Nitrogen-doped carbon dots as a ratiometric fluorescent probe for determination of the activity of acid phosphatase, for inhibitor screening, and for intracellular imaging.

The author describe a method for preparation of green fluorescent nitrogen-doped carbon dots (N-CDs) through hydrothermal treatment of a mixture of lotus leaf juice and ethylenediamine (EDA). The N-CDs have uniform size, good dispersibility and water solubility. Under 316 and 366 nm photoexcitation, they show dual fluorescence with emission peaks at 415 and 509 nm, respectively. They are positively charge and display low cytotoxicity. This makes them an excellent choice for fluorometric assays and for bioimaging. A ratiometric assay was developed for the determination of the activity of acid phosphatase (ACP). It is based on the aggregation- induced quenching (AIQ) of the fluorescence of the N-CDs by sodium hexametaphosphate (NaPO3)6. Enzymatic hydrolysis of (NaPO3)6 by ACP leads to the disintegration of (NaPO3)6 and to the restoration of fluorescence. The measurement of the ratio of fluorescence at two wavelengths (415 and 509 nm), background interference and fluctuating signals can be widely eliminated. The method works in the 1-50 U·L-1 ACP activity range and has a detection limit of 0.43 U·L-1. It was successfully applied (a) to the determination of ACP in spiked serum samples, (b) to ACP inhibitor screening, and (c) to imaging of ACP in HePG2 cells. Graphical abstract Schematic presentation of the synthesis of nitrogen-doped carbon dots (N-CDs), and their application to the ratiometric fluorometric determination of acid phosphatase (ACP) based on the aggregation-induced quenching and enzymatic hydrolysis.

Soil properties influence kinetics of soil acid phosphatase in response to arsenic toxicity.

Soil phosphatase, which plays an important role in phosphorus cycling, is strongly inhibited by Arsenic (As). However, the inhibition mechanism in kinetics is not adequately investigated. In this study, we investigated the kinetic characteristics of soil acid phosphatase (ACP) in 14 soils with varied properties, and also explored how kinetic properties of soil ACP changed with different spiked As concentrations. The results showed that the Michaelis constant (Km) and maximum reaction velocity (Vmax) values of soil ACP ranged from 1.18 to 3.77mM and 0.025-0.133mMh-1 in uncontaminated soils. The kinetic parameters of soil ACP in different soils changed differently with As contamination. The Km remained unchanged and Vmax decreased with increase of As concentration in most acid and neutral soils, indicating a noncompetitive inhibition mechanism. However, in alkaline soils, the Km increased linearly and Vmax decreased with increase of As concentration, indicating a mixed inhibition mechanism that include competitive and noncompetitive. The competitive inhibition constant (Kic) and noncompetitive inhibition constant (Kiu) varied among soils and ranged from 0.38 to 3.65mM and 0.84-7.43mM respectively. The inhibitory effect of As on soil ACP was mostly affected by soil organic matter and cation exchange capacity. Those factors influenced the combination of As with enzyme, which resulted in a difference of As toxicity to soil ACP. Catalytic efficiency (Vmax/Km) of soil ACP was a sensitive kinetic parameter to assess the ecological risks of soil As contamination.

The Protective Effects of Alisol A 24-Acetate from Alisma canaliculatum on Ovariectomy Induced Bone Loss in Vivo.

Alisma canaliculatum is a herb commonly used in traditional Korean medicine, and has been shown in scientific studies to have antitumor, diuretic hepatoprotective, and antibacterial effects. Recently, the anti-osteoclastogenesis of alisol A 24-acetate from Alisma canaliculatum was investigated in vitro. However, the influence of alisol A 24-acetate on osteoporosis in animals has not been investigated. The present study was undertaken to investigate the anti-osteoporotic effect of alisol A 24-acetate on bone mass in ovariectomized (OVX) mice and to identify the mechanism responsible for its effects. OVX mice were treated daily with 0.5 or 2 µg/g of alisol A 24-acetate for a period of six weeks. It was found that these administrations significantly suppressed osteoporosis in OVX mice and improved bone morphometric parameters. The serum estradiol, bone alkaline phosphatase levels, regulatory T/Th17 cell numbers were significantly increased by alisol A 24-acetate as compared with untreated OVX mice. In addition, TRAP activity was inhibited by alisol A 24-acetate in OVX mice. These results suggest alisol A 24-acetate effectively prevents bone loss in OVX mice, and that it can be considered a potential therapeutic for the treatment of postmenopausal osteoporosis.

Therapeutic potentials of naringin on polymethylmethacrylate induced osteoclastogenesis and osteolysis, in vitro and in vivo assessments.

Wear debris associated periprosthetic osteolysis represents a major pathological process associated with the aseptic loosening of joint prostheses. Naringin is a major flavonoid identified in grapefruit. Studies have shown that naringin possesses many pharmacological properties including effects on bone metabolism. The current study evaluated the influence of naringin on wear debris induced osteoclastic bone resorption both in vitro and in vivo. The osteoclast precursor cell line RAW 264.7 was cultured and stimulated with polymethylmethacrylate (PMMA) particles followed by treatment with naringin at several doses. Tartrate resistant acid phosphatase (TRAP), calcium release, and gene expression profiles of TRAP, cathepsin K, and receptor activator of nuclear factor-kappa B were sequentially evaluated. PMMA challenged murine air pouch and the load bearing tibia titanium pin-implantation mouse models were used to evaluate the effects of naringin in controlling PMMA induced bone resorption. Histological analyses and biomechanical pullout tests were performed following the animal experimentation. The in vitro data clearly demonstrated the inhibitory effects of naringin in PMMA induced osteoclastogenesis. The naringin dose of 10 µg/mL exhibited the most significant influence on the suppression of TRAP activities. Naringin treatment also markedly decreased calcium release in the stimulated cell culture medium. The short-term air pouch mouse study revealed that local injection of naringin ameliorated the PMMA induced inflammatory tissue response and subsequent bone resorption. The long-term tibia pin-implantation mouse model study suggested that daily oral gavage of naringin at 300 mg/kg dosage for 30 days significantly alleviated the periprosthetic bone resorption. A significant increase of periprosthetic bone volume and regaining of the pin stability were found in naringin treated mice. Overall, this study suggests that naringin may serve as a potential therapeutic agent to treat wear debris associated osteolysis.

A dietary colorant crocin mitigates arthritis and associated secondary complications by modulating cartilage deteriorating enzymes, inflammatory mediators and antioxidant status.

Articular cartilage degeneration and inflammation are the hallmark of progressive arthritis and is the leading cause of disability in 10-15% of middle aged individuals across the world. Cartilage and synovium are mainly degraded by either enzymatic or non-enzymatic ways. Matrix metalloproteinases (MMPs), hyaluronidases (HAases) and aggrecanases are the enzymatic mediators and inflammatory cytokines and reactive oxygen species being non-enzymatic mediators. In addition, MMPs and HAases generated end-products act as inflammation inducers via CD44 and TLR-4 receptors involved NF-κB pathway. Although several drugs have been used to treat arthritis, numerous reports describe the side effects of these drugs that may turn fatal. On this account several medicinal plants and their isolated molecules have been involved in modern medicine strategies to fight against arthritis. In view of this, the present study investigated the antiarthritic potentiality of Crocin, a dietary colorant carotenoid isolated from stigma of Crocus sativus. Crocin effectively neutralized the augmented serum levels of enzymatic (MMP-13, MMP-3 and MMP-9 and HAases) and non-enzymatic (TNF-α, IL-1ß, NF-κB, IL-6, COX-2, PGE(2) and ROS) inflammatory mediators. Further, Crocin re-established the arthritis altered antioxidant status of the system (GSH, SOD, CAT and GST). It also protected the bone resorption by inhibiting the elevated levels of bone joint exoglycosidases, cathepsin-D and tartrate resistant acid phosphatases. Taken together, Crocin revitalized the arthritis induced cartilage and bone deterioration along with inflammation and oxidative damage that could be accredited to its antioxidant nature. Thus, Crocin could be an effective antiarthritic agent which can equally nullify the arthritis associated secondary complication.

Development of acid phosphatase based amperometric biosensors for the inhibitive determination of As(V).

An enzymatic amperometric procedure for the direct measurement of As(V) in the presence of As(III) was developed. The method is based on the inhibitive action of this species on acid phosphatase enzyme (AcP) activity. Screen-printed carbon electrodes (SPCEs) were used as support for the cross-linking immobilization of the enzyme AcP. 2-Phospho-l-ascorbic acid was used as a novel substrate, in arsenic determination, which amperometric response decreased by the presence of As(V) ions. The optimum working conditions were found using experimental design methodology. Under these conditions, repeatability and reproducibility of the constructed biosensors were determined, reaching values below 8% in terms of residual standard deviation. The capability of detection obtained for As(V) was 0.11 µM for AcP/SPCE biosensors. Analysis of the possible effect of the presence of foreign ions in the solution was performed. The method was successfully applied to the determination of the As(V) content in a ground water sample.

Osteoblastogenesis and osteoprotection enhanced by flavonolignan silibinin in osteoblasts and osteoclasts.

Bone-remodeling imbalance induced by decreased osteoblastogenesis and increased bone resorption is known to cause skeletal diseases such as osteoporosis. Silibinin is the major active constituent of silymarin, the mixture of flavonolignans extracted from blessed milk thistle (Silybum marianum). Numerous studies suggest that silibinin is a powerful antioxidant and has anti-hepatotoxic properties and anti-cancer effects against carcinoma cells. This study investigated that silibinin had bone-forming and osteoprotective effects in in vitro cell systems of murine osteoblastic MC3T3-E1 cells and RAW 264.7 murine macrophages. MC3T3-E1 cells were incubated in osteogenic media in the presence of 1-20 µM silibinin up to 15 days. Silibinin accelerated cell proliferation and promoted matrix mineralization by enhancing bone nodule formation by calcium deposits. In addition, silibinin furthered the induction of osteoblastogenic biomarkers of alkaline phosphatase, collagen type 1, connective tissue growth factor, and bone morphogenetic protein-2. Differentiated MC3T3-E1 cells enhanced secretion of receptor activator of nuclear factor-κB ligand (RANKL) essential for osteoclastogenesis, which was reversed by silibinin. On the other hand, RAW 264.7 cells were pre-incubated with 1-20 µM silibinin for 5 days in the presence of RANKL. Non-toxic silibinin markedly attenuated RANK transcription and intracellular adhesion molecule-1 expression elevated by RANKL, thereby suppressing the differentiation of macrophages to multi-nucleated osteoclasts. It was also found that silibinin retarded tartrate-resistant acid phosphatase and cathepsin K induction and matrix metalloproteinase-9 activity elevated by RANKL through disturbing TRAF6-c-Src signaling pathways. These results demonstrate that silibinin was a potential therapeutic agent promoting bone-forming osteoblastogenesis and encumbering osteoclastic bone resorption.

Inhibitory effect of (-)-saucerneol on osteoclast differentiation and bone pit formation.

In this study, the effect of (-)-saucerneol, one of the lignans isolated from Saururus chinensis, on osteoclast differentiation and bone resorption was evaluated in two in vitro models for osteoclast differentiation, the receptor activator of nuclear factor kappaB (NF-kappaB) ligand (RANKL)-treated RAW264.7 cells and mouse BMMs treated with both RANKL and macrophage-colony stimulating factor. (-)-Saucerneol significantly inhibited the RANKL-induced activity of tartrate-resistance acid phosphatase (TRAP, an early marker of osteoclast formation) and formation of osteoclasts in a dose-dependent manner. Interestingly, (-)-saucerneol was shown to inhibit the RANKL-induced activation of extracellular signal-regulated kinase in both in vitro models. In addition, (-)-saucerneol inhibited the bone resorptive activity and the expression of transcription factors and genes essential for osteoclast formation and bone resorption as well. In conclusion, (-)-saucerneol has a potential to inhibit the osteoclast differentiation via preventing the activation of ERK signaling pathway. In addition, its activity to inhibit the bone resorption activities of osteoclasts could result from its potential to inhibit RANKL-induced expression levels of transcription factors and genes essential for bone resorption.

Acid phosphatase activity may affect the tuber swelling by partially regulating sucrose-mediated sugar resorption in potato.

APase activity is involved in regulating many physiological and developmental events by affecting the resorption process. In this study, we investigate the role of APase activity in tuber development in potato. APase activities were mainly localized in cytoplasm, gaps among cells and stroma of amyloplasts of parenchyma cells at the stage of tuber swelling. AP1, encoding a putative APase, was also highly expressed in swelling tubers and a low level of expression was observed in elongated stolons and matured tubers. Inhibition of APase activity by applying Brefeldin A, an inhibitor of APase production and secretion, significantly suppressed the tuber swelling and moderately affected the stolon elongation and the tuberization frequency. During tuber development, sucrose serves as the main soluble sugar for long-distance transportation and resorption. Moreover, inhibition of APase activity by Brefeldin A markedly reduced the sucrose content in tubers and further decreased the starch accumulation, suggesting that the function of APase in regulating the tuber swelling might be at least partially mediated by the sugar resorption. Exogenous sucrose treatments further indicate the important role of sucrose-mediated sugar resorption in tuber swelling. These results suggest that the APase activity might affect the tuber swelling by partially regulating the sucrose-mediated sugar resorption.

Unfolding and inactivation during thermal denaturation of an enzyme that exhibits phytase and acid phosphatase activities.

The thermostability of an enzyme that exhibits phytase and acid phosphatase activities was studied. Kinetics of inactivation and unfolding during thermal denaturation of the enzyme were compared. The loss of phytase activity on thermal denaturation is most suggestive of a reversible process. As for acid phosphatase activities, an interesting phenomenon was observed; there are two phases in thermal inactivation: when the temperature was between 45 and 50 degrees C, the thermal inactivation could be characterized as an irreversible inactivation which had some residual activity and when the temperature was above 55 degrees C, the thermal inactivation could be characterized as an irreversible process which had no residual activity. The microscopic rate constants for the free enzyme and substrate-enzyme complex were determined by Tsou's method [Adv. Enzymol. Relat. Areas Mol. Biol. 61 (1988) 381]. Fluorescence analyses indicate that when the enzyme was treated at temperatures below 60 degrees C for 60 min, the conformation of the enzyme had no detectable change; when the temperatures were above 60 degrees C, some fluorescence red-shift could be observed with a decrease in emission intensity. The inactivation rates (k(+0)) of free enzymes were faster than those of conformational changes during thermal denaturation at the same temperature. The rapid inactivation and slow conformational changes of phytase during thermal denaturation suggest that inactivation occurs before significant conformational changes of the enzyme, and the active site of this enzyme is situated in a relatively fragile region which makes the active site more flexible than the molecule as a whole.

Modulatory effect of Mentha piperita (Linn.) on serum phosphatases activity in Swiss albino mice against gamma irradiation.

Mentha extract (ME; 1 g/kg body wt) given orally for three consecutive days prior to whole body irradiation (8 Gy) showed modulation of activity of serum phosphatases in albino mice. Values of acid phosphatase activities were significantly higher in untreated irradiated group throughout the experiment. Irradiated animals pretreated with ME showed significant decline in acid phosphatase activity as compared to untreated irradiated animals at all autopsy intervals and attained normalcy at day 5. A marked decrease in serum alkaline phosphatase activity was recorded in both irradiated groups. However, in ME pretreated irradiated group, values of alkaline phosphatase activity remained significantly higher than untreated irradiated animals at all intervals and attained normalcy from day 5 onwards.

Acid phosphatase reaction with peroxyl radicals: inactivation mechanism and behavior of the partially modified ensemble.

Acid phosphatase (AP) is readily inactivated when exposed to the free radicals generated in the pyrolysis of 2, 2'-azobis(2-amidinopropane) hydrochloride (AAPH) under aerobic conditions. On average, a large number of tryptophan groups are modified by each protein molecule that loses its catalytic activity. In spite of this, the enzyme inactivation takes place without induction times, a result that indicates either that damage is progressive or that damage of a critical target is needed to inactivate the enzyme (all-or-nothing mechanism). A Lineweaver-Burk plot of the enzyme activity measured at pH 4.8 is not compatible with an all-or-nothing mechanism, showing that after exposure of the native protein ensemble to the free radical source there are partially damaged molecules whose affinity for the substrate is widely different from that of the native molecules. On the other hand, the partially damaged ensemble shows a normal Michaelis-Menten behavior when the activity is measured at pH 7.0, with only a reduced value of V(M), relative to that of the unmodified ensemble. These results show that the native protein and modified proteins that remain active constitute different populations, with different responses to pH changes. Comparative heat denaturation studies of the native and pretreated proteins support this proposal.

Viper venom-induced inflammation and inhibition of free radical formation by pure compound (2-hydroxy-4-methoxy benzoic acid) isolated and purified from anantamul (Hemidesmus indicus R. BR) root extract.

The present investigation explored the possible venom neutralizing effect of a pure compound (2-hydroxy-4-methoxy benzoic acid) isolated and purified from the methanolic root extract of Hemidesmus indicus R.Rr. 2-OH-4-MeO benzoic acid possessed potent anti-inflammatory, antipyretic and antioxidant properties. The compound effectively neutralized inflammation induced by Vipera russelli venom in male albino mice and reduced cotton pellet-induced granuloma in rats. The compound produced a significant fall in body temperature in yeast-induced pyrexia in rats but did not change the normothermic body temperature. The compound effectively neutralized viper venom-induced changes in serum phosphatase and transaminase activity in male albino rats. It also neutralized free radical formation as estimated by TBAPS and superoxide dismutase activities. The antisnake venom activity of the pure compound is partly mediated through the above physiological process.

Stability and activity of potato acid phosphatase in aqueous surfactant media.

The effect of three different classes of surfactants viz., anionic, cationic and neutral on catalytic activity of potato acid phosphatase (AcPase) was studied. Anionic surfactants bis(2-ethylhexyl) sodium sulfosuccinate (AOT) and sodium dodecyl sulfate (SDS) inhibited AcPase activity completely at 3 mM concentration. The cationic surfactant cetyltrimethylammonium bromide (CTAB), at 3.33 mM enhanced the activity by 20 per cent. Increase in CTAB concentration decreased the activity, so that only 50 per cent was retained at 27 mM concentration. Brij 35, a neutral surfactant also decreased the activity, whereas TritonX-100 had little or no effect. At low CTAB concentration, an increase in Vmax and a decrease in Km was observed and the V/K ratio increased. TritonX-100 enhanced Vmax without changing Km. The V/K ratio was also increased. The UV-difference spectra of AcPase in presence of CTAB showed perturbations. The intensity of intrinsic fluorescence also enhanced in presence of CTAB.

Mechanism of Fe(III)-Zn(II) purple acid phosphatase based on crystal structures.

Purple acid phosphatase is a widely distributed non-specific phosphomonoesterase. X-ray structures of the dimeric 111-kDa Fe(III)-Zn(II) kidney bean purple acid phosphatase (kbPAP) complexed with phosphate, the product of the reaction, and with tungstate, a strong inhibitor of the phosphatase activity, were determined at 2.7 and 3.0 angstroms resolution, respectively. Furthermore the resolution of the unligated enzyme, recently solved at 2.9 angstroms could be extended to 2.65 angstroms with completely new data. The binding of both oxoanions is not accompanied by larger conformational changes in the enzyme structure. Small movements with a maximal coordinate shift of 1 angstroms are only observed for the active site residues His295 and His296. In the inhibitor complex as well as in the product complex, the oxoanion binds in a bidentate bridging mode to the two metal ions, replacing two of the presumed solvent ligands present in the unligated enzyme form. As also proposed for the unligated structure a bridging hydroxide ion completes the coordination spheres of both metal ions to octahedral arrangements. All three structures reported herein support a mechanism of phosphate ester hydrolysis involving interaction of the substrate with Zn(II) followed by a nucleophilic attack on the phosphorus by an Fe(III)-coordinated hydroxide ion. The negative charge evolving at the pentacoordinated transition state is probably stabilized by interactions with the divalent zinc and the imidazole groups of His202, His295, and His296, the latter protonating the leaving alcohol group.

Acid phosphatase synthesis in Aspergillus flavus.

Proteins with phosphatase activity were produced during the growth of Aspergillus flavus in a phosphate-supplemented liquid synthetic medium. The best carbon and nitrogen sources for the synthesis of phosphatase were glucose and ammonium sulfate, respectively. The proteins were separated by molecular exclusion and ion exclusion chromatography (IEC) into three components one of which showed phosphatase activity. The molar mass of the enzyme was approximately 62 kDa. The purified enzyme exhibited an optimum activity at pH 4.0 and at 45 degrees C. The activity of the enzyme was stimulated by Ca2+ and Mg2+ but inhibited by fluoride, iodoacetic acid, ethylenediaminetetraacetic acid and 2,4-dinitrophenol, and exhibited an apparent KM of approximately 420 mumol/L.

Inhibition of ATPase activity in pea plasma membranes by fungal suppressors from Mycosphaerella pinodes and their peptide moieties.

The effects of two suppressors of the defense reactions of host plants, which had been purified from the pea pathogen Mycosphaerella pinodes, as well as the effects of peptide moieties, on the ATPase activity in pea plasma membranes were examined in vitro. One of the suppressors, Supprescin B, inhibited the ATPase activity in a non-competitive manner, but the other suppressor, Supprescin A, did not. Supprescin A was observed to reduce the inhibitory effect of Supprescin B. A tripeptide, Ser-Ser-Gly, and a hexapeptide, Ser-Ser-Gly-Asp-Glu-Thr, which were the respective peptide moieties of Supprescin A and B, inhibited the ATPase activity in a competitive manner. Supprescin B and fragments of the hexapeptide, such as Asp-Glu-Thr and Gly-Asp-Glu, inhibited not only the ATPase activity but also the acid phosphatase activity of plasma membranes in vitro. These results indicate that the acidic amino-acid residues of the "Asp-Glu" moiety seem to act as inhibitors of the phosphatase activity. Thus, the peptide moiety of Supprescin B consists of at least two functional elements.

[The effect of alloxan on activity of various enzymes of phosphorus metabolism in vitro]. / Vliianie alloksana in vitro na aktivnost' nekotorykh fermentov fosfornogo obmena.

Effect of alloxan on activity of acetylphosphatase, alkaline and acid phosphatases, phenylpyrophosphatase was studied in vitro. Alloxan at concentrations 100 microM-10 mM inhibited acid phosphatase and especially alkaline phosphatase. Reducing agents (pyrocatechol, hydroquinone, ascorbic acid), which are easily converted into corresponding oxidized forms, at concentrations 100 microM-1.0 mM activated alkaline phosphatase in rat kidney and small intestine.

A therapeutic profile of metal chelators in the detoxication of methylmercury chloride inhibited acid and alkaline phosphatases in different areas of the central nervous system of rats.

Acid and alkaline phosphatase changes in various parts of the central nervous system (olfactory bulbs, cerebral hemispheres, cerebellum, medulla oblongata, and spinal cord) were analyzed during methylmercury chloride (MMC) treatment with a dose of 5 mg/kg/day body weight. The drug was subcutaneously introduced into the animals and the enzymes were analyzed after 2, 7, and 15 days' treatment. One group of animals was treated for seven days and kept without drug for another seven days (withdrawal group). The antagonizing capacities of four chelators, namely, N-acetyl-DL-homocysteine thiolactone (NAHT), D-penicillamine (DPA), glutathione (GSH), and sodium selenite (SEL), were also analyzed in relation to the restoration of enzymes. Study results show a linear inhibition of acid and alkaline phosphatases with increasing duration of MMC treatment. However, the magnitude of enzymatic inhibition is different in different brain areas. After 15 days' treatment, maximum inhibition of acid and alkaline phosphatases was recorded in the spinal cord and cerebellum, respectively. Chelators also exhibited differential recovery of the enzymes in various animal groups, as well as in discrete brain areas. No uniformity in the recovery of the enzymes with chelators was observed. However, study results show that biochemical parameters are good indicators of early recognition of neurotoxicity.