Morphological evaluation of macrophage infected with Toxoplasma Gondii / Avaliação morfológica de macrófagos infectados com Toxoplasma Gondii

O Toxoplasma gondii é o parasita causador da toxoplasmose, doença negligenciada que ainda carece de estudos que visem elucidar a relação entre a parasitemia e o sistema imunológico. Uma das principais células do sistema imunológico são os macrófagos e estes possuem linhagens celulares imortalizadas que fornecem um modelo de estudo acessível para experimentos in vitro . Com isso, este trabalho pretende enfatizar a importância da linhagem de macrófagos J774G8 em estudos com protozoários, destacando a viabilidade e alterações morfológicas da cultura de células infectadas com Toxoplasma gondii

The Functional co-operativity of Tissue-Nonspecific Alkaline Phosphatase (TNAP) and PHOSPHO1 during initiation of Skeletal Mineralization

AbstractPhosphatases are recognized to have important functions in the initiation of skeletal mineralization. Tissue-nonspecific alkaline phosphatase (TNAP) and PHOSPHO1 are indispensable for bone and cartilage mineralization but their functional relationship in the mineralization process remains unclear. In this study, we have used osteoblast and ex-vivo metatarsal cultures to obtain biochemical evidence for co-operativity and cross-talk between PHOSPHO1 and TNAP in the initiation of mineralization. Clones 14 and 24 of the MC3T3-E1 cell line were used in the initial studies. Clone 14 cells expressed high levels of PHOSPHO1 and low levels of TNAP and in the presence of β-glycerol phosphate (βGP) or phosphocholine (P-Cho) as substrates and they mineralized their matrix strongly. In contrast clone 24 cells expressed high levels of TNAP and low levels of PHOSPHO1 and mineralized their matrix poorly. Lentiviral Phospho1 overexpression in clone 24 cells resulted in higher PHOSPHO1 and TNAP protein expression and increased levels of matrix mineralization. To uncouple the roles of PHOSPHO1 and TNAP in promoting matrix mineralization we used PHOSPHO1 (MLS-0263839) and TNAP (MLS-0038949) specific inhibitors, which individually reduced mineralization levels of Phospho1 overexpressing C24 cells, whereas the simultaneous addition of both inhibitors essentially abolished matrix mineralization (85%; P<0.001). Using metatarsals from E15 mice as a physiological ex vivo model of mineralization, the response to both TNAP and PHOSPHO1 inhibitors appeared to be substrate dependent. Nevertheless, in the presence of βGP, mineralization was reduced by the TNAP inhibitor alone and almost completely eliminated by the co-incubation of both inhibitors. These data suggest critical non-redundant roles for PHOSPHO1 and TNAP during the initiation of osteoblast and chondrocyte mineralization

Skeletal Mineralization Deficits and Impaired Biogenesis and Function of Chondrocyte-Derived Matrix Vesicles in Phospho1(-/-) and Phospho1/Pit1 Double Knockout Mice

International audienceWe have previously shown that ablation of either the Phospho1 or Alpl gene, encoding PHOSPHO1 and tissue-nonspecific alkaline phosphatase (TNAP) respectively, lead to hyperosteoidosis, but that their chondrocyte-derived and osteoblast-derived matrix vesicles (MVs) are able to initiate mineralization. In contrast, the double ablation of Phospho1 and Alpl completely abolish initiation and progression of skeletal mineralization. We argued that MVs initiate mineralization by a dual mechanism: PHOSPHO1-mediated intravesicular generation of inorganic phosphate (Pi ) and phosphate transporter-mediated influx of Pi . To test this hypothesis, we generated mice with col2a1-driven Cre-mediated ablation of Slc20a1, hereafter referred to as Pi t1, alone or in combination with a Phospho1 gene deletion. Pi t1(col2/col2) mice did not show any major phenotypic abnormalities, whereas severe skeletal deformities were observed in the [Phospho1(-/-) ; Pi t1(col2/col2) ] double knockout mice that were more pronounced than those observed in the Phospho1(-/-) mice. Histological analysis of [Phospho1(-/-) ; Pi t1(col2/col2) ] bones showed growth plate abnormalities with a shorter hypertrophic chondrocyte zone and extensive hyperosteoidosis. The [Phospho1(-/-) ; Pi t1(col2/col2) ] skeleton displayed significant decreases in BV/TV%, trabecular number, and bone mineral density, as well as decreased stiffness, decreased strength, and increased postyield deflection compared to Phospho1(-/-) mice. Using atomic force microscopy we found that ∼80% of [Phospho1(-/-) ; Pi t1(col2/col2) ] MVs were devoid of mineral in comparison to ∼50% for the Phospho1(-/-) MVs and ∼25% for the WT and Pi t1(col2/col2) MVs. We also found a significant decrease in the number of MVs produced by both Phospho1(-/-) and [Phospho1(-/-) ; Pi t1(col2/col2) ] chondrocytes. These data support the involvement of phosphate transporter 1, hereafter referred to as Pi T-1, in the initiation of skeletal mineralization and provide compelling evidence that PHOSPHO1 function is involved in MV biogenesis. © 2016 American Society for Bone and Mineral Research

How to build a bone: PHOSPHO1, biomineralization and beyond

Since its characterization two decades ago, the phosphatase PHOSPHO1 has been the subject of an increasing focus of research. This work has elucidated PHOSPHO1’s central role in the biomineralization of bone and other hard tissues, but has also implicated the enzyme in other biological processes in health and disease. During mineralization PHOSPHO1 liberates inorganic phosphate (Pi) to be incorporated into the mineral phase through hydrolysis of its substrates phosphocholine (PCho) and phosphoethanolamine (PEA). Localization of PHOSPHO1 within matrix vesicles allows accumulation of Pi within a protected environment where mineral crystals may nucleate and subsequently invade the organic collagenous scaffold. Here, we examine the evidence for this process, first discussing the discovery and characterization of PHOSPHO1, before considering experimental evidence for its canonical role in matrix vesicle-mediated biomineralization. We also contemplate roles for PHOSPHO1 in disorders of dysregulated mineralization such as vascular calcification, along with emerging evidence of its activity in other systems including choline synthesis and homeostasis, and energy metabolism

Functional Significance of Calcium Binding to Tissue-Nonspecific Alkaline Phosphatase

<div><p>The conserved active site of alkaline phosphatases (AP) contains catalytically important Zn²⁺ (M1 and M2) and Mg²⁺-sites (M3) and a fourth peripheral Ca²⁺ site (M4) of unknown significance. We have studied Ca²⁺ binding to M1-4 of tissue-nonspecific AP (TNAP), an enzyme crucial for skeletal mineralization, using recombinant TNAP and a series of M4 mutants. Ca²⁺ could substitute for Mg²⁺ at M3, with maximal activity for Ca²⁺/Zn²⁺-TNAP around 40% that of Mg²⁺/Zn²⁺-TNAP at pH 9.8 and 7.4. At pH 7.4, allosteric TNAP-activation at M3 by Ca²⁺ occurred faster than by Mg²⁺. Several TNAP M4 mutations eradicated TNAP activity, while others mildly influenced the affinity of Ca²⁺ and Mg²⁺ for M3 similarly, excluding a catalytic role for Ca²⁺ in the TNAP M4 site. At pH 9.8, Ca²⁺ competed with soluble Zn²⁺ for binding to M1 and M2 up to 1 mM and at higher concentrations, it even displaced M1- and M2-bound Zn²⁺, forming Ca²⁺/Ca²⁺-TNAP with a catalytic activity only 4–6% that of Mg²⁺/Zn²⁺-TNAP. At pH 7.4, competition with Zn²⁺ and its displacement from M1 and M2 required >10-fold higher Ca²⁺ concentrations, to generate weakly active Ca²⁺/Ca²⁺-TNAP. Thus, in a Ca²⁺-rich environment, such as during skeletal mineralization at pH 7.4, Ca²⁺ adequately activates Zn²⁺-TNAP at M3, but very high Ca²⁺ concentrations compete with available Zn²⁺ for binding to M1 and M2 and ultimately displace Zn²⁺ from the active site, virtually inactivating TNAP. Those <i>ALPL</i> mutations that substitute critical TNAP amino acids involved in coordinating Ca²⁺ to M4 cause hypophosphatasia because of their 3D-structural impact, but M4-bound Ca²⁺ is catalytically inactive. In conclusion, during skeletal mineralization, the building Ca²⁺ gradient first activates TNAP, but gradually inactivates it at high Ca²⁺ concentrations, toward completion of mineralization.</p></div

Kinetic Parameters of PLAP, TNAP and the M4-site mutants, measured in 1M DEA buffer, pH 9.8, with pNPP as a substrate.

<p>* based on historical values [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119874#pone.0119874.ref013" target="_blank">13</a>]</p><p>Kinetic Parameters of PLAP, TNAP and the M4-site mutants, measured in 1M DEA buffer, pH 9.8, with pNPP as a substrate.</p

Allosteric activation of Zn²⁺-TNAP by CaCl₂.

<p>a. Progressive AbM2-bound Zn²⁺-TNAP activation, measured as A405 nm <i>vs</i>. time, for the indicated [Ca²⁺], added to Chelex-pretreated pNPP (10 mM) at pH 9.8, showing dose-dependent activation (left panel) and inhibition at high concentrations (right panel); b. Dose-response of generated AP activity (mean mA405nm/min) in steady-state (i.e. the slope measured between 60–90 min in Figs. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119874#pone.0119874.g001" target="_blank">1A</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119874#pone.0119874.g002" target="_blank">2A</a>) for increasing [MgCl₂] and [CaCl₂] at identical AbM2-bound [Zn²⁺-TNAP], reflecting the plateau and pseudo-plateau at high [MgCl₂] and [CaCl₂] respectively, followed by a steep drop of the TNAP activity in the case of CaCl₂ (right panel). Activities were measured in Chelex-treated pNPP (10 mM) at pH 9.8, supplemented with MgCl₂ and CaCl₂, as indicated. Experiments representative of at least three replicates with variable enzyme and MgCl₂ concentrations.</p

TNAP inactivation by high [CaCl₂] at pH 7.4.

<p><b>a</b>. Reconstitution of TNAP activity by 20 μM Zn²⁺ + 1 mM Mg²⁺, but not by the individual metal ions, added to fully demetalated holo-TNAP, starting after 60 min (left panel); comparison of specific TNAP activity of (holo)-TNAP, after reconstitution with the indicated metal ion composition, after overnight treatment with TBS, with or without added EDTA (250 μM); b. Dose-dependency of holo-TNAP reconstitution by CaCl₂ (0–10 mM), in the absence or presence of the indicated [ZnCl₂] (0–20 μM, left panel); competition between the indicated concentrations of Zn²⁺ (2 μM and 20 μM) and increasing concentrations of CaCl₂; minor displacement of TNAP-bound Zn²⁺ (overnight Zn²⁺ preloading indicated as “Pre”) by increasing concentrations of CaCl₂, independently of the presence of free Zn²⁺.(0–20 μM). Results represent mean ± SD for 3 identical experiments (*p<0.003, **p<0.0001,).</p

Activation <i>vs</i>. inhibition of Zn²⁺-TNAP by MgCl₂ and CaCl₂.

<p><b>a</b>. Dose-response of generated AP activity (mean mA405nm/min) in steady-state (i.e. the slope measured between 60–90 min) for increasing [MgCl₂] and [CaCl₂] at identical AbM2-bound [Zn²⁺-TNAP], measured in Chelex-treated pNPP (1 mM) at pH 9.8; <b>b</b>. TNAP inhibition at high [MgCl₂] and [CaCl₂], measured in Chelex-treated pNPP (1 or 10 mM as indicated) at pH 9.8, in the presence of the indicated concentrations of MgCl₂. Results represent mean ± SD for 3 identical experiments or are representative examples of experiments, performed in 3-fold (b, middle and right panel).</p