasites have to overcome the microbicide repertoire of the cells of the immune system to successfully establish the infection. In this context, the expression of protein phosphatases is of particular interest. Several members of the serine/threonine-specific protein phosphatase (STP), protein tyrosine phosphatase (PTP), and histidine acid phosphatase (HAcP) families have been described in different Leishmania species. Although their physiological roles have not been fully elucidated, many studies suggest they have an involvement with parasite biology and pathogeny. Phosphatases play a role in adaptation to nutrient starvation during parasite passage through the sand fly midgut. They are also important to parasite virulence, mainly due to the modulation of host cytokine production and impairment of the microbiocidal potential of macrophages. Furthermore, recent whole-genome expression analyses have shown that different phosphatases are upregulated in metacyclic promastigotes, the infective form of the mammalian host. Leishmania phosphatases are also upregulated in drug-resistant strains, probably due to the increase in drug efflux related to the activation of ABC transporters. Throughout this review, we will describe the physiological roles that have been attributed to Leishmania endogenous phosphatases, including their involvement in the adaptation, survival, and proliferation of the parasites inside their hosts.

Prostaglandin E2 induces dual-specificity phosphatase-1, thereby attenuating inflammatory genes expression in human osteoarthritic synovial fibroblasts

Characteristic features of osteoarthritis (OA) are joint pain and cartilage degeneration. The degeneration is caused by excess induction of matrix metalloproteinases (MMPs) and the pain is caused by nerve growth factor (NGF)-dependent nerve invasion into synovial tissue in addition to nociceptive pain by prostaglandin (PG)E2. The objective of this study was to clarify the suppressive mechanism of PGE2 on the regulation of MMPs and NGF by focusing on mitogen-activated protein kinases (MAPKs) and their endogenous phosphatase, dual-specificity phosphatase (DUSP)-1 in human synovial fibroblasts. PGE2 strongly increased DUSP-1 and suppressed IL-1β-induced MAPKs phosphorylation. Inhibition of MAPKs by selective inhibitors differentially regulated the IL-1β-induced expression of MMPs and NGF expression. IL-1β-induced MAPKs phosphorylation was prolonged and enhanced in DUSP-1 knockdown cells and the expression of MMPs and NGF was also increased. This study revealed that PGE2 has novel biological activity that suppresses NGF and MMPs expression by inducing DUSP-1 expression.

Sialidase of Glaesserella parasuis Augments Inflammatory Response via Desialylation and Abrogation of Negative Regulation of Siglec-5.

Siglecs are sialic acid-binding immunoglobulin-like lectins that play an important role in tissue homeostasis, immune response, and pathogen infection. Bacterial sialidases act on natural ligands of Siglecs, interfering with the Siglec-mediated immune response. Glaesserella parasuis is a porcine bacterial pathogen that secretes sialidase. However, little is known about the sialidase of G. parasuis and its impact on immune regulation. Here, we used wild-type G. parasuis, a sialidase-deficient mutant, and complementary strains to investigate the role of sialidase in porcine alveolar macrophage infection. Sialidase induced the release of proinflammatory cytokines, such as interleukin-1α (IL-1α), IL-6, and tumor necrosis factor alpha, from porcine alveolar macrophages. Moreover, sialidase desialylated the surface of porcine alveolar macrophages and altered the expression of Siglecs (the expression of Siglec-5 was reduced). Furthermore, sialidase led to a reduction in endogenous SH2 domain-containing protein tyrosine phosphatase (SHP-2) recruitment to Siglec-5 and simultaneously activated the inflammatory response via the mitogen-activated protein kinase and nuclear factor kappa light chain enhancer of activated B cell signaling pathways. This desialylation occurred before the release of proinflammatory cytokines, suggesting that the sialidase-induced inflammatory response was followed by reduced recruitment of SHP-2 to Siglec-5. Thus, this study is the first to demonstrate the role of sialidase in the inflammatory response of G. parasuis. This role resulted from the abrogation of negative regulation of Siglec-5 on proinflammatory cytokine release. This study helps to understand the molecular mechanism underlying the inflammatory response induced by sialidase secreted by G. parasuis and the acute inflammation caused by G. parasuis.

Tropism of Severe Acute Respiratory Syndrome Coronavirus 2 for Barrett’s Esophagus May Increase Susceptibility to Developing Coronavirus Disease 2019

Tropism of Severe Acute Respiratory Syndrome Coronavirus 2 for Barrett’s Esophagus May Increase Susceptibility to Developing Coronavirus Disease 2019

A turn-on near-infrared fluorescent probe for visualization of endogenous alkaline phosphatase activity in living cells and zebrafish.

Alkaline phosphatase (ALP) is an essential hydrolase and widely distributed in living organisms. It plays important roles in various physiological and pathological processes. Herein, a turn-on near-infrared (NIR) fluorescent probe (DXMP) was developed for sensitive detection of ALP activity both in vitro and in vivo based on the intramolecular charge transfer (ICT) mechanism. Upon incubation with ALP, DXMP exhibited a strong fluorescence increment at 640 nm, which was attributed to the fact that ALP-catalyzed cleavage of the phosphate group in DXMP induced the transformation of DXMP into DXM-OH. The probe exhibited prominent features including outstanding selectivity, high sensitivity, and excellent biocompatibility. More importantly, it has been successfully used to detect and image endogenous ALP in living cells and zebrafish.

A novel aggregation-induced dual emission probe for in situ light-up detection of endogenous alkaline phosphatase

Abnormal level of alkaline phosphatase (ALP) activity has been linked to many diseases in human. The development of fluorescent molecular probes that can report the expression and activity of ALP in various biological systems will be extremely valuable. However, the in vivo monitoring for ALP in living cells and more complex biological systems remains a great challenge. The excited-state intramolecular proton transfer (ESIPT) probe with proportional fluorescence has low background noise, while the aggregation induced emission (AIE) probe has the advantages of signal amplification and good light stability. Herein, an “AIE + ESIPT” fluorescent probe 2-(benzo[d]thiazol-2-yl)-4-(1,4,5-triphenyl-1H-imidazole-2-yl)phenyl dihydrogen phosphate (THP) was constructed for the highly selective and sensitive detection of ALP. By introducing a phosphate ester at the hydroxyl position of the solid fluorophore 2-(benzo[d]thiazol-2-yl)-4-(1,4,5-triphenyl-1H-imidazole-2-yl)phenol, ESIPT was hindered and the probe present a faint blue fluorescence in DMSO solution. While ALP was introduced, causing the phosphate in THP hydrolyzed, and the ESIPT process was restored to yield a yellow fluorescence at 550 nm, thereby achieving proportionality detection. THP exhibited high selectivity and sensitively to ALP with low limit of detection (1.21228 U/L), and the reaction completed within 20 min. In addition, with its outstanding advantages of low biological toxicity and enzyme conversion characteristics, THP has been successfully applied to ALP imaging in living cells (Hela cells, A549 cells and Hek293 cells), and can provide in situ information on the reaction site. Therefore, THP has the potential for detecting ALP activity in biomedical application.

Antioxidant, Anti-Inflammatory and Anti-Angiogenic Properties of Citrus lumia Juice.

Citrus juices are a rich source of bioactive compounds with various and well-known health benefits. The aim of this study was to investigate the polyphenols and ascorbic acid content as well as to investigate the antioxidant, anti-inflammatory and anti-angiogenic properties of the juice of an ancient Mediterranean species, Citrus lumia Risso (CLJ). The antioxidant and anti-inflammatory activities were evaluated by several in vitro cell-free and cell-based assays, whereas two different in vivo models, the chick chorioallantoic membrane (CAM) and the zebrafish embryos, were used to characterize the anti-angiogenic properties. Twenty-eight polyphenols were identified by RP-LC-DAD-ESI-MS analysis (flavonoids 68.82% and phenolic acids 31.18%) with 1-caffeoyl-5-feruloylquinic acid and kaempferol 3′-rhamnoside, which represent the most abundant compounds (25.70 and 23.12%, respectively). HPLC-DAD analysis showed a high ascorbic acid content (352 mg/kg of CLJ), which contributes with polyphenols to the marked and dose-dependent antioxidant and anti-inflammatory properties observed. CLJ showed strong and dose-dependent anti-angiogenic activity as highlighted by the inhibition of blood vessel formation on CAMs and the decrease of endogenous alkaline phosphatase on zebrafish embryos. Moreover, within the concentration range tested, no dead or malformed embryos were recorded. Certainly, further studies are needed to investigate the molecular mechanisms underlying these promising biological effects, but considering the evidence of the present study, the use of CLJ as a ready-to drink safe prevention strategy for inflammatory-based diseases correlated to angiogenesis could be justified.

Evaluation of sporadic bovine alkaline phosphatase interference in the Beckman Access unconjugated estriol (uE3) assay affecting maternal serum screening results

ObjectivesBovine alkaline phosphatase (BALP) mediated interference is a potential issue in the Beckman Access unconjugated estriol (uE3) assay. As the uE3 assay is a component of second trimester maternal serum screening characterizing this interference is essential for delivering accurate trisomy 18 and trisomy 21 risks. Design and methodsResidual serum samples (n = 517) were measured by two different lots of uE3 assay. Scavenger BALP (sBALP) was added to all samples to remove potential BALP dependent interference and assessed using both lots of uE3 reagent. ResultsBALP mediated interference was observed in similar frequency in both lots of reagent (~3%), although the patterns of positive and negative interference differed between the lots. Pretreatment with sBALP improved lot-to-lot comparison. The presence of BALP related interference was not related to the concentration of endogenous human alkaline phosphatase. The use of polyethylene glycol and sBALP treatment appeared to mitigate BALP mediated interference equally well, and resulted in concordance in measured uE3 concentrations between reagent lots. Additionally, heterophile antibody interference was observed in two samples affected with BALP interference, and the heterophile antibody interference was resolved by both PEG and heterophile antibody blocking reagent treatment, but not sBALP treatment. While the maternal screen numeric risk for affected samples changed, the risk classification changed from a negative to positive screen in two samples. ConclusionsInterference in the uE3 assay has the potential to affect maternal serum risk calculations in different reagent lots, and pretreatment of samples with scavenger BALP or PEG should be considered in cases of unexplained uE3 concentrations.

Calnexin mediates the maturation of GPI-anchors through ER retention

The protein folding and lipid moiety status of glycosylphosphatidylinositol-anchored proteins (GPI-APs) are monitored in the endoplasmic reticulum (ER), with calnexin playing dual roles in the maturation of GPI-APs. In the present study, we investigated the functions of calnexin in the quality control and lipid remodeling of GPI-APs in the ER. By directly binding the N-glycan on proteins, calnexin was observed to efficiently retain GPI-APs in the ER until they were correctly folded. In addition, sufficient ER retention time was crucial for GPI-inositol deacylation, which is mediated by post-GPI attachment protein 1 (PGAP1). Once the calnexin/calreticulin cycle was disrupted, misfolded and inositol-acylated GPI-APs could not be retained in the ER and were exposed on the plasma membrane. In calnexin/calreticulin-deficient cells, endogenous GPI-anchored alkaline phosphatase was expressed on the cell surface, but its activity was significantly decreased. ER stress induced surface expression of misfolded GPI-APs, but proper GPI-inositol deacylation occurred due to the extended time that they were retained in the ER. Our results indicate that calnexin-mediated ER quality control systems for GPI-APs are necessary for both protein folding and GPI-inositol deacylation.

Estrogen receptor-dependent and independent roles of benzo[a]pyrene in Ishikawa cells.

Polycyclic aromatic hydrocarbons (PAHs) are environmental pollutants generated from the incomplete combustion of organic material. PAHs have been studied as genotoxicants, but some also act via non-genotoxic mechanisms in estrogen-dependent malignancies, such as breast cancer. PAHs require metabolic activation to electrophilic metabolites to exert their genotoxicity but non-genotoxic properties may also contribute to their carcinogenicity. The role of PAHs in endometrial cancer, a cancer associated with unopposed estrogen action is unknown. We assessed the metabolism of the representative PAH, benzo[a]pyrene (B[a]P), to estrogenic compounds in Ishikawa human endometrial cells in the presence and absence of cytochrome P450 induction. Using stable-isotope dilution high-performance liquid chromatography and APCI tandem mass spectrometry in the selected reaction monitoring mode, we analyzed B[a]P metabolism in Ishikawa cells. Estrogenic activity of B[a]P metabolites was determined by the endogenous estrogen inducible alkaline phosphatase reporter gene and an exogenous estrogen response element (ERE) luciferase reporter gene construct. We also assessed whether PAHs can induce a proliferative phenotype via estrogen receptor (ER)- and non-ER-regulated pathways. We demonstrate that B[a]P can be metabolized in human endometrial cells into 3-OH-B[a]P and B[a]P-7,8-dione in sufficient amounts to activate ERs. We also show that only B[a]P-7,8-dione induces endometrial cell proliferation at concentrations lower than required to activate the ER; instead non-genomic signaling by the EGF receptor (EGFR) and activation of the mitogen-activated protein kinase (MAPK) pathway was responsible. This work indicates that human endometrial cells can metabolize PAHs into estrogenic metabolites, which may induce cell proliferation through non-ER-regulated pathways.

Multivalent, Stabilized Mannose-6-Phosphates for the Targeted Delivery of Toll-Like Receptor Ligands and Peptide Antigens.

Mannose‐6‐phosphate (M6P) is recognized by the mannose‐6‐phosphate receptor and plays an important role in the transport of cargo to the endosomes, making it an attractive tool to improve endosomal trafficking of vaccines. We describe herein the assembly of peptide antigen conjugates carrying clusters of mannose‐6‐C‐phosphonates (M6Po). The M6Po's are stable M6P mimics that are resistant to cleavage of the phosphate group by endogenous phosphatases. Two different strategies for the incorporation of the M6Po clusters in the conjugate have been developed: the first relies on a “post‐assembly” click approach employing an M6Po bearing an alkyne functionality; the second hinges on an M6Po C‐glycoside amino acid building block that can be used in solid‐phase peptide synthesis. The generated conjugates were further equipped with a TLR7 ligand to stimulate dendritic cell (DC) maturation. While antigen presentation is hindered by the presence of the M6Po clusters, the incorporation of the M6Po clusters leads to increased activation of DCs, thus demonstrating their potential in improving vaccine adjuvanticity by intraendosomally active TLR ligands.

A dual-mode strategy for sensing and bio-imaging of endogenous alkaline phosphatase based on the combination of photoinduced electron transfer and hyperchromic effect

Benefit from the additional correction of the output signal in dual-mode detection, traditional dual signal readout strategies are performed by constructing the ratiometric fluorescent probe through excitation energy transfer (EET) or fluorescence resonance energy transfer (FRET). To avoid the complicated modification process and obtain the results rapidly, a simple dual-mode sensing strategy based on the electronic effects of p-nitrophenol (PNP) is described to monitor the activities of alkaline phosphatase (ALP). In the sensing platform, p-nitrophenylphosphate was used as a substrate to produce the PNP using ALP as the catalyst. Due to the PNP possesses negative effect of induction and conjugation, photoinduced electron transfer and hyperchromic effect have been achieved between PNP and polyethyleneimine-protected copper nanoclusters (PEI-Cu NCs), which caused the changes of the fluorescence intensity and UV–visible absorption. The dual-mode signal sensing system showed the satisfactory linear results of ALP from 1 to 100 U/L for fluorescent sensing strategy and 1–70 U/L for the absorption method with a competitive LOD of 0.27 and 0.87 U/L (signal-to-noise ratio of 3). This strategy detected biological ALP in human serum and bio-imaging of endogenous ALP in A549 cells successfully, which verifies a certain potential of the strategy for the practical applications.

Использование левамизола для блокирования кишечной формы щелочной фосфатазы при проведении иммуногистохимических реакций

Introduction. Immunohistochemical staining by an indirect method with a chromogenic label requires en-zymes, among which is alkaline phosphatase (AP). AP is also present in human tissues. It can break down the molecules of the immunohistochemical substrate, which leads to significant background staining. It is necessary to block endogenous enzymes prior to immunohistochemical staining to reduce this effect. One of the ways to block endogenous alkaline phosphatase is to use levamisole solutions. The aim of the study was to describe the technique of levamisole use to block the alkaline phosphatase intestinal form during immunohistochemical assays. Materials and methods. This article provides the calculations for 0.001M working levamisole solution preparation from a 10% officinal veterinary levamisole hydrochloride produced by Livisto Invesa Industrial Veterinaria S. A., Spain. The inactivation of AP intestinal form was checked by a reaction with one marker (PDGFRb) and two markers (FAP and SMA) on the colon cancer specimens. We used the Abcam ab210061 DoubleStain IHC Kit: M&R on human tissue (HRP/Green&AP/Red, Great Britain) according to the method recommended by the manufacturer with some changes to identify two markers on the same slide. Results. During the immunohistochemical assay, a complete absence of background staining and a bright contrast reaction with antibodies (both with one and two in the same section) using 1mМ levamisole was achieved. It indicates sufficient inactivation of the AP intestinal isoform in the colon cancer specimens. A comparative cost-benefit analysis for one slide using ready-to-use commercial blocking reagents and officinal levamisole solution shows a significant economic advantage of the latter. Conclusion. The high reaction quality and the palpable economic profits open up opportunities for using 1mM levamisole solution for immunohistochemical studies in laboratory practice and research work. Keywords: levamisole, immunochistochemistry, alkaline phosphatase

Platelet polyphosphate level is elevated in patients with chronic primary thrombocytopenia: A preliminary study

Platelets are key players in hemostasis. These blood cells contain different types of granules. Recently, there has been a growing interest in the role of inorganic polyphosphate (polyP) structures stored in dense granules of platelets and secreted during platelet activation. To measure platelet polyP levels in patients with thrombocytopenia and thrombocythemia, and to examine the relationship of this indicator with platelet aggregation. The study included 36 patients with hematological disorders (26 with primary chronic thrombocytopenia and 10 with essential thrombocythemia (ET)) and 40 healthy subjects. Platelet reactivity was measured using whole blood impedance aggregometry. The polyP levels were isolated from lysed platelets, which were obtained from citrated platelet-rich plasma. The procedure included inactivating endogenous phosphatases, removing phosphate units derived from DNA and proteins, and finally hydrolyzing them into monophosphate units. A colorimetric assay using malachite green and ammonium molybdate was performed in order to quantify polyP levels. The polyP concentrations were significantly higher in the patients with thrombocytopenia than in the patients with thrombocythemia or the controls. The polyP level was not correlated with the level of aggregation. The higher polyP levels observed in the patients with low platelet counts may indicate the existence of a compensatory mechanism that prevents excessive bleeding in such patients. Our study provides evidence of an essential role of polyP in platelet function and the coagulation process.

Research Note: Jejunum phosphatases and systemic myo-inositol in broiler chickens fed without or with supplemented phytase

As a constituent of animal cells, myo-inositol (MI) has been hypothesized to be crucial in several metabolic and regulatory pathways. Recently, it was shown that dietary phytase contributes to release of MI from phytate in the poultry digestive tract, increasing its systemic concentrations. This study investigated the activities of phosphatases in the jejunum and systemic plasma MI concentration in broilers not supplemented or supplemented with phytase through analyses based on modifications from commercial enzyme activity kits. Three hundred sixty male Ross 308 broilers were randomly allocated to 24 pens (15 birds per pen) in 4 dietary groups. The positive control group was fed with an adequate basal diet. The negative control group (NC) was fed with a reduced level of P and Ca. Groups Phy1500 and Phy3000 were fed with the NC diet plus 1,500 or 3,000 FTU of phytase per kilogram of feed, respectively. One bird per pen was selected for the measurement of jejunal phosphatase activity; MI concentration in plasma, the liver, and the kidney; and key MI enzyme concentrations (liver inositol monophosphatase 1 [IMPase 1] and kidney myo-inositol oxygenase [MIOX]). Endogenous phytase and alkaline phosphatase activity as well as IMPase 1 and MIOX expression were not statistically different among the dietary groups. The supplementation of 1500 FTU of phytase per kilogram of feed resulted in increase of plasma (P < 0.001) and kidney (P < 0.05) but not liver MI concentrations. The results indicated that systemic MI might reflect MI released from dietary sources; however, it did not appear to change expression of enzymes related to endogenous MI synthesis in the liver and catabolism in the kidney. New and larger studies are necessary to reach stronger evidence on the effects of dietary phytase on intestinal and systemic MI concentrations in broilers.

DUSP5 (dual-specificity protein phosphatase 5) suppresses BCG-induced autophagy via ERK 1/2 signaling pathway

Autophagy is considered as an effective strategy for host cells to eliminate intracellular Mycobacterium tuberculosis (Mtb). Dual-specificity phosphatase 5 (DUSP5) is an endogenous phosphatase of ERK1/2, and plays an important role in host innate immune responses, its function in autophagy regulation however remains unexplored. In the present study, the function of DUSP5 in autophagy in Mycobacterium bovis Bacillus Calmette-Guerin (BCG)-infected RAW264.7 cells, a murine macrophage-like cell line, was examined by assessing the alteration of the cell morphology, expression of autophagy markers, and ERK1/2 signaling activation. The results demonstrated that the BCG infection could induce DUSP5 expression and activate ERK1/2 signaling in RAW264.7 cells; an activation of ERK1/2 signaling contributed to autophagic process in RAW264.7 cells. Moreover, DUSP5 knockdown increased the expression of autophagy-related proteins (Atgs), including LC3-II, Beclin1, Atg5 and Atg7. However, an overexpression of DUSP5 exhibited an opposite effect. Mechanistically, DUSP5 could inhibit the formation of autophagosome by suppressing the phosphorylation of signaling molecules in ERK1/2 signaling cascade. This study thus demonstrated a novel role of DUSP5 in modulating autophagy inRAW264.7 cells in response to BCG infection in particular, and autophagy macrophage to Mtb in general.

Interspecific competition between Cylindrospermopsis raciborskii and Microcystis aeruginosa on different phosphorus substrates.

Phosphorus (P) is responsible for algal growth and the structural changes in algal communities. Therefore, it is essential to know whether the different phosphorus availability to different algae can change the community structure. In this study, the interspecific competition was investigated at two bloom-forming cyanobacterium, Cylindrospermopsis raciborskii and Microcystis aeruginosa, when both were treated with five different phosphate compounds, including K2HPO4, β-glycerol phosphate, (2-aminoethyl)-phosphinic acid, glyphosate, and P-free. The results of mono-culture experiments showed that the two species could utilize the dissolved organic phosphorus (DOP) and K2HPO4 (DIP) as the sole P resource. Moreover, the specific growth rates and the endogenous alkaline phosphatase activity in M. aeruginosa cells were much lower than those in C. raciborskii under DOP and DIP treatments. In the co-cultured experiments, however, a significant biomass increase in C. raciborskii was observed in all experimental P treatments, except for glyphosate, regardless of its initial cell density proportion. A 31.8-63.4% increase in cell number of C. raciborskii was found after incubated into K2HPO4, while the highest biomass of mixed samples, 17.72 × 106 cell mL-1, was observed in the (2-aminoethyl)-phosphinic acid treatment (50C50M). Additionally, higher specific growth rate was also found in C. raciborskii when compared with M. aeruginosa under P-free; the increasing proportion of C. raciborskii were 29.1% (50C50M), 16.4% (75C25M), and 36.7% (25C75M), respectively. When the mixed samples were co-cultivated under glyphosate, C. raciborskii cells appeared to be depressed, whereas the cell density of M. aeruginosa increased rapidly. The findings indicated that an excellent P competition might give some advantages for C. raciborskii dominance in natural waters with DIP limitation or DOP abundance.