Search Results (57)

ABCC6, a key regulator in ectopic calcification, plays a crucial role in mineralization through the modulation of extracellular purinergic pathways and production of inorganic pyrophosphate (PPi), which inhibits calcification. Inherited deficiencies in ABCC6 lead to pseudoxanthoma elasticum (PXE) and related conditions, characterized by calcification in various tissues, particularly affecting the skin, eyes, and cardiovascular system. Although PXE does not directly impact the nervous system, secondary neurological issues arise from cerebrovascular complications, increasing the risk of strokes linked to arterial blockages resembling atherosclerosis. This review investigates the connection between ABCC6 mutations and cerebral small vessel disease (SVD), expanding the understanding of PXE and related phenotypes. Mutations in ABCC6, identified as causing PXE, contribute to systemic metabolic dysfunction, with significant implications for cerebrovascular health. An association between ABCC6 mutations and cerebral SVD has been suggested in various studies, particularly in populations with distinct genetic backgrounds. Emerging evidence indicates that pathogenic mutations increase the risk of ischemic strokes, with both homozygous and heterozygous carriers showing susceptibility. Mechanistically, ABCC6 deficiency is implicated in dyslipidemia and atherosclerosis, further exacerbating cerebrovascular risks. Increased arterial pulsatility, linked to carotid siphon calcification, may also contribute to microvascular damage and subsequent brain injury. Understanding these mechanisms is vital for developing targeted diagnostic and therapeutic strategies for managing cerebrovascular risks in PXE patients. This review emphasizes the need for comprehensive genetic screening and the consideration of traditional vascular risk factors in patient management, highlighting the complex interplay between genetic mutations and environmental influences affecting cerebrovascular health. Future research should focus on longitudinal studies to elucidate the causal pathways linking arterial calcification, pulsatility, and brain damage in PXE. Full article

Fluorine accounts for 0.3 g/kg of the Earth’s crust, being widely distributed in the environment as fluoride. The toxic effects of this anion in humans and other organisms have been known for a long time. Fluoride has been reported to alter several cellular processes although the mechanisms involved are largely unknown. Inorganic pyrophosphatases (PPases) are ubiquitous enzymes that hydrolyze inorganic pyrophosphate (PPi), a metabolite generated from ATP. In Saccharomyces cerevisiae, the enzyme responsible for PPi hydrolysis in the cytosol (IPP1) is strongly inhibited by fluoride in vitro. The essentiality of IPP1 for growth has been previously demonstrated using YPC3, a yeast mutant with conditional expression of the corresponding gene. Here, YPC3 was used to generate cells that tolerate high concentrations of fluoride by (a) the overexpression of IPP1 or its human ortholog, or (b) the substitution of IPP1 by the fluoride-insensitive PPase from Streptococcus mutans. The results obtained suggest that maintaining appropriate levels of PPase activity in the cytosol is essential for the adaptation of S. cerevisiae to high fluoride concentrations. The increase in fluoride tolerance allows YPC3 cells transformed with suitable plasmids to be selected on rich non-selective medium supplemented with this anion. Full article

A novel organic–inorganic eutectic phase change material (PCM) based on sodium acetate trihydrate (SAT) and polyethylene glycol (PEG) was developed to meet the needs of heat recovery and building heating. Three kinds of PEG with different molecular weights were selected to form organic–inorganic eutectic PCM with SAT. The thermal properties of three series of SAT-PEG eutectic PCM were compared based on DSC results, focusing on the impact of PEG addition on the phase change temperature and enthalpy of SAT, as well as the melting uniformity. The inhibitory effects of two nucleating agents on the supercooling of SAT-PEG eutectic PCM were systematically investigated. The effect of PEG on the crystallization behavior of SAT was studied using a metallographic microscope. To evaluate the thermal reliability of the SAT-PEG eutectic PCM, 600 cycles of melting–solidification experiments were conducted. Experimental results show that SAT can form eutectic PCMs with PEG200, PEG600, and PEG6000, respectively, with high enthalpy and excellent melting uniformity. The phase change temperature ranged from 55 °C to 60 °C and the enthalpy was as high as 250–280 kJ/kg. The results of the cooling curves show that 10 wt% tetrasodium pyrophosphate decahydrate (TPD) can reduce the supercooling degree to less than 1 °C. Significantly, all three series of SAT-PEG eutectic PCMs exhibit exceptional thermal reliability after 600 cycles of melting–solidification, with shifts in the phase change temperatures and enthalpies of less than 4%. XRD diffraction patterns showed that SAT, PEG, and TPD were physically mixed without a chemical reaction to form new substances. Microscopic images reveal that the addition of PEG preserves the original needle-shaped crystal morphology of SAT while reducing its crystal size. The rapid formation of small crystals can provide more nucleation points and expedite crystallization, thereby enhancing the heat release capabilities of the PCM. Full article

This study explored the effect of the combined application of chemical and organic fertilizers on phosphorus morphology and its conversion to an active state. A long-term field positioning experiment comprising five treatments was conducted in black soil. The results concluded that the soil total phosphorus (TP), available phosphorus (AP), inorganic phosphorus, and organic phosphorus contents of all treatments ranked as follows: 1.5M1NPK > M2NPK > M1NPK > NPK > CK. The long-term application of chemical and organic fertilizers increased the proportion of soil reactive phosphorus and moderately reactive phosphorus but decreased the proportion of mildly active phosphorus and residual phosphorus. A phosphorus-31 nuclear magnetic resonance (³¹P NMR) spectral analysis showed that the contents of orthophosphate, pyrophosphate, phosphoric acid diesters, and orthophosphate acid monoesters increased with the application of chemical and organic fertilizers, of which 1.5M1NPK usually resulted in the highest increases. In conclusion, the long-term application of chemical fertilizers could promote the conversion of soil phosphorus into active phosphorus and improve the effectiveness of soil phosphorus, and the long-term use of organic and chemical fertilizers was more effective than the use of chemical fertilizers only, with 1.5M1NPK providing the best effects. Full article

Phosphatases are enzymes that catalyze the hydrolysis of phosphate esters. They play critical roles in diverse biological processes such as extracellular nucleotide homeostasis, transport of molecules across membranes, intracellular signaling pathways, or vertebrate mineralization. Among them, tissue-nonspecific alkaline phosphatase (TNAP) is today increasingly studied, due to its ubiquitous expression and its ability to dephosphorylate a very broad range of substrates and participate in several different biological functions. For instance, TNAP hydrolyzes inorganic pyrophosphate (PP_i) to allow skeletal and dental mineralization. Additionally, TNAP hydrolyzes pyridoxal phosphate to allow cellular pyridoxal uptake, and stimulate vitamin B6-dependent reactions. Furthermore, TNAP has been identified as a key enzyme in non-shivering adaptive thermogenesis, by dephosphorylating phosphocreatine in the mitochondrial creatine futile cycle. This latter recent discovery and others suggest that the list of substrates and functions of TNAP may be much longer than previously thought. In the present review, we sought to examine TNAP within the alkaline phosphatase (AP) superfamily, comparing its sequence, structure, and evolutionary trajectory. The AP superfamily, characterized by a conserved central folding motif of a mixed beta-sheet flanked by alpha-helices, includes six subfamilies: AP, arylsulfatases (ARS), ectonucleotide pyrophosphatases/phosphodiesterases (ENPP), phosphoglycerate mutases (PGM), phosphonoacetate hydrolases, and phosphopentomutases. Interestingly, TNAP and several ENPP family members appear to participate in the same metabolic pathways and functions. For instance, extra-skeletal mineralization in vertebrates is inhibited by ENPP1-mediated ATP hydrolysis into the mineralization inhibitor PP_i, which is hydrolyzed by TNAP expressed in the skeleton. Better understanding how TNAP and other AP family members differ structurally will be very useful to clarify their complementary functions. Structurally, TNAP shares the conserved catalytic core with other AP superfamily members but has unique features affecting substrate specificity and activity. The review also aims to highlight the importance of oligomerization in enzyme stability and function, and the role of conserved metal ion coordination, particularly magnesium, in APs. By exploring the structural and functional diversity within the AP superfamily, and discussing to which extent its members exert redundant, complementary, or specific functions, this review illuminates the evolutionary pressures shaping these enzymes and their broad physiological roles, offering insights into TNAP’s multifunctionality and its implications for health and disease. Full article

Soluble inorganic pyrophosphatase (s-PPase), a pyrophosphate hydrolase, is crucial for various physiological processes including plant growth and development, metabolic functions, and responses to abiotic stresses. However, research on s-PPase in woody plants is limited. To investigate the potential role of soluble inorganic pyrophosphatase in Eucommia ulmoides Oliver (E. ulmoides) in drought stress, the E. ulmoides soluble inorganic pyrophosphatase 5 (EuSIP5) cDNA sequence was amplified via RT-PCR. A bioinformatic analysis suggested that EuSIP5 may be an unstable amphipathic protein predominantly localized in the cytoplasm. In E. ulmoides, the highest expression of the EuSIP5 gene was detected in the leaves and pericarp of male plants from April to October, and in the leaves in July and September. Under drought conditions, the expression of EuSIP5 in E. ulmoides leaves was significantly greater than that in the control. An overexpression vector containing EuSIP5 was constructed and introduced into Nicotiana tabacum L. cv. Xanthi (N. tabacum L.). Compared with that in wild-type (WT) plants, wilting in N. tabacum L. EuSIP5-overexpressing (OE) plants was delayed by 4 days under drought stress. Additionally, the expression levels of the drought-related genes DET2, CYP85A1, P5CS, ERF1, F-box, and NCED1 were elevated in the leaves of transgenic N. tabacum L. Moreover, the activities of the protective enzymes peroxidase, superoxide dismutase, and catalase were significantly greater, whereas the malondialdehyde content was lower in the transgenic plants than in the WT plants. These findings suggest that the introduction of the EuSIP5 gene into N. tabacum L. enhances drought-related gene expression, increases antioxidant capacity, and reduces oxidative stress damage, thereby improving drought resistance. Full article

Craniometaphyseal Dysplasia (CMD) is a rare skeletal disorder that can result from mutations in the ANKH gene. This gene encodes progressive anksylosis (ANK), which is responsible for transporting inorganic pyrophosphate (PPi) and ATP from the intracellular to the extracellular environment, where PPi inhibits bone mineralization. When ANK is dysfunctional, as in patients with CMD, the passage of PPi to the extracellular environment is reduced, leading to excess mineralization, particularly in bones of the skull. Zebrafish may serve as a promising model to study the mechanistic basis of CMD. Here, we provide a detailed analysis of the zebrafish Ankh paralogs, Ankha and Ankhb, in terms of their phylogenic relationship with ANK in other vertebrates as well as their spatiotemporal expression patterns during zebrafish development. We found that a closer evolutionary relationship exists between the zebrafish Ankhb protein and its human and other vertebrate counterparts, and stronger promoter activity was predicted for ankhb compared to ankha. Furthermore, we noted distinct temporal expression patterns, with ankha more prominently expressed in early development stages, and both paralogs also being expressed at larval growth stages. Whole-mount in situ hybridization was used to compare the spatial expression patterns of each paralog during bone development, and both showed strong expression in the craniofacial region as well as the notochord and somites. Given the substantial overlap in spatiotemporal expression but only subtle patterning differences, the exact roles of these genes remain speculative. In silico analyses predicted that Ankha and Ankhb have the same function in transporting PPi across the membrane. Nevertheless, this study lays the groundwork for functional analyses of each ankh paralog and highlights the potential of using zebrafish to find possible targeted therapies for CMD. Full article

Phosphorus (P) is a crucial nutrient in lake ecosystems and organic phosphorus (Po) is a significant component. However, the distribution characteristics and migration behaviour of Po in ice–water–sediment systems under freezing and thawing conditions in cold and arid regions remain unclear. This study aims to investigate the forms of Po and its contribution to endogenous P pollution. We selected three lakes (Dai, Hu, and Wu Lake) and employed phosphorus nuclear magnetic resonance (³¹P-NMR) techniques to analyse the following: (1) The total phosphorus (TP) content, which was the highest in the water from Dai Lake (0.16 mg/L), with substantial seasonal variation observed in Wu Lake, where P content was four times higher in summer than in winter because of farmland drainage. (2) Eutrophication analysis, which indicated that Dai Lake had significantly higher eutrophication levels than Wu Lake, with P being the controlling factor in Dai Lake and both N and P in Wu Lake. The proportion of Po in the TP content was 90%, 70%, and 55% for Wu, Dai, and Hu Lake, respectively, indicating that Po was the main component of eutrophic lakes. (3) ³¹P-NMR, which revealed that orthophosphate (Ortho-P) and monoester phosphate (Mon-P) were the main P components in the winter, with a higher P content in Dai Lake. Ortho-P has a higher content in ice, indicating that inorganic phosphorus (Pi) migration is the main factor in ice–water media. Mon-P showed multiple peaks in Dai Lake, indicating a complex composition of adenosine monophosphate and glucose-1-phosphate. (4) The ice–water phase change simulation experiments, which showed that phosphate was the least repelled in ice, while pyrophosphate (Pyro-P) and macromolecular P were more repelled. Adding sediment enhanced the migration of P but did not change the repulsion of macromolecular P, suggesting the molecular structure as the main influencing factor. These results provide important scientific evidence for the quantitative analysis of Po pollution in lake water environments, aiding in P load reduction and risk prevention and control. Full article

Pseudoxanthoma elasticum (PXE) is a rare inherited systemic disease responsible for a juvenile peripheral arterial calcification disease. The clinical diagnosis of PXE is only based on a complex multi-organ phenotypic score and/or genetical analysis. Reduced plasma inorganic pyrophosphate concentration [PPi]p has been linked to PXE. In this study, we used a novel and accurate method to measure [PPi]p in one of the largest cohorts of PXE patients, and we reported the valuable contribution of a cutoff value to PXE diagnosis. Plasma samples and clinical records from two French reference centers for PXE (PXE Consultation Center, Angers, and FAVA-MULTI South Competent Center, Nice) were assessed. Plasma PPi were measured in 153 PXE and 46 non-PXE patients. The PPi concentrations in the plasma samples were determined by a new method combining enzymatic and ion chromatography approaches. The best match between the sensitivity and specificity (Youden index) for diagnosing PXE was determined by ROC analysis. [PPi]p were lower in PXE patients (0.92 ± 0.30 µmol/L) than in non-PXE patients (1.61 ± 0.33 µmol/L, p < 0.0001), corresponding to a mean reduction of 43 ± 19% (SD). The PPi cutoff value for diagnosing PXE in all patients was 1.2 µmol/L, with a sensitivity of 83.3% and a specificity of 91.1% (AUC = 0.93), without sex differences. In patients aged <50 years (i.e., the age period for PXE diagnosis), the cutoff PPi was 1.2 µmol/L (sensitivity, specificity, and AUC of 93%, 96%, and 0.97, respectively). The [PPi]p shows high accuracy for diagnosing PXE; thus, quantifying plasma PPi represents the first blood assay for diagnosing PXE. Full article

Calcium pyrophosphate dehydrate (CPPD) crystals are found in the synovial fluid of patients with articular chondrocalcinosis or sometimes with osteoarthritis. In inflammatory conditions, the synovial membrane (SM) is subjected to transient hypoxia, especially during movement. CPPD formation is supported by an increase in extracellular inorganic pyrophosphate (ePPi) levels, which are mainly controlled by the transporter Ank and ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1). We demonstrated previously that transforming growth factor (TGF)-β1 increased ePPi production by inducing Ank and Enpp1 expression in chondrocytes. As the TGF-β1 level raises in synovial fluid under hypoxic conditions, we investigated whether hypoxia may transform SM as a major source of ePPi production. Synovial fibroblasts and SM explants were exposed to 10 ng/mL of TGF-β1 in normoxic or hypoxic (5% O₂) culture conditions. Ank and Enpp1 expression were assessed by quantitative PCR, Western blot and immunohistochemistry. ePPi was quantified in culture supernatants. RNA silencing was used to define the respective roles of Ank and Enpp1 in TGF-β1-induced ePPi generation. The molecular mechanisms involved in hypoxia were investigated using an Ank promoter reporter plasmid for transactivation studies, as well as gene overexpression and RNA silencing, the respective role of hypoxia-induced factor (HIF)-1 and HIF-2. Our results showed that TGF-β1 increased Ank, Enpp1, and therefore ePPi production in synovial fibroblasts and SM explants. Ank was the major contributor in ePPi production compared to ENPP1. Hypoxia increased ePPi levels on its own and enhanced the stimulating effect of TGF-β1. Hypoxic conditions enhanced Ank promoter transactivation in an HIF-1-dependent/HIF-2-independent fashion. We demonstrated that under hypoxia, SM is an important contributor to ePPi production in the joint through the induction of Enpp1 and Ank. These findings are of interest as a rationale for the beneficial effect of anti-inflammatory drugs on SM in crystal depositions. Full article

In Saccharomyces cerevisiae, pH homeostasis is reliant on ATP due to the use of proton-translocating ATPase (H⁺-ATPase) which constitutes a major drain within cellular ATP supply. Here, an exogenous proton-translocating pyrophosphatase (H⁺-PPase) from Arabidopsis thaliana, which uses inorganic pyrophosphate (PP_i) rather than ATP, was evaluated for its effect on reducing the ATP burden. The H⁺-Ppase was localized to the vacuolar membrane or to the cell membrane, and their impact was studied under acetate stress at a low pH. Biosensors (pHluorin and mQueen-2m) were used to observe changes in intracellular pH (pH_i) and ATP levels during growth on either glucose or xylose. A significant improvement of 35% in the growth rate at a pH of 3.7 and 6 g·L⁻¹ acetic acid stress was observed in the vacuolar membrane H⁺-PPase strain compared to the parent strain. ATP levels were elevated in the same strain during anaerobic glucose and xylose fermentations. During anaerobic xylose fermentations, co-expression of pHluorin and a vacuolar membrane H⁺-PPase improved the growth characteristics by means of an improved growth rate (11.4%) and elongated logarithmic growth duration. Our study identified a potential method for improving productivity in the use of S. cerevisiae as a cell factory under the harsh conditions present in industry. Full article

Inorganic phosphate (Pi) is a critical determinant of calcification, and its concentration is regulated by alkaline phosphatase (ALP) and Pit1. ALP is a key regulator of osteogenic calcification and acts by modulating local inorganic phosphate (Pi) concentrations through hydrolyzing pyrophosphate in the extracellular matrix (ECM). Pit1, a sodium-dependent phosphate transporter, regulates calcification via facilitating phosphate uptake within the cells. To investigate whether zinc differentially regulates osteoblastic and vascular calcifications, we examined ALP activity and Pit1 in osteoblastic and vascular smooth muscle cells (VSMCs). Our findings demonstrate that calcification in osteoblastic MC3T3-E1 cells is decreased via diminished ALP action under zinc deficiency. In contrast, zinc-deficiency-induced calcification in VSMCs is independent of ALP action, as demonstrated by very weak ALP activity and expression in calcified VSMCs. In zinc-deficient A7r5 VSMC, P accumulation increased with increasing Na phosphate concentration (3–7 mM) but not with β-GP treatment, which requires ALP activity to generate Pi. Ca deposition also increased with Na phosphate in a dose-dependent manner; in contrast, β-GP did not affect Ca deposition. In osteoblastic cells, Pit1 expression was not affected by zinc treatments. In contrast, Pit1 expression is highly upregulated in A7r5 VSMC under zinc deficiency. Using phosphonoformic acid, a competitive inhibitor of Pit1, we showed that calcification is inhibited in both A7r5 and MC3T3-E1 cells, indicating a requirement for Pit1 in both calcifications. Moreover, the downregulation of VSMC markers under zinc deficiency was restored by blocking Pit1. Taken together, our results imply that zinc-deficiency-induced calcification in VSMC is independent of ALP action in contrast to osteoblastic calcification. Moreover, Pit1 expression in VSMCs is a target for zinc deficiency and may mediate the inhibition of VSMC marker expression under zinc deficiency. Full article

Bone growth plate abnormalities and skull shape defects are seen in hypophosphatasia, a heritable disorder in humans that occurs due to the deficiency of tissue nonspecific alkaline phosphatase (TNAP, Alpl) enzyme activity. The abnormal development of the cranial base growth plates (synchondroses) and abnormal skull shapes have also been demonstrated in global Alpl^−/− mice. To distinguish local vs. systemic effects of TNAP on skull development, we utilized P0-Cre to knockout Alpl only in cranial neural crest-derived tissues using Alpl flox mice. Here, we show that Alpl deficiency using P0-Cre in cranial neural crest leads to skull shape defects and the deficient growth of the intersphenoid synchondrosis (ISS). ISS chondrocyte abnormalities included increased proliferation in resting and proliferative zones with decreased apoptosis in hypertrophic zones. ColX expression was increased, which is indicative of premature differentiation in the absence of Alpl. Sox9 expression was increased in both the resting and prehypertrophic zones of mutant mice. The expression of Parathyroid hormone related protein (PTHrP) and Indian hedgehog homolog (IHH) were also increased. Finally, cranial base organ culture revealed that inorganic phosphate (P_i) and pyrophosphate (PP_i) have specific effects on cell signaling and phenotype changes in the ISS. Together, these results demonstrate that the TNAP expression downstream of Alpl in growth plate chondrocytes is essential for normal development, and that the mechanism likely involves Sox9, PTHrP, IHH and PP_i. Full article

Pseudoxanthoma elasticum (PXE) is a heritable multisystem ectopic calcification disorder. The gene responsible for PXE, ABCC6, encodes ABCC6, a hepatic efflux transporter regulating extracellular inorganic pyrophosphate (PPi), a potent endogenous calcification inhibitor. Recent studies demonstrated that in addition to the deficiency of plasma PPi, the activated DDR/PARP signaling in calcified tissues provides an additional possible mechanism of ectopic calcification in PXE. This study examined the effects of etidronate (ETD), a stable PPi analog, and its combination with minocycline (Mino), a potent inhibitor of DDR/PARP, on ectopic calcification in an Abcc6^-/- mouse model of PXE. Abcc6^-/- mice, at 4 weeks of age, before the development of ectopic calcification, were treated with ETD, Mino, or both for 18 weeks. Micro-computed tomography, histopathologic examination, and quantification of the calcium content in Abcc6^-/- mice treated with both ETD and Mino revealed further reduced calcification than either treatment alone. The effects were associated with reduced serum alkaline phosphatase activity without changes in plasma PPi concentrations. These results suggest that ETD and Mino combination therapy might provide an effective therapeutic approach for PXE, a currently intractable disease. Full article

Protein histidine phosphorylation (pHis) is a posttranslational modification involved in cell cycle regulation, ion channel activity and phagocytosis. Using novel monoclonal antibodies to detect pHis, we previously reported that the loss of the histidine phosphatase LHPP (phospholysine phosphohistidine inorganic pyrophosphate phosphatase) results in elevated pHis levels in hepatocellular carcinoma. Here, we show that intestinal inflammation correlates with the loss of LHPP in dextran sulfate sodium (DSS)-treated mice and in inflammatory bowel disease (IBD) patients. Increased histidine phosphorylation was observed in intestinal epithelial cells (IECs), as determined by pHis immunofluorescence staining of colon samples from a colitis mouse model. However, the ablation of Lhpp did not cause increased pHis or promote intestinal inflammation under physiological conditions or after DSS treatment. Our observations suggest that increased histidine phosphorylation plays a role in colitis, but the loss of LHPP is not sufficient to increase pHis or to cause inflammation in the intestine. Full article