Extracellular Acid Phosphatase Research Articles

Presence of pharmaceutical contaminants of emerging concerns in two rivers of western Cuba and their relationship with the extracellular enzymatic activity of microbial communities

In recent years, the concern derived from the presence of emerging contaminants in the environment and the possible effects on the One Health trilogy has increased. This study determined the concentration of pharmaceutical contaminants of emerging concern and their relationship with the extracellular enzymatic activity of microbial communities from two rivers in western Cuba. Two sampling stations were analyzed; one in the Almendares River (urban) and the other in the San Juan River (rural), taking into account the pollution sources that arrive at these stations and previous physicochemical characterizations. Extracellular protease, acid phosphatase, alkaline phosphatase, lipase, and catalase activities in water and sediments were determined and correlated with contaminants of emerging concern determined by liquid chromatography with mass spectrometry. This study evidenced the presence of different pharmaceutical contaminants found in the categories of antihypertensives, stimulants, anti-inflammatories, and antibiotics in both rivers. Concentrations of contaminants of emerging concern were greater in the Almendares River compared to the San Juan River. In addition, through the canonical redundancy analysis, the influence of these contaminants on the extracellular enzymatic activities of microbial communities was documented, where in most cases they inhibit protease, phosphatase, and lipase activities and enhance catalase activity in response to oxidative stress. The present investigation constitutes the first report in Cuba of the presence of pharmaceutical contaminants of emerging concern and one of the few works that exist in the Latin American region.

Plant Diversity Changes and Its Driving Factors of Abandoned Land at Different Restoration Stages in the Middle of the Qinling Mountains

The process of vegetation restoration is often accompanied by significant changes in aboveground plant diversity. To explore the driving mechanism of litter nutrient-soil nutrient-enzyme activity stoichiometry on aboveground vegetation change is of great importance for maintaining regional biodiversity conservation and ecological stability. Taking typical abandoned farmland of different restoration years （1， 8， 16， 31， and 50 a） in the Qinling Mountains as the research object， the variation characteristics of plant community diversity during vegetation restoration were analyzed through field investigation. Litter nutrients， soil nutrients， and the activities of five extracellular enzymes， including β-1，4-glucosidase （BG）， cellobiohydrolase （CBH）， β-1，4-N-acetylglucosaminidase （NAG）， leucine aminopeptidase （LAP）， and acid phosphatase （AP）， were determined. The characteristics of litter nutrients， soil nutrients， and enzyme stoichiometric ratios during vegetation restoration and the driving mechanism of plant diversity changes were discussed. The results showed that the plant community diversity index firstly decreased and then increased with the increase in vegetation restoration years， and the minimum was reached at 16 years after restoration. The results of principal component analysis showed that there were significant differences between total plant community diversity index and litter-soil-enzyme stoichiometric characteristics in different years of vegetation restoration. The plant community diversity index had a strong positive correlation with litter C∶P ratio and litter N∶P ratio but had a negative correlation with soil enzyme C∶P ratio （EEA C∶P）. The results of redundancy analysis showed that soil EEA C∶P had the highest explanation rate of plant diversity changes during vegetation restoration （25.93%）， followed by soil TP （5.94%）， which was the key factor regulating plant diversity changes. In conclusion， plant species and quantity increased significantly in abandoned farmland in the middle part of the Qinling Mountains at the late stage of vegetation restoration. Changes in the soil environment affected microbial metabolic activities and thus changed enzyme activities. Litter-soil-soil extracellular enzymes affected the community environment and plant diversity through feedback and regulation. EEA C∶P and TP were the main driving factors of aboveground plant diversity change during vegetation restoration.

Assessment of degradation mechanism of imidacloprid residues in grape rhizosphere soil by UHPLC-Orbitrap™-MS and its residual impact on soil enzyme activity.

Imidacloprid (IM) is a systemic insecticide persistent in the environment and possesses a negative impact on the non-targeted ecosystem. The objective of the present study was to evaluate the dissipation and degradation mechanism of IM residues in grape rhizosphere soil and to investigate its residual effect on soil enzyme activity at different IM spiking levels. The half-life of IM residue in soil was 27, 36, and 43.5days at a spiking level of 1, 10, and 50mgkg-1, respectively following a bi-phasic first + first-order dissipation kinetics. UHPLC-Orbitrap™-MS analysis by targeted metabolomics approach revealed that IM metabolites such as IM-amine analogue, guanidine (reduction), 5-hydroxy IM (hydroxylation), IM-Urea (oxidation), reduced NO analogue of IM (oxidation), and olefin of guanidine IM (dehydrogenation) were identified and proposed the degradation mechanism in grape rhizosphere soil. Toxicity of IM residues on five extracellular enzymes, viz., dehydrogenase, acid phosphatase, alkaline phosphatase, β-glucosidase, and urease revealed that activity of dehydrogenase, acid phosphatase, and alkaline phosphatase remained unaffected at 60th day of sampling. The β-glucosidase and urease were negatively affected throughout the incubation period indicating the influence of IM residues on carbon and nitrogen mineralization in soil. Thus, long-term exposure of IM to grape rhizosphere through soil drenching could affect soil enzyme activity which has a negative effect on the soil nutrient cycle and soil microbiome.

Integration of osteoclastogenesis through addition of PBMCs in human osteochondral explants cultured ex vivo

The preservation of tissue specific cells in their native 3D extracellular matrix in bone explants provides a unique platform to study remodeling. Thus far, studies involving bone explant cultures showed a clear focus on achieving bone formation and neglected osteoclast activity and resorption. To simulate the homeostatic bone environment ex vivo, both key elements of bone remodeling need to be represented. This study aimed to assess and include osteoclastogenesis in human osteochondral explants through medium supplementation with RANKL and M-CSF and addition of peripheral blood mononuclear cells (PBMCs), providing osteoclast precursors.Osteochondral explants were freshly harvested from human femoral heads obtained from hip surgeries and cultured for 20 days in a two-compartment culture system. Osteochondral explants preserved viability and cellular abundance over the culture period, but histology demonstrated that resident osteoclasts were no longer present after 4 days of culture. Quantitative extracellular tartrate resistant acid phosphatase (TRAP) analysis confirmed depletion of osteoclast activity on day 4 even when stimulated with RANKL and M-CSF. Upon addition of PBMCs, a significant upregulation of TRAP activity was measured from day 10 onwards. Evaluation of bone loss trough μCT registration and measurement of extracellular cathepsin K activity revealed indications of enhanced resorption upon addition of PBMCs. Based on the results we suggest that an external source of osteoclast precursors, such as PBMCs, needs to be added in long-term bone explant cultures to maintain osteoclastic activity, and bone remodeling.

Changes in Soil Properties and Enzyme Stoichiometry in Three Different Forest Types Changed to Tea Plantations

Understanding the characteristics and driving factors of soil carbon, nitrogen, phosphorus, and enzyme stoichiometry during land use/cover change is of great significance for assessing microbial nutrient restriction and sustainable land development during the process. China, the world’s largest tea producer, is witnessing a significant expansion of tea plantations into previously forested areas. We performed field sampling in three forest types with the area partially converted to tea plantations in Wuyishan National Park. We examined the changes in soil carbon (TC), nitrogen (TN), phosphorus (TP), and three kinds of extracellular enzyme activities, β-glucosidase (BG), β-n-acetylglucosidase (NAG), and acid phosphatase (ACP). By analyzing the enzyme stoichiometric ratio, vector length (VL), and vector angle (VA), the relative nutrient limitations of soil microorganisms were explored. The results showed that soil TC and TN decreased significantly (p < 0.05), TP increased significantly, and soil carbon (C):nitrogen (N), carbon (C):phosphorus (P), and nitrogen (N):phosphorus (P) ratios decreased significantly after the conversion of forest land to tea plantation. Soil BG, NAG, and ACP contents decreased significantly (p < 0.05). There were no significant differences in enzyme carbon:nitrogen ratios (EC/N), enzyme carbon:phosphorus ratios (EC/P), enzyme nitrogen:phosphorus ratios (EN/P), VL, or VA (p > 0.05). Through the analysis of soil enzyme stoichiometry, it was found that forest soil was generally limited by P, which was, to some extent, relieved after the conversion to tea plantation. Redundancy analysis showed that TC, TN, and the C:N ratio were the main factors influencing enzyme activity and stoichiometry. These results indicated that land use/cover change had significant effects on soil nutrient status, enzyme activity, and stoichiometry. Soil enzyme activity is very sensitive to the changes in soil nutrients and can reflect the restriction of soil nutrients more accurately.

The ecl family gene ecl3+ is induced by phosphate starvation and contributes to sexual differentiation in fission yeast.

In Schizosaccharomyces pombe, ecl family genes are induced by several signals, such as starvation of various nutrients, including sulfur, amino acids and Mg2+, and environmental stress, including heat or oxidative stress. These genes mediate appropriate cellular responses and contribute to the maintenance of cell viability and induction of sexual differentiation. Although this yeast has three ecl family genes with overlapping functions, any environmental conditions that induce ecl3+ remain unidentified. We demonstrate that ecl3+ is induced by phosphate starvation, similar to its chromosomally neighboring genes, pho1+ and pho84+, which respectively encode an extracellular acid phosphatase and an inorganic phosphate transporter. ecl3+ expression was induced by the transcription factor Pho7 and affected by the cyclin-dependent kinase (CDK)-activating kinase Csk1. Phosphate starvation induced G1 arrest and sexual differentiation via ecl family genes. Biochemical analyses suggested that this G1 arrest was mediated by the stabilization of the CDK inhibitor Rum1, which was dependent on ecl family genes. This study shows that ecl family genes are required for appropriate responses to phosphate starvation and provides novel insights into the diversity and similarity of starvation responses.

The wheat secreted root proteome: Implications for phosphorus mobilisation and biotic interactions

Root secreted acid phosphatases and organic anions are widely perceived as major players of plant phosphorus (P) mobilisation from the rhizosphere under P limiting growth conditions. Previous research indicated that other mechanisms play a role, especially in species with fine roots, such as wheat. In this study we characterised the plant-derived extracellular proteome of wheat roots by profiling root tip mucilage, soluble root secreted and root tip proteomes. Extracellular acid phosphatases and enzymes of the central carbon metabolism were targeted using selected reaction monitoring. More than 140 proteins with extracellular localisation prediction were identified in mucilage. P starvation induced proteins predicted to be localised to the apoplast which are related to cell wall modification and defence in both, root tip and soluble root-secreted proteomes. Glycolytic enzymes were strongly increased in abundance by P limitation in root tips, as were PEPC and plastidial MDH. Soluble acid phosphatases were not identified in extracellular protein samples. Our results indicate that root tip mucilage contains proteins with the functional potential to actively shape their immediate environment by modification of plant structural components and biotic interactions. Wheat acid phosphatases appear to play a minor role in P mobilisation beyond the immediate root surface. SignificancePhosphorus (P) is a plant growth limiting nutrient in many agricultural situations and the development of phosphorus efficient crops is of paramount importance for future agricultural management practices. As P is relatively immobile in soils, processes occurring at the root-soil interface, the rhizosphere, are suspected to play a key role in plant-induced P mobilisation. According to the current view, the secretion of extracellular acid phosphatases and organic anions enhances P mobilisation within several millimetres beyond the root surface, either directly or indirectly through the selection and appropriate soil microbes. However, the mechanisms of P mobilisation in species with fine roots, such as wheat, and the role of other secreted root proteins are poorly understood. Here, we carried out the profiling of wheat root tip mucilage, soluble root secreted and root tip proteomes. We analysed proteome changes in response to P starvation. We found that proteins with a predicted localisation to the apoplast made up a major proportion of stress-responsive proteins. Acid phosphatases were not identified within extracellular protein samples, which were enriched in proteins with predicted extracellular localisation. The absence of extracellular APases was further validated by multiple reaction monitoring. Our data indicates that wheat acid phosphatases play a minor role in P mobilisation beyond the immediate root surface and provides a resource for breeding strategies and further investigations of the functional roles of root tip-released proteins in the rhizosphere under P limitation.

Impact of zinc and plant growth-promoting bacteria on soil health as well as aboveground biomass of desi and kabuli chickpea under arid conditions.

Zinc (Zn) deficiency and low soil fertility are the major factors responsible for low yield in chickpea. This study was conducted to evaluate the effect of Zn application and plant growth-promoting bacteria (PGPB) (endophyte Enterobacter sp. MN17) on soil health and aboveground biomass of desi and kabuli chickpea under natural field conditions. Zn was applied as seed priming (0.001 mol L-1 ) and soil application (10 kg Zn ha-1 ) with and without PGPB. To determine the impacts of Zn and PGPB on soil biological health, soil microbial biomass carbon (MBC) and soil extracellular enzyme activities were analyzed at two growth stages: vegetative (90 days after sowing) and maturity (163 days after sowing). The highest aboveground biomass (5.1 t ha-1 ) was recorded with Zn seed priming + PGPB in kabuli chickpea and in desi chickpea (4.8 t ha-1 ) with Zn seed priming only. The application of Zn significantly increased soil MBC, which was higher in kabuli (795 and 731 μg C g-1 ) compared to desi chickpea (655 and 533 μg C g-1 ) at both vegetative and reproductive growth stages, respectively. The highest extracellular soil enzyme activities, - β-glucosidase (4758 nmol g-1 h-1 ), acid phosphatase (5508 nmol g-1 h-1 ), chitinase (5997 nmol g-1 h-1 ) and leucine aminopeptidase (993 nmol g-1 h-1 ) - were recorded with Zn seed priming. Of the chickpea types, kabuli chickpea had higher soil extracellular enzyme activities in the rhizosphere than desi chickpea. Zn seed priming along with PGPB application may improve soil health and chickpea biomass in marginal soils. © 2021 Society of Chemical Industry.

Potato root and leaf phosphatase activity in response to P deprivation

AbstractBackgroundPlants are not able to take up organically bound phosphorus (P) before it is hydrolyzed by extracellular phosphatases while intracellular phosphatases play a role in P remobilization.AimsThe aim of this study was to evaluate intra‐ and extracellular acid phosphatase activity as well as root and shoot growth of two modern starch potato cultivars at different levels of P deprivation.MethodsIn vitro propagated potato plantlets (cultivars Kuba and Cardoso) were grown in sand and fertigated with five different P concentrations, ranging from 1 to 0.05 mM P. Growth parameters, plant P concentrations, and acid phosphatase activities were determined after 40 days.ResultsShoot and root biomass decreased gradually with decreasing P supply, while the root‐to‐shoot ratio increased. Shoot P concentration decreased steadily, but root P concentration remained constant at P levels below 0.5 mM. Root‐associated and root intracellular acid phosphatase activity increased in both cultivars with rising levels of P deprivation. The activity of acid phosphatases in old but not in young leaves was increased with P deprivation. Intracellular acid phosphatase activity was genotype dependent, with higher activity in the cultivar Kuba than in Cardoso.ConclusionsOur results point to a tissue specific regulation of acid phosphatase activity depending on plant P status and potato genotype. Increasing root‐associated acid phosphatase activities may help P starved potato plants to ensure a sufficient P nutrition. Further research is needed to unravel the roles of leaf intracellular acid phosphatases in potato.

Characterization of Phosphate Solubilizing Bacterial Endophytes and Plant Growth Promotion In Vitro and in Greenhouse.

Phosphate is one of the most important nutrients for plant growth and development, and only 0.1% of the phosphate in soils is available to plants. Currently, the use of excess phosphate fertilizer has caused surface and ground water pollution and water eutrophication, resulting in algal blooms in lakes and oceans. Therefore, it is imperative to explore alternative ways to solve these problems for sustainable agricultural production and improvement of soil fertility, while protecting the environment. Microorganisms from the rhizosphere and within plants are able to solubilize insoluble soil phosphate, making it available to plants. Five high phosphate solubilizing bacteria from our bacterial endophyte library were chosen for this study and identified as Pantoea vagans IALR611, Pseudomonas psychrotolerans IALR632, Bacillus subtilis IALR1033, Bacillus safensis IALR1035 and Pantoea agglomerans IALR1325. All five bacteria significantly promoted tall fescue growth in vitro. Greenhouse experiments showed that IALR1325 significantly promoted pepper and tomato growth, and IALR632 was the best in promoting tomato growth. In addition, all these bacteria had extracellular acid phosphatase and phytase activities. One of the mechanisms for phosphate solubilization by bacteria is pH reduction caused by gluconic acid production. Our results indicate that P. agglomerans IALR1325 is a promising bacterium for future applications.

Phosphorus Deficiency Promoted Hydrolysis of Organophosphate Esters in Plants: Mechanisms and Transformation Pathways.

The biotransformation of organophosphate esters (OPEs) in white lupin (Lupinus albus) and wheat (Triticum aestivum L.) was investigated in hydroponic experiments with different phosphorus (P)-containing conditions. The hydrolysis rates of OPEs followed the order of triphenyl phosphate (TPHP) > tri-n-butyl phosphate (TnBP) > tris(1,3-dichloro-2-propyl) phosphate (TDCPP). Hydrolysis of OPEs was accelerated at P-deficient conditions, and faster hydrolysis took place in white lupin than in wheat. Coincidingly, the production of acid phosphatase (ACP) in both plants was promoted, and much higher intracellular and extracellular ACPs were observed in white lupin under P-deficient conditions. In vitro experiments revealed that ACP was a key enzyme to hydrolyze OPEs. The hydrolysis rates of OPEs were significantly correlated with the Hirshfeld charges, calculated by density functional theory, of the oxygen atom in the single P-O bond. Using ultra-high-performance liquid chromatography coupled with Orbitrap Fusion mass spectrometer, 30 metabolites were successfully identified. Some of these metabolites, such as sulfate-conjugated products, hydration of cysteine-conjugated products of TPHP, and reductively dechlorinated metabolites of TDCPP, were observed for the first time in plants. It is noteworthy that OPEs may transform into many hydroxylated metabolites, and special attention should be paid to their potential environmental effects.

Responses of maize yield, nitrogen and phosphorus runoff losses and soil properties to biochar and organic fertilizer application in a light-loamy fluvo-aquic soil

Nitrogen (N) and phosphorus (P) are the primary nutrients to the promoting of crop yield, however, the losses of N and P by runoff dominated the non-point pollution. In the context of maintaining high farmland productivity and mitigating negative impacts on the environment, it is important to understand how biochar and organic fertilizer application influence maize yield, N and P use efficiency and losses by runoff, as well as soil quality. To address this, the current study was conducted based on a three-year field experiment that includes five fertilized treatments: No fertilizer (CK), chemical fertilizer (CF), CF + biochar (CFB), 20% chemical N was replaced by organic fertilizer (OF) and OF + biochar (OFB). The amendments of biochar and organic fertilizer significantly increased maize yield, N and P use efficiency, and meanwhile, decreased the N and P losses by runoff. From 2017 to 2019, the yield of OFB treatment was 4.4–161.9% higher than that of other treatments, The N and P use efficiencies of OFB treatment was 3.0–83.7% and 9.9–195.1% higher than that of other treatments respectively. N losses by runoff were decreased by 35.1–59.7% in OFB than CF, and −0.6–42.0% in OFB than other fertilized treatments. P losses by runoff were decreased by 39.9–48.8% in OFB than CF, and 5.6–32.0% in OFB than other fertilized treatments in 2018 and 2019. Moreover, biochar amendment significantly improved soil aggregate stability, and increased extracellular urease and acid phosphatase activities. Soil alkaline hydrolysable nitrogen and available potassium contents were significantly increased by the biochar amendment, but no significant impact on soil available P. These findings indicate that the integrated use of chemical fertilizer, organic fertilizer and biochar has the potential to maintain high grain yield with mitigating environmental impact.

A Previously Undescribed Helotialean Fungus That Is Superabundant in Soil Under Maritime Antarctic Higher Plants.

We report a previously undescribed member of the Helotiales that is superabundant in soils at two maritime Antarctic islands under Antarctic Hairgrass (Deschampsia antarctica Desv.). High throughput sequencing showed that up to 92% of DNA reads, and 68% of RNA reads, in soils from the islands were accounted for by the fungus. Sequencing of the large subunit region of ribosomal (r)DNA places the fungus close to the Pezizellaceae, Porodiplodiaceae, and Sclerotiniaceae, with analyses of internal transcribed spacer regions of rDNA indicating that it has affinities to previously unnamed soil and root fungi from alpine, cool temperate and Low Arctic regions. The fungus was found to be most frequent in soils containing C aged to 1,000–1,200 years before present. The relative abundances of its DNA and RNA reads were positively associated with soil carbon and nitrogen concentrations and δ13C values, with the relative abundance of its DNA being negatively associated with soil pH value. An isolate of the fungus produces flask-shaped phialides with a pronounced venter bearing masses of conidia measuring 4.5–6(7) × 1.8–2.5 μm, suggestive of anamorphic Chalara. Enzymatic studies indicate that the isolate strongly synthesizes the extracellular enzyme acid phosphatase, and also exhibits alkaline phosphatase and naphthol-AS-BI-phosphohydrolase activities. Ecophysiological measurements indicate optimal hyphal growth of the isolate at a pH of 4.2–4.5 and a water potential of −0.66 MPa. The isolate is a psychrotroph, exhibiting measureable hyphal growth at −2°C, optimal hyphal extension rate at 15°C and negligible growth at 25°C. It is proposed that the rising temperatures that are predicted to occur in maritime Antarctica later this century will increase the growth rate of the fungus, with the potential loss of ancient C from soils. Analyses using the GlobalFungi Database indicate that the fungus is present in cold, acidic soils on all continents. We advocate further studies to identify whether it is superabundant in soils under D. antarctica elsewhere in maritime Antarctica, and for further isolates to be obtained so that the species can be formally described.

Degradation of diclofenac by new bacterial strains and its influence on the physiological status of cells

Diclofenac (DCF) is one of the most commonly utilized non-steroidal anti-inflammatory drugs (NSAIDs), which is known to pose an ecotoxicological threat. In this study, from activated sludge and contaminated soil, we isolated four new bacterial strains able to degrade DCF under mono-substrate and co-metabolic conditions with glucose supplementation. We found that the effectiveness of DCF removal is strictly strain-specific and the addition of the primary substrate is not always beneficial. To assess the multidirectional influence of DCF on bacterial cells we evaluated the alterations of increasing concentrations of this drug on membrane structure. A significant increase was observed in the content of 17:0 cyclo fatty acid, which is responsible for reduced fluidity and profound changes in membrane rigidity. The cell injury and oxidative stress were assessed with biomarkers used as endpoints of toxicity, i.e. catalase (CAT), superoxide dismutase (SOD), lipids peroxidation (LPX), and both intra- and extracellular alkaline and acid phosphatase activity. Results indicated that DCF induced oxidative stress, frequently intensified by the addition of glucose. However, the response of the microbial cells to the presence of DCF should not be generalized, since the overall picture of the particular alterations greatly varied for each of the examined strains.

ATP as Phosphorus and Nitrogen Source for Nutrient Uptake by Fagus sylvatica and Populus x canescens Roots.

The present study elucidated whether roots of temperate forest trees can take up organic phosphorus in the form of ATP. Detached non-mycorrhizal roots of beech (Fagus sylvatica) and gray poplar (Populus x canescens) were exposed under controlled conditions to 33P-ATP and/or 13C/15N labeled ATP in the presence and absence of the acid phosphatase inhibitor MoO42-. Accumulation of the respective label in the roots was used to calculate 33P, 13C and 15N uptake rates in ATP equivalents for comparison reason. The present data shown that a significant part of ATP was cleaved outside the roots before phosphate (Pi) was taken up. Furthermore, nucleotide uptake seems more reasonable after cleavage of at least one Pi unit as ADP, AMP and/or as the nucleoside adenosine. Similar results were obtained when still attached mycorrhizal roots of adult beech trees and their natural regeneration of two forest stands were exposed to ATP in the presence or absence of MoO42-. Cleavage of Pi from ATP by enzymes commonly present in the rhizosphere, such as extracellular acid phosphatases, ecto-apyrase and/or nucleotidases, prior ADP/AMP/adenosine uptake is highly probable but depended on the soil type and the pH of the soil solution. Although uptake of ATP/ADP/AMP cannot be excluded, uptake of the nucleoside adenosine without breakdown into its constituents ribose and adenine is highly evident. Based on the 33P, 13C, and 15N uptake rates calculated as equivalents of ATP the ‘pro and contra’ for the uptake of nucleotides and nucleosides is discussed.Short SummaryRoots take up phosphorus from ATP as Pi after cleavage but might also take up ADP and/or AMP by yet unknown nucleotide transporter(s) because at least the nucleoside adenosine as N source is taken up without cleavage into its constituents ribose and adenine.

Measurement of Acid Ecto-phosphatase Activity in Live Leishmania donovani Parasites

Acid ecto-phosphatases are enzymes that hydrolyze phosphomonoesters in the acidic pH range with their active sites facing the extacellular medium. Their activities can be measured in living cells. In bacteria and protozoan pathogens, acid ecto-phosphatases have been associated with the survival of intracellular pathogens within phagocytes through inhibition of the respiratory burst, suggesting that they act as virulence factors. Extracellular acid phosphatase activity in Leishmania (L.) donovani has been associated with the degree of promastigote virulence/infectivity. The levels of acid ecto-phosphatase activity in different Leishmania sp or even strains of the same species vary and this has been linked to their virulence. It may also be related to their ability to survive and multiply in the insect host. Acid phosphatase enzymatic activity can be measured in crude membrane fractions and in membrane fractions enriched in plasma membrane, however, in these cases, the intracellular acid phosphatases, mainly localized in lysosomes, contribute to the final result. Therefore, measuring phosphatase activity at the surface of live cells in acidic pH range is the only accurate way to measure acid ecto-phosphatase activity. This assay is performed at 25 °C or 37 °C for 30 min using as substrate the generic phosphatase substrate p-nitrophenyl phosphate (pNPP), in a citrate buffer, with or without sodium tartrate (L(+)-tartaric acid), as histidine acid phosphatases are classified according to their sensitivity to tartate inhibition. The steps of the protocol consist of pelleting cells in suspension, in this case Leishmania promastigotes, washing twice with HEPES buffer, resuspending the cells in the substrate reaction mixture and terminating the reaction by the addition of 0.5 N NaOH. The cells are removed by centrifugation and the absorbance of the reaction product (p-nitrophenolate=pNP) in the supernatant is measured at 405 nm. The enzymatic activity (A405 values) is normalized for the mean number of cells/ml used for each independent experiment.

How understory vegetation affects the catalytic properties of soil extracellular hydrolases in a Chinese fir (Cunninghamia lanceolata) forest

To study how the understory vegetation influences the catalytic properties of extracellular hydrolases, we established a paired treatment experiment, with understory vegetation and litter removed from one treatment (understory removal, UR) and litter removed and the understory vegetation left intact in the other (control, CK), in a subtropical Chinese fir (Cunninghamia lanceolata) plantation. We used fluorescent substrates to determine the maximum velocity (Vmax), the affinity of hydrolases to the substrates (Km), and the catalytic efficiency (Vmax/Km) of three extracellular hydrolases (β-1,4-glucosidase (βG), β-1,4-N-acetylglucosaminidase (NAG) and acid phosphatase (AP)). We found that the Vmax values for βG and NAG were 23.0% and 16.8% lower, respectively, in the UR treatment than in the CK treatment, and that the Vmax for AP was similar in both treatments. The Km and Vmax/Km for all the three hydrolases remained steady after the understory vegetation was removed. The soil C and N contents and the bacterial and fungal biomass were generally positively correlated with the Vmax values. Thus, understory vegetation had more influence on the activities (Vmax) than on the substrate affinities of extracellular hydrolases (Km). Microbes tended to concentrate on maintaining the catalytic efficiency when the SOC contents were between 15.8 and 20.3 g kg−1 when the understory vegetation was removed from a subtropical Chinese fir forest, and the catalytic efficiency could decrease under extremely low soil C contents. We suggest that understory vegetation should be maintained to sustain the potential microbial activity in subtropical Chinese fir forests.

Mechanisms of oat (Avena sativa L.) acclimation to phosphate deficiency.

BackgroundDeficiency of available forms of phosphorus is common in most soils and causes reduction of crop plants growth and yield. Recently, model plants responses to phosphate (Pi) deficiency have been intensively studied. However, acclimation mechanisms of cereals like oat (Avena sativa L.), to low Pi stress remains not fully understood. Oat plants have been usually cultured on poor soils, with a low nutrient content, but their responses to such conditions are not well known, therefore the main goal of the study was to investigate the mechanisms that enable oat plants to grow under low Pi conditions.MethodsFour oat cultivars (A. sativa, cv. Arab, Krezus, Rajtar and Szakal) were grown for three weeks in a nutrient media with various P sources: inorganic—KH2PO4 (control), organic—phytate (PA) and with no phosphate (−P). The effects of Pi deficiency on the level of P, oat growth parameters, intensity of photosynthesis, plant productivity, root exudation ability, localization, activity and isoforms of acid phosphatases, enzymes involved in Pi mobilization, were estimated. In addition, the effect of mycorrhization on plant growth was also observed.ResultsAll studied oat cultivars grown on Pi-deficient media had significantly decreased Pi content in the tissues. Pi deficiency caused inhibition of shoot growth, but generally it did not affect root elongation; root diameter was decreased, root/shoot ratios increased, whereas PA plants showed a similar growth to control. Photosynthesis rate and productivity parameters decreased under low Pi nutrition, however, sugar content generally increased. Studied oat cultivars did not respond to low Pi via increased exudation of carboxylates from the roots, as pH changes in the growth media were not observed. Pi starvation significantly increased the activity of extracellular and intracellular acid phosphatases (APases) in comparison to the control plants. Three major APase isoforms were detected in oat tissues and the isoform pattern was similar in all studied conditions, usually with a higher level of one of the isoforms under Pi starvation. Generally no significant effects of mycorrhizal colonization on growth of oat cultivars were observed.DiscussionWe postulated that acid phosphatases played the most important role in oat cultivars acclimation to Pi deficiency, especially extracellular enzymes involved in Pi acquisition from soil organic P esters. These APases are mainly located in the epidermis of young roots, and may be released to the rhizosphere. On the other hand, intracellular APases could be involved in fast Pi remobilization from internal sources. Our study showed that oat, in contrast to other plants, can use phytates as the sole source of P. The studied oat cultivars demonstrated similar acclimation mechanisms to Pi deficiency, however, depending on stress level, they can use different pools of acid phosphatases.

Acid Phosphatases Activity and Growth of Barley, Oat, Rye and Wheat Plants as Affected by Pi Deficiency

Objective: The influence of phosphorus deficit on the growth of plants and acid phosphatases activity in leaves and roots of barley seedlings (Hordeum vulgare L.), as well as oat (Avena sativa L.), rye (Secale cereale L.) and wheat plants (Triticum vulgare L.) was studied. Method: Plants were cultured three weeks in a nutrient media: complete (control, +P) or without phosphorus (-P). The growth on -P medium significantly affected the inorganic phosphate (Pi) content in plants tissues. Pi deficit decreased shoots growth but ratio of root/shoot was higher for -P plants when compared to control. The root elongation was enhanced under Pi deficiency - in -P oat and barley more intensive elongation was observed than in other plants. On the other hand, inhibition of shoot growth was more pronounced for -P rye and wheat. Pi-deficient plants showed higher activity of acid phosphatases in tissue extracts and in exudates from roots than +P plants. Result: Extracellular acid phosphatases activity increased the most for -P rye and wheat plants. Acid phosphatases secretion was intensive in growing parts of Pi-deficient roots. The activity of enzymes secreted by -P roots of all studied plants was higher than intracellular acid phosphatases. Conclusion: Our results indicated that wheat is more sensitive to the Pi deficiency at the early stage of growth than other plants, whereas oat is rather resistant to Pi deficit. The results suggested that acid phosphatases played an important role in acclimation of studied crop plants to moderate Pi deficiency.

Increased amount of phosphorylated proinflammatory osteopontin in rheumatoid arthritis synovia is associated to decreased tartrate-resistant acid phosphatase 5B/5A ratio.

BackgroundOsteopontin (OPN) is an immunoregulatory protein which production increases in both rheumatoid arthritis (RA) and osteoarthritis (OA). Phosphorylated osteopontin (Phospho-OPN) is known to increase macrophage and osteoclast activation, this process is controlled by extracellular tartrate-resistant acid phosphatase (TRAcP), also a biomarker for RA. Here, we evaluated the phosphorylation status of OPN in RA and OA synovia, as well as its correlation with TRAcP isoforms.MethodsSynovial tissue and fluid were obtained from 24 RA (14 seropositive and 10 seronegative) and 24 OA patients. Western blotting was used to analyze the extent of OPN phosphorylation. TRAcP isoforms were measured in synovial fluid using ELISA; immunohistochemistry assessed the distribution of OPN and TRAcP expressing cells in the synovial tissue, especially distinguishing between the TRAcP isoforms.ResultsFull-length OPN was more phosphorylated in RA than in OA (p<0.05). The thrombin cleaved C-terminal end of OPN was also more phosphorylated in RA (p<0.05). RA patients had a lower concentration of TRAcP 5B and higher concentration of less active 5A in their synovial fluid compared to OA patients. The TRAcP 5B/5A ratio was decreased in RA and correlated negatively with the amount of phospho-OPN (p<0.05). TRAcP positive cells for both isoforms were found all along the synovial lining; OPN antibody staining was localized in the extracellular matrix.ConclusionOur data suggests that in RA the synovial fluid contains insufficient amounts of TRAcP 5B which increase levels of the proinflammatory phospho-OPN. This may lead to increased macrophage and osteoclast activation, resulting in the increased local inflammation and bone resorption present in RA joints.