Preparation and structural properties of starch phosphate modified by alkaline phosphatase.

In this study, a method for the synthesis of starch phosphate using the transferase properties of alkaline phosphatase was explored. Maize starch was treated with a pyrophosphate solution containing alkaline phosphatase and catalytic ions under pH 8 at 37 °C. The synthesis of starch phosphate was evaluated and compared with untreated and treated starch controls. The phosphorus content of the samples increased up to 8500% with the catalytic ion concentration, whereas the peak viscosity by up to 41.4% decreased. The crystallinity and enthalpy of the phosphorylated samples were reduced by up to 26.8% and 23.3%, respectively; however, no significant was observed by Fourier-transform infrared spectrometer. The roughness of the starch surface and the distribution of elemental phosphorus were observed by scanning electron microscopy and energy dispersive Spectrometry. X-ray photoelectron spectroscopy and time of flight secondary ion mass spectrometry results further indicated the grafting of the phosphate radical.

Enzymatic Approach in Calcium Phosphate Biomineralization: A Contribution to Reconcile the Physicochemical with the Physiological View.

Biomineralization is the process by which organisms produce hard inorganic matter from soft tissues with outstanding control of mineral deposition in time and space. For this purpose, organisms deploy a sophisticated "toolkit" that has resulted in significant evolutionary innovations, for which calcium phosphate (CaP) is the biomineral selected for the skeleton of vertebrates. While CaP mineral formation in aqueous media can be investigated by studying thermodynamics and kinetics of phase transitions in supersaturated solutions, biogenic mineralization requires coping with the inherent complexity of biological systems. This mainly includes compartmentalization and homeostatic processes used by organisms to regulate key physiological factors, including temperature, pH and ion concentration. A detailed analysis of the literature shows the emergence of two main views describing the mechanism of CaP biomineralization. The first one, more dedicated to the study of in vivo systems and supported by researchers in physiology, often involves matrix vesicles (MVs). The second one, more investigated by the physicochemistry community, involves collagen intrafibrillar mineralization particularly through in vitro acellular models. Herein, we show that there is an obvious need in the biological systems to control both where and when the mineral forms through an in-depth survey of the mechanism of CaP mineralization. This necessity could gather both communities of physiologists and physicochemists under a common interest for an enzymatic approach to better describe CaP biomineralization. Both homogeneous and heterogeneous enzymatic catalyses are conceivable for these systems, and a few preliminary promising results on CaP mineralization for both types of enzymatic catalysis are reported in this work. Through them, we aim to describe the relevance of our point of view and the likely findings that could be obtained when adding an enzymatic approach to the already rich and creative research field dealing with CaP mineralization. This complementary approach could lead to a better understanding of the biomineralization mechanism and inspire the biomimetic design of new materials.

Antihyaluronidase and Alkaline Phosphatase (ALP) Activities of Medicinal Plants to Combat Echis carinatus Venom-Induced Toxicities.

Snakebite is one of the most neglected diseases of developing countries. Deaths due to snakebite envenoming are quite high in Pakistan, and many deaths are caused by Echis carinatus envenomation. Traditional use of medicinal plants against snakebites is a common practice in Pakistan due to countless benefits. The current study was performed with the objective to evaluate eighteen Pakistani medicinal plants inhibitory potential against hyaluronidase and alkaline phosphatase enzymes of Pakistani Echis carinatus venom. Hyaluronidase activity (0.2-1.6 mg/0.1 mL) and alkaline phosphatase activity (0.1-0.8 mg/0.1 mL) were measured in dose-dependent manner. Crude methanolic extracts of medicinal plants were used for in vitro investigation of their inhibitory activity against toxic enzymes. All active plants were fractioned using different solvents and were again analyzed for inhibitory activity of same enzymes. Results indicated all plants were able to neutralize hyaluronidase that Swertia chirayita (Roxb. ex Flem.) Karst., Terminalia arjuna Wight and Arn, Rubia cordifolia Thumb., and Matthiola incana (L.) R.Br. inhibited maximum hyaluronidase activity equivalent to standard reference (p > 0.5). Pakistani medicinal plants are dense with natural neutralizing metabolites and other active phytochemicals which could inhibit hyaluronidase activity of Pakistani Echis carinatus venom. Further advanced studies at molecular level could lead us to an alternative for envenoming of Pakistani Echis carinatus venom.

Fluorometric and Colorimetric Dual-Readout Immunoassay Based on an Alkaline Phosphatase-Triggered Reaction.

Alkaline phosphatase (ALP) usually acts as a signal transmitter in enzyme-linked immunosorbent assay (ELISA); therefore, developing an attractive ALP activity assay, especially using a preferable substrate, would help improve the efficiency and convenience of ELISA in practical applications. Herein we have first prepared an original and creative substrate, named m-hydroxyphenyl phosphate sodium salt ( m-HPP), with a desirable dephosphorylation site for ALP. On the basis of the ALP-catalyzed hydrolysis of m-HPP to resorcinol and its subsequent specific nucleophilic reaction with dopamine, we have exploited a fluorometric and colorimetric dual-readout ALP activity assay and ALP-based ELISA system. Under the employed experimental conditions, highly sensitive and specific assay of ALP and cardiac troponin I (cTnI) has been accomplished in a straightforward way. Furthermore, the commendable sensing performance of our proposed ELISA in the determination of the cTnI level in diluted human serum unambiguously illustrates great potential in the early diagnosis of acute myocardial infarction.

Plasmonic and Photothermal Immunoassay via Enzyme-Triggered Crystal Growth on Gold Nanostars.

Immunoassay is commonly used for the detection of disease biomarkers, but advanced instruments and professional operating are often needed with current techniques. The facile readout strategy for immunoassay is mainly limited to the gold nanoparticles-based colorimetric detection. Here, we show that photothermal nanoparticles can be applied for biosensing and immunoassay with temperature as readout. We develop a plasmonic and photothermal immunoassay that allows straightforward readout by color and temperature based on crystal growth, without advanced equipment. It is demonstrated that alkaline phosphatase-triggered silver deposition on the surface of gold nanostars causes a large blue shift in the localized surface plasmon resonance of the nanosensor, accompanied by photothermal conversion efficiency changes. This approach also allows dual-readout of immunoassays with high sensitivity and great accuracy for the detection of prostate-specific antigen in complex samples. Our strategy provides a promising way for point-of-care testing and may broaden the applicability of programmable nanomaterials for diagnostics.

Effects of modified sediments from a eutrophic lake in removing phosphorus and inhibiting phosphatase activity.

Phosphorus is one of the main limiting and strong influencing factors of eutrophication, and phosphorus controlling in lake is of great significance for eutrophication. To do this, sediment materials were taken from Dianchi Lake, a typically eutrophic lake, and modified by hexadecyltrimethylammonium bromide (CTAB) and ZnSO4 to remove phosphorus and inhibit alkaline phosphatase activity (APA). Results indicated that phosphorus removal efficiencies of sediments modified by CTAB (S-CTAB), ZnSO4 (S-Zn), and oxidized sediments (OS) were higher than that of the raw sediment (RS). Ability to absorb phosphorus varied, following the order S-Zn>S-CTAB>OS>RS. Sorption was influenced by ionic strength, with the former decreasing with the increase of the latter. Freundlich model well described the sorption isotherm, with an R2 ranging from 0.9168 to 0.9958. Furthermore, compared with the raw sediments, the maximum phosphorus sorption capacities of S-Zn and S-CTAB increased by 12.2% and 124.5%, respectively. Results of desorption studies suggest that the desorption rate of S-Zn was from 3.88 to 13.76%, lower than that of other sediment materials. APA was inhibited by S-CTAB and S-Zn at the same time, with inhibition rates from 29.6% and 61.0% when the concentrations of S-CTAB and S-Zn were 10 nmol L-1 and 0.2 nmol L-1, respectively. This study provides new insights into phosphorus removal and phosphatase activity inhibition in water treatment.

Towards the identification of alkaline phosphatase binding ligands in Li-Dan-Hua-Shi pills: A Box-Behnken design optimized affinity selection approach tandem with UHPLC-Q-TOF/MS analysis.

Alkaline phosphatase conjugated magnetic microspheres were synthesized via amide reaction, and employed as an effective adsorbent in affinity selection of binding ligands followed by UHPLC-Q-TOF/MS analysis. The analytical validity of the developed approach was evaluated under optimized conditions and the following figures of merit were obtained: linearity, 0.01-0.5 g L-1 with good determination coefficients (R2 = 0.9992); limits of detection (LODs), 0.003 g L-1; and limits of quantitation (LOQ), 0.01 g L-1. The precision (RSD%) of the proposed affinity selection approach was studied based on intra-day (0.8%) and inter-day (1.3%) precisions. Finally, the adsorbent was successfully applied to identification of binding ligands in Li-Dan-Hua-Shi pills and good recoveries were obtained in the range from 96.9 to 99.4% (RSDs 1.6-3.0%).

Comparisons of the spectroscopic and microbiological activities among coumarin-3-carboxylate, o-phenanthroline and zinc(II) complexes.

Coumarins (2H-chromen-2-one) are oxygen-containing heterocyclic compounds that belong to the benzopyranones family. In this work we have synthesized different coordination complexes with coumarin-3-carboxylic acid (HCCA), o-phenanthroline (phen) and zinc(II). In the reported [Zn(CCA)2(H2O)2] complex, coumarin-3-carboxylate (CCA) is acting as a bidentate ligand while in the two prepared complexes, [Zn(phen)3]CCA(NO3) (obtained as a single crystal) and [Zn(CCA)2phen].4H2O, CCA is acting as a counterion of the complex cation [Zn(phen)3]+2 or coordinated to the metal center along with phen, respectively. These compounds were characterized on the basis of elemental analysis and thermogravimetry. NMR, FTIR and Raman spectroscopies of the compounds and the CCA potassium salt (KCCA) allow to determine several similarities and differences among them. Finally, their behavior against alkaline phosphatase enzyme and their antimicrobial activities were also measured.

Temperature enhances the affinity of soil alkaline phosphatase to Cd.

Both elevated temperature and heavy metal contamination can have profound effects on microbial function and soil biogeochemical cycling. However, the interactive effects of heavy metal toxicity and temperature on microbial activity have been poorly understood. The aim of this study was to quantify the effect of temperature and cadmium (Cd) toxicity on alkaline phosphatase (ALP) produced by microbes to acquire phosphorus. To determine whether these effects were dependent on soil properties, we utilized 11 soil types from cropland throughout China. We measured ALP activities and kinetics across a temperature (17, 27, 37, and 47 °C) and Cd concentration gradient (0, 0.6, 5, 25, 50, 100, 200, 300, and 500 mg kg-1). We found that the half saturation constant (Km) and the velocity constant (k) of ALP increased nonlinearly with temperature across all soil types. However, the maximum reaction velocity (Vmax) increased linearly with temperature. Regardless of soil type and temperature, Cd had a non-competitive inhibitory mechanism. Soil pH, TOC, and clay content were the major factors controlling the affinity of ALP for Cd (Ki). The ecology dose (ED50) for Vmax and k, and Ki were negatively related to temperature, indicating that the toxicity of Cd on ALP is temperature-dependent. Additionally, higher temperatures led to more inhibition of Cd on ALP activity in alkaline soils than that in acidic and neutral soils. Our results suggest that global warming might accelerate the deficiency of available phosphorus in Cd contaminated soils due to higher inhibition of Cd on ALP activity, particularly in alkaline soils.

Isonicotinohydrazones as inhibitors of alkaline phosphatase and ecto-5'-nucleotidase.

A series of isonicotinohydrazide derivatives was synthesized and tested against recombinant human and rat ecto-5'-nucleotidases (h-e5'NT and r-e5'NT) and alkaline phosphatase isozymes including both bovine tissue-non-specific alkaline phosphatase (b-TNAP) and tissue-specific calf intestinal alkaline phosphatase (c-IAP). These enzymes are implicated in vascular calcifications, hypophosphatasia, solid tumors, and cancers, such as colon, lung, breast, pancreas, and ovary. All tested compounds were active against both enzymes. The most potent inhibitor of h-e5'NT was derivative (E)-N'-(1-(3-(4-fluorophenyl)-5-phenyl-4,5-dihydro-1H-pyrazol-1-yl)ethylidene)isonicotinohydrazide (3j), whereas derivative (E)-N'-(4-hydroxy-3-methoxybenzylidene)isonicotinohydrazide (3g) exhibited significant inhibitory activity against r-e5'NT. In addition, the derivative (E)-N'-(4'-chlorobenzylidene)isonicotinohydrazide (3a) was most potent inhibitor against calf intestinal alkaline phosphatase and the derivative (E)-N'-(4-hydroxy-3-methoxybenzylidene)isonicotinohydrazide (3g) was found to be most potent inhibitor of bovine tissue-non-specific alkaline phosphatase. Furthermore, putative binding modes of potent compounds against e5'NT (human and rat e5'NT) and AP (including b-TNAP and c-IAP) were determined computationally.

Enhanced cell adhesion on bioinert ceramics mediated by the osteogenic cell membrane enzyme alkaline phosphatase.

Functional bone and dental implant materials are required to guide cell response, offering cues that provide specific instructions to cells at the implant/tissue interface while maintaining full biocompatibility as well as the desired structural requirements and functions. In this work we investigate the influence of covalently immobilized alkaline phosphatase (ALP), an enzyme involved in bone mineralization, on the first contact and initial cell adhesion. To this end, ALP is covalently immobilized by carbodiimide-mediated chemoligation on two highly bioinert ceramics, alpha-alumina (Al2O3) and yttria-stabilized zirconia (Y-TZP) that are well-established for load-bearing applications. The physicochemical surface properties are evaluated by profilometry, zeta potential and water contact angle measurements. The initial cell adhesion of human osteoblasts (HOBs), human osteoblast-like cells (MG-63) and mesenchymal stromal cells (hMSCs) was investigated. Cell adhesion was assessed at serum free condition via quantification of percentage of adherent cells, adhesion area and staining of the focal adhesion protein vinculin. Our findings show that after ALP immobilization, the Al2O3 and Y-TZP surfaces gained a negative charge and their hydrophilicity was increased. In the presence of surface-immobilized ALP, a higher cell adhesion, more pronounced cell spreading and a higher number of focal contact points were found. Thereby, this work gives evidence that surface functionalization with ALP can be utilized to modify inert materials for biological conversion and faster bone regeneration on inert and potentially load-bearing implant materials.

Zein as biodegradable material for effective delivery of alkaline phosphatase and substrates in biokits and biosensors.

A biodegradable material, zein, is proposed as a reagent delivery platform for biokits and biosensors based on alkaline phosphatase (ALP) activity/inhibition in the presence of phosphatase substrates. The immobilization and release of both the substrate and/or the active ALP, in a biodegradable and low-cost material such as zein, a prolamin from maize, and in combination with glycerol as plasticizer have been investigated. Three zein-based devices are proposed for several applications: (1) inorganic phosphorus estimation in water of different sources (river, lake, coastal water and tap water) with a detection limit of 0.2mg/L - compared to at least 1mg/L required by legislation, (2) estimation of ALP in saliva and (3) chlorpyrifos control in commercial preparations. The single-use kits developed are low cost, easy and fast to manufacture and are stable for at least 20 days at -20°C, so the zein film can preserve and deliver both the enzyme and substrates.

Changes in microbial dynamics during vermicomposting of fresh and composted sewage sludge.

Municipal sewage sludge is a waste with high organic load generated in large quantities that can be treated by biodegradation techniques to reduce its risk to the environment. This research studies vermicomposting and vermicomposting after composting of sewage sludge with the earthworm specie Eisenia andrei. In order to determine the effect that earthworms cause on the microbial dynamics depending on the treatment, the structure and activity of the microbial community was assessed using phospholipid fatty acid analysis and enzyme activities, during 112days of vermicomposting of fresh and composted sewage sludge, with and without earthworms. The presence of earthworms significantly reduced microbial biomass and all microbial groups (Gram+ bacteria, Gram- bacteria and fungi), as well as cellulase and alkaline phosphatase activities. Combined composting-vermicomposting treatment showed a lesser development of earthworms, higher bacterial and fungal biomass than vermicomposting treatment and greater differences, compared with the control without earthworms, in cellulase, ß-glucosidase, alkaline and acid phosphatase. Both treatments were suitable for the stabilization of municipal sewage sludge and the combined composting-vermicomposting treatment can be a viable process for maturation of fresh compost.

Enzyme-assisted calcium phosphate biomineralization on an inert alumina surface.

In this study a bioinspired approach to induce self-mineralization of bone-like material on alumina surfaces is presented. The mineralizing enzyme alkaline phosphatase (ALP) is covalently immobilized by a carbodiimide-mediated chemoligation method. The enzymatic activity of immobilized ALP and its mineralization capability are investigated under acellular conditions as well as in the presence of human bone cells. Analytical, biochemical and immunohistochemical characterization show that ALP is efficiently immobilized, retains its activity and can trigger calcium phosphate mineralization on alumina at acellular conditions. In vitro cell tests demonstrate that ALP-functionalized alumina clearly boosts and enhances bone cell mineralization. Our results underpin the great potential of ALP-functionalized alumina for the development of bioactive surfaces for applications such as orthopaedic and dental implants, enabling a fast and firm implant osseointegration.

An annual survey of bacterial production, respiration and ectoenzyme activity in coastal NW Mediterranean waters: temperature and resource controls.

We simultaneously measured bacterial production (BP), bacterial respiration (BR), alkaline phosphatase activity (phos) and ectoaminopeptidase activity (prot) in relation to biogeochemical parameters, nutritive resources and in situ temperature over a 1-year survey at the long-term observatory the SOLEMIO station (Marseille bay, NW Mediterranean Sea). Despite its proximity to the coast, oligotrophic conditions prevailed at this station (yearly mean of Chl a = 0.43 µg dm(-3), NO3 = 0.55 µmol dm(-3) and PO4 = 0.04 µmol dm(-3)). Episodic meteorological events (dominant winds, inputs from the Rhone River) induced rapid oscillations (within 15 days) in temperature and sometimes salinity that resulted in rapid changes in phytoplankton succession and a high variability in C/P ratios within the particulate and dissolved organic matter. Throughout the year, BP ranged from 0.01 to 0.82 µg C dm-(3) h-(1) and bacterial growth efficiency varied from 1 to 39%, with higher values in summer. Enrichment experiments showed that BP was limited most of the year by phosphorus availability (except in winter). A significant positive correlation was found between in situ temperature, BP, BR and phos. Finally, we found that temperature and phosphate availability were the main factors driving heterotrophic bacterial activity and thus play a fundamental role in carbon fluxes within the marine ecosystem.

Metformin rescues the MG63 osteoblasts against the effect of high glucose on proliferation.

AIMS. To study the proliferation of osteoblasts and genes expression under normal glucose, high glucose, and metformin (Met). METHODS. MG63 osteoblast-like cells were cultured in osteogenic medium supplemented with normal glucose (glucose 5.5 mmol/L) or high glucose (glucose 16.7 mmol/L) and metformin + high glucose (Met 300 µmol/L + glucose 16.7 mmol/L). Proliferation was detected with CCK-8 assay at days 1, 3, and 7. Real-time PCR and Western blot were performed to compare the expression of collagen I (Col I), osteocalcin (OCN), osteoprotegerin (OPG), receptor activator for NF- κB ligand (RANKL), and metal matrix proteinases 1 and 2 (MMP1, MMP2). Alkaline phosphatase (ALP) activity was also detected at days 6, 12, and 18. RESULTS. Exposure to high glucose inhibited the proliferation of osteoblasts (P < 0.05), with suppressed OCN and OPG. Meanwhile, Col I, RANKL, MMP1, and MMP2 were unaffected. Metformin attenuated the suppression on proliferation with increased expression of Col I, OCN, and OPG, meanwhile suppressing MMP1 and MMP2. High glucose lowered the intracellular ALP, while metformin raised it. Metformin attenuated the downregulation of ALP completely at day 6, partly at day 12, but not at day 18. CONCLUSIONS. Metformin attenuated the suppression effect of high glucose to the osteoblast proliferation and gene expression, more prominently in earlier stage.

The effects of prebiotics on the digestive enzymes and gut histomorphology of red drum (Sciaenops ocellatus) and hybrid striped bass (Morone chrysops × M. saxatilis).

The effects of four prebiotics (fructo-oligosaccharide, Bio-MOS, transgalacto-oligosaccharide and GroBiotic-A) on digestive enzymes and intestinal morphology were studied in juvenile hybrid striped bass (Morone chrysops × M. saxatilis) and red drum (Sciaenops ocellatus) using two separate 8-week feeding trials. Red drum were fed experimental diets with the four prebiotics each individually supplemented at 1% and hybrid striped bass were fed diets supplemented with GroBiotic-A at 1 and 2%. Both trials were conducted with each diet fed to apparent satiation twice per d to three replicate groups of fifteen juvenile fish. For histomorphological analysis, gastrointestinal tract (GIT) samples from three randomly selected fish per tank were taken at 4 and 8 weeks for hybrid striped bass and at 8 weeks for red drum. For both trials, GIT samples from two randomly selected fish per tank were taken at 4 and 8 weeks and analysed for pepsin, trypsin, chymotrypsin, aminopeptidase, α-amylase, lipase, and both acid and alkaline phosphatase activities. The results of the histological evaluation indicated that the inclusion of prebiotics was adequate to elicit structural changes in the GIT of both species. On the other hand, no significant changes in the enzyme activities were detected at week 8 in both species. However, there was a transient effect of Bio-MOS supplementation on the activities of aminopeptidase, α-amylase and alkaline phosphatase at week 4 in red drum only. Thus, previously observed improvements in nutrient digestibility by these fish in response to prebiotic supplementation appear to be mostly related to changes in GIT structure as opposed to the enhancement of digestive enzyme activity.

Role of sea water DIP and DOP in controlling bulk alkaline phosphatase activity in N.W. Mediterranean Sea (Toulon, France).

The regulation of alkaline phosphatase activity by dissolved inorganic (DIP) and organic phosphorus (DOP) and the contribution of DOP as phosphorus source were studied monthly in Toulon Bay (NW Mediterranean, France) in 2005-2006. The concentrations of DIP and DOP varied respectively from 0 to 0.185µM and from 0 to 0.329µM. The bulk activities (Vm, Km, Vm/Km) were measured using MUFP as substrate. Its high affinity component (Km: 0.05-1.00µM) was negatively correlated with the sum of the concentrations of DIP and DOP but not with these compounds taken independently. A negative correlation with DIP was found when the concentrations of DOP were lower than 0.08µM. A negative correlation with DOP was shown when the concentrations of DIP were lower than 0.05µM. This high affinity component can be considered as a valuable indicator for the potential utilization of the compounds which contribute to the intracellular phosphorus pool.

Compatibility of garlic (Allium sativum L.) leaf agglutinin and Cry1Ac δ-endotoxin for gene pyramiding.

δ-Endotoxins produced by Bacillus thuringiensis (Bt) have been used as bio-pesticides for the control of lepidopteran insect pests. Garlic (Allium sativum L.) leaf agglutinin (ASAL), being toxic to several sap-sucking pests and some lepidopteran pests, may be a good candidate for pyramiding with δ-endotoxins in transgenic plants for enhancing the range of resistance to insect pests. Since ASAL shares the midgut receptors with Cry1Ac in Helicoverpa armigera, there is possibility of antagonism in their toxicity. Our study demonstrated that ASAL increased the toxicity of Cry1Ac against H. armigera while Cry1Ac did not alter the toxicity of ASAL against cotton aphids. The two toxins interacted and increased binding of each other to brush border membrane vesicle (BBMV) proteins and to the two important receptors, alkaline phosphatase (ALP) and aminopeptidase N (APN). The results indicated that the toxins had different binding sites on the ALP and APN but influenced mutual binding. We conclude that ASAL can be safely employed with Cry1Ac for developing transgenic crops for wider insect resistance.