Effects of fluorine on crops, soil exoenzyme activities, and earthworms in terrestrial ecosystems.

Fluorine can flow into the environment after leakage or spill accidents and these excessive amounts can cause adverse effects on terrestrial ecosystems. Using three media (filter paper, soil, and filter-paper-on-soil), we investigated the toxic effects of fluorine on the germination and growth of crops (barley, mung bean, sorghum, and wheat), on the activities of soil exoenzymes (acid phosphatase, arylsulfatase, fluorescein diacetate hydrolase, and urease) and on the survival, abnormality, and cytotoxicity of Eisenia andrei earthworms. The germination and growth of crops were affected by fluorine as exposure concentration increased. The activities of the four enzymes after 0-, 3-, 10-, and 20-day periods varied as exposure concentration increased. According to in vivo and in vitro earthworm assays, E. andrei mortality, abnormality, and cytotoxicity increased with increasing fluorine concentration. Overall, fluorine significantly affected each tested species in the concentration ranges used in this study. The activities of soil exoenzymes were also affected by soil fluorine concentration, although in an inconsistent manner. Albeit the abnormally high concentrations of fluorine in soil compared to that observed under natural conditions, its toxicity was much restrained possibly due to the adsorption of fluorine on soil particles and its combination with soil cations.

High-throughput estimation of specific activities of enzyme/mutants in cell lysates through immunoturbidimetric assay of proteins.

High-throughput estimation of specific activities of an enzyme and its mutants in a group (enzyme/mutants) in cell lysates via high-throughput assay of their activities and separate immunoturbidimetric assay (ITA) of their proteins was proposed. Pseudomonas aeruginosa arylsulfatase (PAAS) and Bacillus fastidious uricase (BFU) served as two models. ITA employed 0.75 mg of antisera against PAAS or BFU as the reference in 96-well microplates to measure the difference of extinction at 340 and 700 nm. According to the calibration curve, ITA quantified the reference from 0.40 to about 2.4 µg. The consistency among the abundance of enzyme/mutants through ITA of proteins in cell lysates prepared under the same conditions supported their consistent immunological reactivity to the antisera. Specific activities of PAAS/mutants or BFU/mutants in cell lysates through ITA of proteins showed excellent proportionality to those carefully determined after purification. Receiver-operating-characteristic (ROC) analysis of specific activities through ITA of proteins gave a higher area-under-curve than those for ROC analyses of other activity indices, which allowed the recognition of a PAAS/mutant of 50% higher activity after cell amplification in high-throughput mode. Therefore, ITA of enzyme/mutants as proteins is promising to estimate their specific activities in cell lysates in high-throughput mode for quantitative comparison.

Exoenzyme activity in contaminated soils before and after soil washing: ß-glucosidase activity as a biological indicator of soil health.

It is essential to remediate or amend soils contaminated with various heavy metals or pollutants so that the soils may be used again safely. Verifying that the remediated or amended soils meet soil quality standards is an important part of the process. We estimated the activity levels of eight soil exoenzymes (acid phosphatase, arylsulfatase, catalase, dehydrogenase, fluorescein diacetate hydrolase, protease, urease, and ß-glucosidase) in contaminated and remediated soils from two sites near a non-ferrous metal smelter, using colorimetric and titrimetric determination methods. Our results provided the levels of activity of soil exoenzymes that indicate soil health. Most enzymes showed lower activity levels in remediated soils than in contaminated soils, with the exception of protease and urease, which showed higher activity after remediation in some soils, perhaps due to the limited nutrients available in remediated soils. Soil exoenzymes showed significantly higher activity in soils from one of the sites than from the other, due to improper conditions at the second site, including high pH, poor nutrient levels, and a high proportion of sand in the latter soil. Principal component analysis revealed that ß-glucosidase was the best indicator of soil ecosystem health, among the enzymes evaluated. We recommend using ß-glucosidase enzyme activity as a prior indicator in estimating soil ecosystem health.

A comparative study on the influence of different organic amendments on trace element mobility and microbial functionality of a polluted mine soil.

A mine soil heavily polluted with zinc and cadmium was employed to evaluate the capacity of organic amendments of different origin to simultaneously reduce soil trace element mobility and enhance soil microbial functionality. With this aim, four organic products, namely olive processing solid waste (OPSW), municipal solid waste compost (MSWC), leonardite and peat, were applied individually at different doses (0, 1, 2 and 5%) to mine soil under controlled laboratory conditions. Extraction studies and analysis of soil microbiological parameters (basal soil respiration and dehydrogenase, ß-glucosidase, urease, arylsulfatase and acid and alkaline phosphatase activities) were performed to assess the effect of such amendments on soil restoration. Their ability to decrease mine soil mobile trace element contents followed the sequence MSWC > OPSW > peat > leonardite, with the former achieving reduction levels of 78 and 73% for Zn and Cd, respectively, when applied at a dose of 5%. This amendment also showed a good performance to restore soil microbial functionality. Thus, basal soil respiration and dehydrogenase, urease and alkaline phosphatase activities experienced increases of 187, 79, 42 and 26%, respectively, when mine soil was treated with 5% MSWC. Among tested organic products, MSWC proved to be the best amendment to perform both the chemical and the microbial soil remediation.

Detection, production, and application of microbial arylsulfatases.

Arylsulfatases are enzymes which catalyze the hydrolysis of arylsulfate ester bonds to release a free sulfonate. They are widespread in nature and are found in microorganisms, most animal and human tissues, and plant seeds. However, this review focuses on arylsulfatases from microbial origin and gives an overview of different assays and substrates used to determine the arylsulfatase activity. Furthermore, the production of microbial arylsulfatases using wild-type organisms as well as the recombinant production using Escherichia coli and Kluyveromyces lactis as expression hosts is discussed. Finally, various potential applications of these enzymes are reviewed.

Microbiological functioning, diversity, and structure of bacterial communities in ultramafic soils from a tropical savanna.

Ultramafic soils are characterized by high levels of metals, and have been studied because of their geochemistry and its relation to their biological component. This study evaluated soil microbiological functioning (SMF), richness, diversity, and structure of bacterial communities from two ultramafic soils and from a non-ultramafic soil in the Brazilian Cerrado, a tropical savanna. SMF was represented according to simultaneous analysis of microbial biomass C (MBC) and activities of the enzymes ß-glucosidase, acid phosphomonoesterase and arylsulfatase, linked to the C, P and S cycles. Bacterial community diversity and structure were studied by sequencing of 16S rRNA gene clone libraries. MBC and enzyme activities were not affected by high Ni contents. Changes in SMF were more related to the organic matter content of soils (SOM) than to their available Ni. Phylogeny-based methods detected qualitative and quantitative differences in pairwise comparisons of bacterial community structures of the three sites. However, no correlations between community structure differences and SOM or SMF were detected. We believe this work presents benchmark information on SMF, diversity, and structure of bacterial communities for a unique type of environment within the Cerrado biome.

Impact of PAH on biological health parameters of soils of an Indian refinery and adjoining agricultural area--a case study.

The present study is aimed at analysing and comparing different soil enzymes in soil samples of native contaminated sites of a Mathura refinery and adjoining agricultural land. Enzyme activities are considered as indicators of soil quality and changes in biogeochemical function due to management or perturbations. Soil samples were collected from the premises and nearby area of Mathura refinery, India. Biological health parameters (dehydrogenase, aryl esterase, aryl sulphatase, [Formula: see text]-glucosidase, alkaline phosphatase, acid phosphatase, lipase, laccase and catalase activity) were estimated in the soil samples. Among all the samples, sewage sludge soil showed maximum activity of enzymes, microbial biomass carbon and most probable number of polycyclic aromatic hydrocarbon (PAH) degraders in soils spiked with three- to four-ring PAHs at 50 ppm. Available phosphorus, potassium and nitrogen was also exceptionally high in this sample, indicating maximum microbial bioconversion due to presence of nutrients stimulating potent PAH-degrading microorganisms.

The effects of high metal concentrations in soil-compost mixtures on soil enzymes.

The study was undertaken to determine the impact of high-metal composts on the activities of four soil enzymes. High concentrations of metal salts (Cr, Cu, Ni or a Co-Mo-Pb combination) were added to feedstocks during the thermophilic stage of composting. These four metal-enriched composts and an unamended control compost were then mixed with soil collected from long-term agriculture plots under organic management or conventional management. The compost-soil mixtures were prepared at two rates (1:1 or 1:3 compost:soil, v/v) and incubated at 20 degrees C for three weeks. These 20 combinations plus the five composts and the two soils were added to pots and incubated for three weeks. Following incubation, soil enzyme activities (acid phosphatase, arysulfatase, dehydrogenase, phosphodiesterase) were measured using traditional assay procedures. Compared to the control, none of the high-metal composts inhibited soil enzyme activity. Notably, the Cu compost treatment produced significantly higher activity of all four enzymes in the soil compared to the control. Previous soil management influenced the activity of three enzymes, arysulfatase and dehydrogenase had greater activity in the organic soil while phosphatase activity was greater in the conventional soil. Increasing the proportion of compost in the pot had a positive effect on phosphodiesterase activity only. In conclusion, the high-metal compost treatments either enhanced or caused no adverse effects on soil enzyme activity.

The corpus luteum of the guinea pig. III. Cytochemical studies on the Golgi complex and GERL during normal postpartum regression of luteal cells, emphasizing the origin of lysosomes and autophagic vacuoles.

The postpartum involution of corpora lutea was examined by electron microscope cytochemistry of guinea pig ovaries previously fixed by vascular perfusion, a method which produces optimal preservation of steroid-secreting cells and yet maintains enzyme activity. The intracellular digestive apparatus was identified through the localization of two acid hydrolases, acid phosphatase (ACPase) and arylsulfatase. Other marker enzymes localized were thiamine pyrophosphatase (in Golgi cisternae) and alkaline phosphatase (along plasma membranes). Prolonged osmication was used to mark the outer Golgi cisterna. The results demonstrate that luteal cell regression is characterized by a striking increase in the number of lysosomes and the appearance of numerous, double-walled autophagic vacuoles. Both lysosomes and the space between the double walls of autophagic vacuoles exhibit ACPase and arylsulfatase activity. In contrast to earlier periods, just before and during regression, Golgi complex-endoplasmic reticulum-lysosomes (GERL) is markedly hypertrophied, displaying intense acid hydrolase activity. On the basis of various criteria, GERL is proposed to function in the formation of lysosomes and autophagic vacuoles. Lysosomes seem to develop from GERL as focal protuberances of varying size and shape, which detach from the parent structure. Double-walled autophagic vacuoles, often large and complex in structure, initially are produced as GERL cisternae envelop small areas of cytoplasm. Lytic enzymes, perhaps furnished by the engulfing membranes and trapped lysosomes, presumably bring about digestion of the contents of these vacuoles, producing first aggregate-type inclusions, then, as the contents are further degraded, myelin figure-filled residual bodies. ACPase activity occasionally appears within smooth endoplasmic reticulum tubules and cisternae in advanced regression, possibly suggesting that lytic enzymes utilize this membrane system as an access route to GERL. These data indicate that cellular autophagy is a prominent mechanism underlying luteal cell involution during normal postpartum degeneration of guinea pig corpora lutea. Furthermore they suggest that in regressing luteal cells GERL is responsible for packaging acid hydrolases into lytic bodies.

Enrichment and isolation of endosulfan-degrading microorganism from tropical acid soil.

Endosulfan (6,7,8,9,10,10-hexachloro-1,5,5a,6,9,9a-hexahydro-6,9-methano-2,3,4-benzo-dioxathiepin-3-oxide) is a cyclodiene organochlorine currently used as an insecticide all over the world and its residues are posing a serious environmental threat. This study reports the enrichment and isolation of a microbial culture capable of degrading endosulfan with minimal production of endosulfan sulfate, the toxic metabolite of endosulfan, from tropical acid soil. Enrichment was achieved by using the insecticide as sole sulfur source. The enriched microbial culture, SKL-1, later identified as Pseudomonas aeruginosa, degraded up to 50.25 and 69.77 % of alpha and beta endosulfan, respectively in 20 days. Percentage of bioformation of endosulfan sulfate to total formation was 2.12% by the 20th day of incubation. Degradation of the insecticide was concomitant with bacterial growth reaching up to an optical density of 600 nm (OD600) 2.34 and aryl sulfatase activity of the broth reaching up to 23.93 microg pNP/mL/hr. The results of this study suggest that this novel strain is a valuable source of potent endosulfan-degrading enzymes for use in enzymatic bioremediation. Further, the increase in aryl sulfatase activity of the broth with the increase in degradation of endosulfan suggests the probable involvement of the enzyme in the transformation of endosulfan to its metabolites.

Ultrastructural localization of Trypanosoma cruzi lysosomes by aryl sulphatase cytochemistry.

Lysosomes of trypanosomatid protozoa are poorly known. In this work we have cytochemically detected the lysosomal enzyme aryl sulphatase in the trypanosomatids Trypanosoma cruzi and Crithidia fasciculata, by using p-nitrocatecholsulphate as substrate. Positive reaction was located exclusively inside membrane-bound cytoplasmic vesicles distributed throughout the cell body. Electron-dense reaction was either dispersed homogeneously through the vesicular matrix or located at the vesicle periphery, apposed to the membrane, with fine granular deposits occasionally found at the vesicular matrix. Trypomastigote and epimastigote forms of T. cruzi lacked electron-dense deposits at the plasma membrane, thus indicating that aryl sulphatase was not secreted to the environment. Furthermore, no positive reaction was detected in epimastigote reservosomes, which are organelles considered as pre-lysosomal compartments. Thus, our data show that reservosomes and lysosomes are organelles that can be distinguished by the cytochemical localization of aryl sulphatase in T. cruzi epimastigotes and trypomastigotes. Positive reaction in cytoplasmic vesicles of C. fasciculata choanomastigotes confirmed the specificity of the reaction for lysosomes in other trypanosomatid species.

Elevated activities and properties of arylsulfatases A and B and B-variant in human lung tumors.

The activities of arylsulfatases A and B were determined in human primary and secondary tumor tissues (total, 53 cases) of various histological types. Significantly higher activities of these sulfatases were found in almost all the primary lung carcinomas as compared to their corresponding uninvolved tissues. No significant correlation was demonstrated between the enzyme activities and histological figures (stroma amounts, etc.). Lung adenocarcinoma and squamous cell carcinoma showed the presence of an additional arylsulfatase component (B1) which was not detected in normal human lung. The tumor arylsulfatase B1 had an isoelectric point (pI) of 6.7 and was clearly distinguished from arylsulfatase A (pI 4.9) and arylsulfatase B (pI 9.1 to 9.2) in normal lung and lung tumor. The tumor B1 enzyme was demonstrated to be most probably an isoenzyme of arylsulfatase B, since this unusual enzyme was indistinguishable from arylsulfatase B in terms of Ag+ inhibition; its kinetic parameters of Km for p-nitrocatechol sulfate, which was 2.9 mM with B1; optimum pH of 6.3 for B1; heat stability; and substrate specificity for three synthetic and two physiological substrates.

Purification, properties and glycoprotein nature of arylsulfatase A from sheep brain.

A simple and rapid method for the purification of arylsulfatase A (EC 3.1.6.1) from sheep brain has been developed. This includes the concanavalin A-Sepharose affinity chromatography and the pH-dependent polymerization and depolymerization of the enzyme. By these methods a homogeneous enzyme was obtained and the enzyme was purified 7180-fold. Sheep brain arylsulfatase A has been shown to be a glycoprotein containing 25% neutral sugar and 0.5% sialic acid. The constituent neutral sugars were identified as glucose and mannose.

Cholesterol sulfate is not degraded but does not accumulate in Epstein-Barr virus-transformed lymphoid cells from patients with X-linked ichthyosis.

The metabolism of cholesterol sulfate (CS) was investigated in immortalized, Epstein-Barr virus-transformed lymphoid cell lines derived from normal individuals and patients affected with recessive X-linked ichthyosis (XLI). Normal lymphoid cells expressed arylsulfatase C and steroid sulfatase (including cholesterol sulfatase) activities, and these two sulfohydrolases showed the same enzyme properties as in other human cells, e.g., leukocytes or skin fibroblasts. XLI-derived lymphoid cell lines exhibited extremely deficient activity of both arylsulfatase C and steroid sulfatase. While normal and XLI intact, living lymphoid cells could take up exogenous radiolabelled CS through a non-receptor-mediated process. XLI cells were completely unable to degrade CS to cholesterol. However, despite their defect in CS degradation, steroid sulfatase-deficient cells did not accumulate CS because of outflux of this sterol. The potential implications of these findings to the pathogenesis of increased CS content in plasma and epidermis of XLI patients are discussed. This study also demonstrates that immortalized lymphoid cell lines may represent a useful experimental model system for the study of XLI.

Microbial and enzymatic activity of soil contaminated with a mixture of diflufenican + mesosulfuron-methyl + iodosulfuron-methyl-sodium.

The aim of this study was to determine the effect of three active substances, diflufenican, mesosulfuron-methyl and iodosulfuron-methyl-sodium, applied in combination, on soil microbial counts, the structure of soil microbial communities, activity of soil enzymes and their resistance to the tested product, the biochemical indicator of soil fertility, and spring wheat yield. Soil samples with the granulometric composition of sandy loam with pHKCl 7.0 were used in a pot experiment. The herbicide was applied to soil at seven doses: 0.057 (dose recommended by the manufacturer), 1.140, 2.280, 4.560, 9.120, 18.240 and 36.480 mg kg(-1) soil DM. Uncontaminated soil served as the control treatment. It was found that a mixture of the tested active substances increased the counts of total oligotrophic bacteria and spore-forming oligotrophic bacteria, organotrophic bacteria and actinomycetes, decreased the counts of Azotobacter and fungi, and modified the structure of soil microbial communities. The highest values of the colony development (CD) index and the ecophysiological (EP) index were observed in fungi and organotrophic bacteria, respectively. The herbicide applied in the recommended dose stimulated the activity of catalase, urease and acid phosphatase, but it had no effect on the activity of dehydrogenases, alkaline phosphatase, arylsulfatase and ß-glucosidase. The highest dose of the analyzed substances (36.480 mg kg(-1)) significantly inhibited the activity of dehydrogenases, acid phosphatase, alkaline phosphatase and arylsulfatase. The values of the biochemical soil fertility indicator (BA21) decreased in response to high doses of the herbicide. Urease was most resistant and dehydrogenases were least resistant to soil contamination with a mixture of diflufenican + mesosulfuron-methyl + iodosulfuron-methyl-sodium. The analyzed herbicide had an adverse influence on spring wheat yield, and doses of 18.240 and 36.480 mg kg(-1) led to eventual death of plants.

Isolation of cytolytic granules from sea urchin amoebocytes.

Calcium-dependent, heat-labile lytic activity was detected in whole Paracentrotus lividus coelomocytes. The molecules responsible for this activity were mainly expressed by an enriched amoebocyte population and were contained in cytoplasmic granules which could be isolated by Percoll density gradient centrifugation. The isolated cytoplasmic granules were small (0.1-0.25 micron) organelles containing the lysosomal marker arylsulfatase. The functional implication of our results is that P. lividus amoebocytes represent cytotoxic cells which mediate the killing event by a secretory phenomenon involving the release of cytolytic material from cytoplasmic granules.

Steroid sulfatase in the human hair follicle concentrates in the dermal papilla.

5 alpha-dihydrotestosterone is known to play a crucial part in the regulation of hair growth and in the development of androgenetic alopecia. 5 alpha-dihydrotestosterone is formed locally within the hair follicle from the systemic precursor testosterone by cutaneous steroid 5 alpha-reductase. Moreover, adrenal steroids such as dehydroepiandrosterone are converted to 5 alpha-dihydrotestosterone by isolated hair follicles, which may provide an additional source of intrafollicular 5 alpha-dihydrotestosterone levels. Elevated urinary dehydroepiandrosterone and serum dehydroepiandrosterone sulfate have been reported to be present in balding young men. These reports suggest that dehydroepiandrosterone sulfate may act as an important endocrine factor in the development of androgenetic alopecia. Hence the question arises whether the dehydroepiandrosterone sulfate can be metabolized within the hair follicles to yield dehydroepiandrosterone by the microsomal enzyme steroid sulfatase, and where steroid sulfatase might be localized. We therefore performed immunostaining for steroid sulfatase on human scalp biopsies as well as analysis of steroid sulfatase enzyme activity in defined compartments of human beard and occipital hair follicles ex vivo. Using both methods steroid sulfatase was primarily detected in the dermal papilla. Steroid sulfatase activity was inhibited by estrone-3-O-sulfamate, a specific inhibitor of steroid sulfatase, in a concentration-dependent way. Furthermore, we show that dermal papillae are able to utilize dehydroepiandrosterone sulfate to produce 5 alpha-dihydrotestosterone, which lends further support to the hypothesis that dehydroepiandrosterone sulfate contributes to androgenetic alopecia and that steroid sulfatase inhibitors could be novel drugs to treat androgen-dependent disorders of the hair follicle such as androgenetic alopecia or hirsutism.

Epidermal steroid sulfatase and cholesterol sulfotransferase are regulated during late gestation in the fetal rat.

Lipids in the stratum corneum (SC) are organized into lamellar membrane unit structures that provide the permeability barrier. Cholesterol sulfate, a SC membrane lipid, is synthesized by cholesterol sulfotransferase (CSTase) in the lower epidermis and hydrolyzed to cholesterol by steroid sulfatase (SSase) in the SC. To determine whether these enzymes are induced during barrier ontogenesis, we examined their activity in epidermis of fetal rats before (gestational day 17), during (day 19), and after (day 21) barrier formation. CSTase activity increased approximately 10-fold between day 17 and day 19, then declined between day 19 and day 21. In contrast, SSase activity reached its peak activity on day 21, increasing >5-fold. Fetal rat skin explants develop a SC and barrier over the same time course in vitro as in utero. Likewise, CSTase and SSase activities during in vitro ontogenesis precisely mirrored those obtained in utero. Moreover, hormones that accelerate barrier ontogenesis (e.g. glucocorticoids, thyroid hormone, and estrogen) accelerated the increase in CSTase and SSase activities during in vitro ontogenesis. mRNA levels of SSase increased in parallel with enzymatic activity, suggesting that these developmental changes are regulated at the genomic level. Finally, addition of exogenous cholesterol sulfate to explants in vitro did not accelerate either SC development or barrier formation. These studies suggest that induction of the cholesterol sulfate cycle enzymes during SC ontogenesis is a component of the fetal epidermal differentiation program and that the synthetic and degradative enzymes of this pathway are differentially regulated.

Lead aspartate, an en bloc contrast stain particularly useful for ultrastructural enzymology.

Lead aspartate is a new en bloc stain for electron microscopy. Its predictable staining depends on chelation that results from the interaction of the two stain components, lead nitrate and aspartic acid, which must be present in a specific ratio. Lead aspartate stain is 0.02 M in lead nitrate and 0.03 M in aspartic acid, adjusted to pH 5.5. Cells or tissues are stained at 60 degrees C for 30 to 60 min. Cells stained en bloc with lead aspartate closely resemble cells stained on grids by lead citrate, except that the former seldom have contamination. En bloc staining with lead aspartate bypasses the grid-staining step so that samples can be viewed and photographed immediately after they are thin-sectioned. The lower pH of the lead aspartate solution allows counterstaining of enzyme reaction products that dissolve in the highly alkaline lead citrate stain. Lead aspartate en bloc staining to enhance contrast should especially benefit studies of ultrastructure requiring a clean and predictably lead stain.

A monoclonal antibody to rat liver arylsulfatase C and its application in immunohistochemistry.

We purified arylsulfatase C from rat liver microsomes and prepared a monoclonal antibody (P42C2) to the purified enzyme. By SDS-PAGE and immunoblotting analysis using P42C2, the molecular weight of the purified enzyme and of the enzyme in liver and kidney microsomes were estimated at 62,000 daltons. P42C2 caused little inhibition of arylsulfatase C activity, and was bound only slightly to liver microsomes. Localization of arylsulfatase C was studied at the light and electron microscopic level by the indirect immunoperoxidase method using P42C2. In rat liver, arylsulfatase C was detected mainly in the hepatocytes, and less frequently in endothelial cells, Kupffer's cells, and Ito's cells. In rat kidney, strong staining was observed in the straight portions of the proximal tubules. The podocytes, interstitial cells, endothelial cells, and epithelial cells of Henle's thin limbs were stained faintly. By electron microscopy, arylsulfatase C was found localized on the membranes of the endoplasmic reticulum and nuclear envelopes in these cells. These immunohistochemical findings agree with the localization demonstrated by an enzyme-histochemical method which we had previously developed.