Microalgae biomass as a renewable biostimulant: meat processing industry effluent treatment, soil health improvement, and plant growth.

Microalgae biomass contributes to effluent bioremediation. It is a concentrated source of nutrients and organic carbon, making it a potential alternative as a soil biostimulant. In this context, this study aimed to evaluate the soil application of microalgae biomass produced from the meat processing industry effluent treatment. The biomass was applied dry and as a mixture to demonstrate its potential to increase plant production and soil metabolic functions, analyzed short-term. Doses of 0.25%, 0.5%, 1%, and 2% biomass were applied in soils from (i) Horizon A: taken at a depth between 0 and 10 cm and; (ii) Horizon B: taken at a depth between 20 and 40 cm. Corn growth (Zea Mays L.), basal soil respiration, microbial biomass carbon, total organic carbon, ß-glucosidase, acid phosphatase, arylsulfatase, and urease enzymatic activity were evaluated in each sample. It is concluded that applying 2% microalgae biomass led to higher basal soil respiration, microbial biomass carbon, and ß-glucosidase, acid phosphatase, arylsulfatase enzymatic activity in both soils. On the other hand, boron may have contributed to urease activity reduction in Soil A. Although 2% biomass led to higher soils characteristics, that dose did not promote higher plant growth. Hence, considering that plant growth must be in line with changes in soil characteristics, the result that provided the higher plant shoot dry matter mass was by applying 0.55% biomass in both soils. Therefore, the application of microalgae biomass produced from a meat processing industry effluent treatment promoted a biologically active soil and boosted plant growth.

Impacts of corn stover management and fertilizer application on soil nutrient availability and enzymatic activity.

Corn stover is a global resource used in many industrial sectors including bioenergy, fuel, and livestock operations. However, stover removal can negatively impact soil nutrient availability, especially nitrogen (N) and phosphorus (P), biological activity, and soil health. We evaluated the effects of corn stover management combined with N and P fertilization on soil quality, using soil chemical (nitrate, ammonium and Bray-1 P) and biological parameters (ß-glucosidase, alkaline phosphatase, arylsulfatase activities and fluorescein diacetate hydrolysis-FDA). The experiment was performed on a Mollisol (Typic Endoaquoll) in a continuous corn system from 2013 to 2015 in Minnesota, USA. The treatments tested included six N rates (0 to 200 kg N ha-1), five P rates (0 to 100 kg P2O5 ha-1), and two residue management strategies (residue removed or incorporated) totalling 60 treatments. Corn stover management significantly impacted soil mineral-N forms and enzyme activity. In general, plots where residue was incorporated were found to have high NH4+ and enzyme activity compared to plots where residue was removed. In contrast, fields where residue was removed showed higher NO3- than plots where residue was incorporated. Residue management had little effect on soil available P. Soil enzyme activity was affected by both nutrient and residue management. In most cases, activity of the enzymes measured in plots where residue was removed frequently showed a positive response to added N and P. In contrast, soil enzyme responses to applied N and P in plots where residue was incorporated were less evident. Soil available nutrients tended to decrease in plots where residue was removed compared with plots where residue was incorporated. In conclusion, stover removal was found to have significant potential to change soil chemical and biological properties and caution should be taken when significant amounts of stover are removed from continuous corn fields. The residue removal could decrease different enzymes related to C-cycle (ß-glucosidase) and soil microbial activity (FDA) over continuous cropping seasons, impairing soil health.

Microbial communities play important roles in modulating paddy soil fertility.

We studied microbial communities in two paddy soils, which did not receive nitrogen fertilization and were distinguished by the soil properties. The two microbial communities differed in the relative abundance of gram-negative bacteria and total microbial biomass. Variability in microbial communities between the two fields was related to the levels of phosphorus and soil moisture. Redundancy analysis for individual soils showed that the bacterial community dynamics in the high-yield soil were significantly correlated with total carbon, moisture, available potassium, and pH, and those in the low-yield cores were shaped by pH, and nitrogen factors. Biolog Eco-plate data showed a more active microbial community in the high yield soil. The variations of enzymatic activities in the two soils were significantly explained by total nitrogen, total potassium, and moisture. The enzymatic variability in the low-yield soil was significantly explained by potassium, available nitrogen, pH, and total carbon, and that in the high-yield soil was partially explained by potassium and moisture. We found the relative abundances of Gram-negative bacteria and Actinomycetes partially explained the spatial and temporal variations of soil enzymatic activities, respectively. The high-yield soil microbes are probably more active to modulate soil fertility for rice production.

Critical function of a Chlamydomonas reinhardtii putative polyphosphate polymerase subunit during nutrient deprivation.

Forward genetics was used to isolate Chlamydomonas reinhardtii mutants with altered abilities to acclimate to sulfur (S) deficiency. The ars76 mutant has a deletion that eliminates several genes, including VACUOLAR TRANSPORTER CHAPERONE1 (VTC1), which encodes a component of a polyphosphate polymerase complex. The ars76 mutant cannot accumulate arylsulfatase protein or mRNA and shows marked alterations in levels of many transcripts encoded by genes induced during S deprivation. The mutant also shows little acidocalcisome formation compared with wild-type, S-deprived cells and dies more rapidly than wild-type cells following exposure to S-, phosphorus-, or nitrogen (N)-deficient conditions. Furthermore, the mutant does not accumulate periplasmic L-amino acid oxidase during N deprivation. Introduction of the VTC1 gene specifically complements the ars76 phenotypes, suggesting that normal acidocalcisome formation in cells deprived of S requires VTC1. Our data also indicate that a deficiency in acidocalcisome function impacts trafficking of periplasmic proteins, which can then feed back on the transcription of the genes encoding these proteins. These results and the reported function of vacuoles in degradation processes suggest a major role of the acidocalcisome in reshaping the cell during acclimation to changing environmental conditions.

Studies on the metabolism of mitragynine, the main alkaloid of the herbal drug Kratom, in rat and human urine using liquid chromatography-linear ion trap mass spectrometry.

Mitragynine (MG) is an indole alkaloid of the Thai medicinal plant Mitragyna speciosa (Kratom in Thai) and reported to have opioid agonistic properties. Because of its stimulant and euphoric effects, Kratom is used as a herbal drug of abuse. The aim of the presented study is to identify the phase I and II metabolites of MG in rat and human urine after solid-phase extraction (SPE) using liquid chromatography-linear ion trap mass spectrometry providing detailed structure information in the MSn mode particularly with high resolution. The seven identified phase I metabolites indicated that MG was metabolized by hydrolysis of the methylester in position 16, O-demethylation of the 9-methoxy group and of the 17-methoxy group, followed, via the intermediate aldehydes, by oxidation to carboxylic acids or reduction to alcohols and combinations of some steps. In rats, four metabolites were additionally conjugated to glucuronides and one to sulfate, but in humans, three metabolites to glucuronides and three to sulfates.

Genetic interactions between regulators of Chlamydomonas phosphorus and sulfur deprivation responses.

The Chlamydomonas reinhardtii PSR1 gene is required for proper acclimation of the cells to phosphorus (P) deficiency. P-starved psr1 mutants show signs of secondary sulfur (S) starvation, exemplified by the synthesis of extracellular arylsulfatase and the accumulation of transcripts encoding proteins involved in S scavenging and assimilation. Epistasis analysis reveals that induction of the S-starvation responses in P-limited psr1 cells requires the regulatory protein kinase SNRK2.1, but bypasses the membrane-targeted activator, SAC1. The inhibitory kinase SNRK2.2 is necessary for repression of S-starvation responses during both nutrient-replete growth and P limitation; arylsulfatase activity and S deficiency-responsive genes are partially induced in the P-deficient snrk2.2 mutants and become fully activated in the P-deficient psr1snrk2.2 double mutant. During P starvation, the sac1snrk2.2 double mutants or the psr1sac1snrk2.2 triple mutants exhibit reduced arylsulfatase activity compared to snrk2.2 or psr1snrk2.2, respectively, but the sac1 mutation has little effect on the abundance of S deficiency-responsive transcripts in these strains, suggesting a post-transcriptional role for SAC1 in elicitation of S-starvation responses. Interestingly, P-starved psr1snrk2.2 cells bleach and die more rapidly than wild-type or psr1 strains, suggesting that activation of S-starvation responses during P deprivation is deleterious to the cell. From these results we infer that (i) P-deficient growth causes some internal S limitation, but the S-deficiency responses are normally inhibited during acclimation to P deprivation; (ii) the S-deficiency responses are not completely suppressed in P-deficient psr1 cells and consequently these cells synthesize some arylsulfatase and exhibit elevated levels of transcripts for S-deprivation genes; and (iii) this increased expression is controlled by regulators that modulate transcription of S-responsive genes during S-deprivation conditions. Overall, the work strongly suggests integration of the different circuits that control nutrient-deprivation responses in Chlamydomonas.

Soil enzyme activities of long-term reclaimed wastewater-irrigated soils.

Studies have shown that physical and chemical properties of soils may be significantly changed when they are subjected to long-term reclaimed water irrigation. It remains unclear how reclaimed water application may affect nutrient cycling in soils. Soil enzymes are responsible for the biogeochemical cycling of many elements and are more sensitive indicators of the ecological changes. In this study, 17 soil enzymes, including those associated with the C, N, P, and S cycles and two oxidoreductases (catalase and dehydrogenase), were assayed in soils obtained from five long-term reclaimed wastewater irrigation sites in southern California. The soil enzyme activities varied widely among the sampling sites. Compared with their respective controls, the overall activities of enzymes involved in the cycling of the four elements in soil were enhanced by an average of 2.2- to 3.1-fold. Principal component analysis and cluster analysis indicated that the soil microbial functional diversity may be evaluated based on activities of catalase, alkaline phosphatase, acid phosphatase, dehydrogenase, and urease.

Soil microbial communities and enzyme activities under various poultry litter application rates.

The potential excessive nutrient and/or microbial loading from mismanaged land application of organic fertilizers is forcing changes in animal waste management. Currently, it is not clear to what extent different rates of poultry litter impact soil microbial communities, which control nutrient availability, organic matter quality and quantity, and soil degradation potential. From 2002 to 2004, we investigated the microbial community and several enzyme activities in a Vertisol soil (fine, smectitic, thermic, Udic Haplustert) at 0 to 15 cm as affected by different rates of poultry litter application to pasture (0, 6.7, and 13.4 Mg ha(-1)) and cultivated sites (0, 4.5, 6.7, 9.0, 11.2, and 13.4 Mg ha(-1)) in Texas, USA. No differences in soil pH (average: 7.9), total N (pasture: 2.01-3.53, cultivated: 1.09-1.98 g kg(-1) soil) or organic C (pasture average: 25-26.7, cultivated average: 13.9-16.1 g kg(-1) soil) were observed following the first four years of litter application. Microbial biomass carbon (MBC) and nitrogen (MBN) increased at litter rates greater than 6.7 Mg ha(-1) (pasture: MBC = >863, MBN = >88 mg kg(-1) soil) compared to sites with no applied litter (MBC = 722, MBN = 69 mg kg(-1) soil). Enzyme activities of C (beta-glucosidase, alpha-galactosidase, beta-glucosaminidase) or N cycling (beta-glucosaminidase) were increased at litter rates greater than 6.7 Mg ha(-1). Enzyme activities of P (alkaline phosphatase) and S (arylsulfatase) mineralization showed the same response in pasture, but they were only increased at the highest (9.0, 11.2, and 13.4 Mg ha(-1)) litter application rates in cultivated sites. According to fatty acid methyl ester (FAME) analysis, the pasture soils experienced shifts to higher bacterial populations at litter rates of 6.7 Mg ha(-1), and shifts to higher fungal populations at the highest litter application rates in cultivated sites. While rates greater than 6.7 Mg ha(-1) provided rapid enhancement of the soil microbial populations and enzymatic activities, they result in P application in excess of crop needs. Thus, studies will continue to investigate whether litter application at rates below 6.7 Mg ha(-1), previously recommended to maintain water quality, will result in similar improved soil microbial and biochemical functioning with continued annual litter application.

3Beta-sulfamate derivatives of C19 and C21 steroids bearing a t-butylbenzyl or a benzyl group: synthesis and evaluation as non-estrogenic and non-androgenic steroid sulfatase inhibitors.

A series of C19 and C21 steroids bearing one or two inhibiting groups (3beta-sulfamate and 17alpha- or 20(S)-t-butylbenzyl or benzyl) were synthesized and tested for inhibition of steroid sulfatase activity. When only a sulfamate group was added to dehydroepiandrosterone, androst-5-ene-3beta,17beta-diol, pregnenolone and 20-hydroxy-pregnenolone, no significant inhibition of steroid sulfatase occurred at concentrations of 0.3 and 3 microM. With only a t-butylbenzyl or a benzyl group, a stronger steroid sulfatase inhibition was obtained in the androst-5-ene than in the pregn-5-ene series. Comparative results from the screening tests and the IC50 values have shown that the effect of a sulfamate moiety as a second inhibiting group can be combined to the t-butylbenzyl or benzyl effect in the C19 and C21 steroid series. The 3beta-sulfamoyloxy-17alpha-t-butylbenzyl-5-androsten-17beta-ol (10) was thus found to be the most active compound with IC50 values of 46 +/- 8 and 14 +/- 1 nM, respectively for the transformations of E1S to E1 and DHEAS to DHEA. The IC50 values of compound 10 are similar to that of 17alpha-t-butylbenzyl-estradiol, which was previously reported by our group as a good steroid sulfatase reversible inhibitor, but remains higher than that of the potent inactivators estrone-3-O-sulfamate (EMATE) and 17alpha-t-butylbenzyl-EMATE. However, contrary to these two latter inhibitors, compound 10 did not induce any proliferative effect on estrogen-sensitive ZR-75-1 cells nor on androgen-sensitive Shionogi cells at concentrations tested, suggesting that this steroid sulfatase inhibitor is non estrogenic and non androgenic.

In vitro and in vivo models for the evaluation of new inhibitors of human steroid sulfatase, devoid of residual estrogenic activity.

The goal of our research project is to develop a new class of orally active drugs, estrone sulfatase inhibitors, for the treatment of estrogen-dependent (receptor positive) breast cancer. Several compounds were synthesized and their pharmacological potencies explored. Based on encouraging preliminary results, three of them, TX 1299, TX 1492 and TX 1506 were further studied in vitro as well as in vivo. They proved to be strong inhibitors of estrone sulfatase when measured on the whole human JEG-3 choriocarcinoma and MCF-7 breast cancer cells and their IC(50)s found to be in the range of known standard inhibitors. Their residual estrogenic activity was checked as negative in the test of induction of alkaline phosphatase (APase) activity in whole human endometrial adenocarcinoma Ishikawa cells. In addition, their effect on aromatase activity in JEG-3 cells was also examined, since the goal of inhibiting both sulfatase and aromatase activities appears very attractive. However, it has been unsuccessful so far. Then, in vivo potencies of TX 1299, the lead compound in our chemical series, were evaluated in comparison with 6,6,7-COUMATE, a non-steroidal standard, in two different rat models and by oral route. First, the absence of any residual estrogenic activity for these compounds was checked in the uterotrophic model in prepubescent female rats. Second, antiuterotrophic activity in adult ovariectomized rat supplemented with estrone sulfate (E(1)S), showed that both compounds were potent inhibitors, the power of TX 1299 relative to 6,6,7-COUMATE being around 80%. This assay was combined with uterine sulfatase level determination and confirmed the complete inhibition of this enzyme within the target organ. Preliminary studies indicated that other non-steroid compounds in the Théramex series were potent in vitro and in vivo inhibitors of estrone sulfatase in rats and further studies are in progress.

Inhibition of steryl sulfatase activity in LNCaP human prostate cancer cells.

The enzyme steryl sulfatase may help support the growth of hormone-dependent tumors, including prostate cancers, by facilitating the conversion of circulating precursor steroids to active hormones. We sought to determine the presence of steryl sulfatase activity in the androgen-dependent human prostate cancer cell line LNCaP, and to determine if this activity was inhibited by known steryl sulfatase inhibitors. Intact LNCaP cultures had steryl sulfatase activity, as determined by conversion of [3H]estrone sulfate (E(1)S) to unconjugated steroids. The level of steryl sulfatase activity was relatively low (4.6 pmol/18 h/million cells) compared to MDA-MB-231 breast cancer cells (284.0 pmol/18 h/million cells). The observed activity in both cell lines was blocked by addition of 1 microM estrone sulfamate (EMATE), an active-site-directed, steroidal inhibitor of steryl sulfatase. Steryl sulfatase activity was also inhibited by Danazol, and by (p-O-sulfamoyl)-tetradecanoyl tyramine (C2-14), a non-steroidal inhibitor. Microsomes prepared from LNCaP cultures also showed steryl sulfatase activity, as determined by hydrolysis of [3H]E(1)S and [3H]dehydroepiandrosterone sulfate (DHEAS) to unconjugated forms. LNCaP and MDA-MB-231 microsomes both hydrolyzed E(1)S about two times faster than DHEAS. Hydrolysis of E(1)S in LNCaP and MDA-MB-231 microsomes was blocked by steryl sulfatase inhibitors with the following relative potencies: EMATE>C2-14>Danazol. These data demonstrate that LNCaP prostate cancer cells contain a steryl sulfatase with properties similar to that found in human breast cancer cells, and that the activity of this enzyme can be blocked by known steryl sulfatase inhibitors. Steryl sulfatase inhibitors may be useful as an adjuvant to androgen deprivation therapy for prostate cancer.

Ozone therapy and the activity of selected lysosomal enzymes in blood serum of patients with lower limb ischaemia associated with obliterative atheromatosis.

BACKGROUND: The paper compares the effects of ozone therapy and conventional balneological methods on health condition of patients with obliterative atheromatosis and on serum activity of three lysosomal enzymes. MATERIAL/METHODS: Sixty-four patients with lower limb ischaemia in the course of obliterative atheromatosis (without diabetes) were enrolled in the study. Thirty-two patients were treated with ozone administered by intravenous infusions and 30-minute aerosol oxygen-ozone baths. A comparative group was formed of 32 patients treated with traditional balneology. There was also a control group made up of 30 healthy subjects. Ozone therapy as well as traditional balneology were administered daily for the period of 10 days, excluding Saturdays and Sundays. Blood for biochemical analysis was collected from elbow vein in the following time intervals: 24 hours before ozone therapy or classical balneology, one hour after therapy and on the 10th day of treatment. The activity of cathepsin D, acid phosphatase and arylsulphatase as well as the levels of a-1-antitrypsin (protease inhibitor) were determined in blood serum of patients with obliterative atheromatosis. RESULTS: In patients who received ozone therapy the activity of analysed lysosomal hydrolases returned to the values typical for healthy subjects. Patients' general condition also improved. The use of traditional balneological methods did not result in any significant change either in the activity of lysosomal hydrolases, the level of a-1-antitrypsin or general condition of patients. CONCLUSIONS: Ozone therapy administered by intravenous infusions and aerosol oxygen-ozone baths of lower extremities yields much better therapeutic results in comparison with classical balneology.

Effects of heavy metal contamination of soils on micronucleus induction in Tradescantia and on microbial enzyme activities: a comparative investigation.

The aim of this study was to investigate correlation between genotoxic effects and changes of microbial parameters caused by metal contamination in soils. In total, 20 soils from nine locations were examined; metal contents and physicochemical soil parameters were measured with standard methods. In general, a pronounced induction of the frequency of micronuclei (MN) in the Tradescantia micronucleus (Trad-MN) assay was seen with increasing metal concentration in soils from identical locations. However, no correlations were found between metal contents and genotoxicity of soils from different locations. These discrepancies are probably due to differences of the physicochemical characteristics of the samples. Also, the microbial parameters depended on the metal content in soils from identical sampling locations. Inconsistent responses of the individual enzymes were seen in soils from different locations, indicating that it is not possible to define a specific marker enzyme for metal contamination. The most sensitive microbial parameters were dehydrogenase and arylsulfatase activity, biomass C, and biomass N. Statistical analyses showed an overall correlation between genotoxicity in Tradescantia on the one hand and dehydrogenase activity, biomass C, and the metabolic quotient on the other hand. In conclusion, the results of the present study show that the Trad-MN assay is suitable for the detection of genotoxic effects of metal contamination in soils and furthermore, that the DNA-damaging potential of soils from different origin cannot be predicted on the basis of chemical analyses of their metal concentrations.

Sulfur economy and cell wall biosynthesis during sulfur limitation of Chlamydomonas reinhardtii.

We have identified two novel periplasmic/cell wall polypeptides that specifically accumulate during sulfur limitation of Chlamydomonas reinhardtii. These polypeptides, present at high levels in the extracellular polypeptide fraction from a sulfur-deprived, cell wall-minus C. reinhardtii strain, have apparent molecular masses of 76 and 88 kD and are designated Ecp76 and Ecp88. N-terminal sequences of these polypeptides facilitated the isolation of full-length Ecp76 and Ecp88 cDNAs. Ecp76 and Ecp88 polypeptides are deduced to be 583 and 595 amino acids, respectively. Their amino acid sequences are similar to each other, with features characteristic of cell wall-localized hydroxyproline-rich glycoproteins; the N terminus of each polypeptide contains a predicted signal sequence, whereas the C terminus is rich in proline, alanine, and serine. Ecp76 and Ecp88 have either no (Ecp88) or one (Ecp76) sulfur-containing amino acid and transcripts encoding these polypeptides are not detected in cultures maintained on complete medium, but accumulate when cells are deprived of sulfur. This accumulation is temporally delayed relative to the accumulation of sulfur stress-induced arylsulfatase and ATP sulfurylase transcripts. The addition of sulfate back to sulfur-starved cultures caused a rapid decline in Ecp76 and Ecp88 mRNAs (half lives < 10 min). Furthermore, the C. reinhardtii sac1 mutant, which lacks a regulatory protein critical for acclimation to sulfur limitation, does not accumulate Ecp76 or Ecp88 transcripts. These results suggest that the Ecp76 and Ecp88 genes are under SacI control, and that restructuring of the C. reinhardtii cell wall during sulfur limitation may be important for redistribution of internal and efficient utilization of environmental sulfur-containing molecules.

Do dietary phytoestrogens influence susceptibility to hormone-dependent cancer by disrupting the metabolism of endogenous oestrogens?

Phytoestrogens are natural constituents of our diets that have been suggested to protect against hormone-dependent breast cancer. Some of the diverse effects of these compounds may be attributed to ligand-dependent differences in their interaction with oestrogen receptor sub-classes. However, phytoestrogens can also inhibit enzymes that are involved in the generation and removal of endogenous steroid hormones. Among the most potent effects of dietary phytoestrogens is their ability to inhibit the sulphotransferases that sulphate both oestrogenic steroids and a variety of environmental chemicals, including dietary pro-carcinogens. Circulating steroid sulphates are thought to be the major source of oestradiol in post-menopausal breast tumours and sulphation is a key step in the activation of some dietary pro-carcinogens. Hence the inhibition of sulphotransferases by dietary phytoestrogens may have complex effects upon human susceptibility to breast cancer.

Effects of alkaline dust deposits from phosphate fertilizer production on microbial biomass and enzyme activities in grassland soils.

Microbial biomass carbon (Cmic) and soil enzyme activities were measured at 12 sites along a gradient of former emissions of phosphate fertilizer production. Seven years after close down of operation, still moderate to high total concentrations of the dust constituents cadmium (up to 33 mg kg-1 dw), fluoride (5300 mg kg-1 dw) and phosphorous (120,000 mg kg-1 dw) were found in topsoils of contaminated sites. Accumulation of partially decomposed plant matter, soil respiration and dehydrogenase activity paralleled the increase of dust deposits, whereas microbial biomass decreased along the gradient. A significant negative correlation was obtained between the Cmic-to-Corg-ratio and the concentration of contaminants. In contrast, the Cmic-specific respiration (qCO2) and the dehydrogenase activity-to-Cmic-ratio were positively correlated. The low Cmic-values and the enhanced activities in the contaminated soils are suggested as a response of microbial communities to environmental stress or ecosystem disturbances. The apparently missing detrimental effects of the alkaline deposits on soil microbial activities are probably due to the low bioavailability of contaminants in the calcareous soil.

Constitutive expression of the steroid sulfatase gene supports the growth of MCF-7 human breast cancer cells in vitro and in vivo.

Many human breast tumors are driven by high intratumor concentrations of 17beta-estradiol that appear to be locally synthesized. The role of aromatase is well established, but the possible contribution of the steroid sulfatase (STS), which liberates estrogens from their biologically inactive sulfates, has been inadequately assessed and remains unclear. To evaluate the role of STS further, we transduced estrogen-dependent MCF-7 human breast cancer cells with a retroviral vector directing the constitutive expression of the human STS gene. Gene integration was confirmed by Southern hybridization, production of the appropriately sized messenger RNA by Northern hybridization, and expression of functional protein by metabolism of [(3)H]estrone sulfate to [(3)H]estrone. Maximum velocity estimates of estrone formation are 64.2 pmol estrone/mg protein.h in STS-transduced cells (STS Clone 20), levels comparable to those seen in some human breast tumors. Lower levels of endogenous activity are seen in MCF-7 cells (13.0 pmol estrone/mg protein.h) and in cells transduced with vector lacking the STS gene (Vector 3 cells; 12.0 pmol estrone/mg protein.h). 17beta-Estradiol sulfate induces expression of the progesterone receptor messenger RNA only in STS Clone 20 cells, whereas estrone sulfate produces the greatest stimulation of anchorage-independent growth in these cells. STS Clone 20 cells retain responsiveness to antiestrogens, which block the ability of estrogen sulfate to increase the proportion of cells in both the S and G(2)/M phases of the cell cycle. Consistent with these in vitro observations, only STS Clone 20 cells exhibit a significant increase in the proportion of proliferating tumors in nude ovariectomized mice supplemented with 17beta-estradiol sulfate. The primary activity in vivo appears to be from intratumor STS, rather than hepatic STS. Surprisingly, 17beta-estradiol sulfate appears more effective than 17beta-estradiol when both are administered at comparable concentrations. This effect, which is seen only in STS Clone 20 cells, may reflect differences in the cellular pharmacology of exogenous estrogens compared with those released by the activity of intracellular STS. These studies directly demonstrate that intratumor STS activity can support estrogen-dependent tumorigenicity in an experimental model and may contribute to the promotion of human breast tumors.

Organic anion-transporting polypeptide B (OATP-B) and its functional comparison with three other OATPs of human liver.

BACKGROUND & AIMS: Hepatic uptake of cholephilic organic compounds is mediated by members of the organic anion-transporting polypeptide (OATP) family. We aimed to characterize the novel OATP-B with respect to tissue distribution and hepatocellular localization and to compare its substrate specificity with those of OATP-A, OATP-C, and OATP8. METHODS: Tissue distribution and hepatocellular localization of OATP-B were analyzed by Northern blotting and immunofluorescence, respectively. Transport of 16 substrates was measured for each individual human OATP in complementary RNA-injected Xenopus laevis oocytes. RESULTS: Expression of OATP-B was most abundant in human liver, where it is localized at the basolateral membrane of hepatocytes. OATP-B, OATP-C, and OATP8 mediated high-affinity uptake of bromosulphophthalein (K(m), approximately 0.7, 0.3, and 0.4 micromol/L, respectively). OATP-B also transported estrone-3-sulfate but not bile salts. Although OATP-A, OATP-C, and OATP8 exhibit broad overlapping substrate specificities, OATP8 was unique in transporting digoxin and exhibited especially high transport activities for the anionic cyclic peptides [D-penicillamine(2,5)]enkephalin (DPDPE; opioid-receptor agonist) and BQ-123 (endothelin-receptor antagonist). CONCLUSIONS: OATP-B is the third bromosulphophthalein uptake system localized at the basolateral membrane of human hepatocytes. OATP-B, OATP-C, and OATP8 account for the major part of sodium-independent bile salt, organic anion, and drug clearance of human liver.

Molecular analysis of X-linked ichthyosis in Japan.

BACKGROUND: X-linked ichthyosis (XLI) is an inherited skin disorder caused by a deficiency of steroid sulfatase (STS). The gene and protein of STS were examined in 19 Japanese patients with XLI. RESULTS: In Western blotting analysis, no cross-reacting peptide was detected in the patients' placenta, although a single band (63 kD) corresponding to STS in a normal subject was observed. Southern blotting was performed using EcoRI digests of cellular DNA from 13 XLI patients and full-length human STS cDNA as a probe. Normal males had bands of 20, 15, 10, 9.0, 6.1, 4.2, 2.6, and 1.5 kb. Twelve of the 19 patients had only 20- and 1.5-kb bands. Only one patient had the same band pattern as that of normal males. The STS gene was analyzed by PCR in 6 of the 19 patients. PCR amplification products were sequenced to analyze the STS gene. Two cases with one-base change in the STS gene and variation in amino acids H444R and E560P were found. Mutant STS cDNA was transfected into COS-1 cells and the STS enzyme activity was assayed. The enzyme activities were less than the minimum detection value of the detection system. CONCLUSIONS: These results suggest that XLI is mainly caused by an extensive deletion of the STS gene and that the PCR method is useful for detection of STS point mutations.

Steroid sulfatase activity in the rat ovary, cultured granulosa cells, and a granulosa cell line.

Direct production of gonadal steroids from sulfated adrenal androgens may be an important alternative or complementary pathway for ovarian steroidogenesis. The conversion of sulfated adrenal androgens, present in serum at micromolar concentrations in adult women, into unconjugated androgens or estrogens requires steroid sulfatase (STS) activity. STS activity has not been characterized in the rat ovary. Substantial STS activity was present in homogenates of rat ovaries, primary cultures of rat granulosa cells, and a granulosa cell line, as determined by conversion of radiolabeled estrone sulfate (E1S) to unconjugated estrone. The potent inhibitor estrone sulfamate eliminated the STS activity. Using E1S as a substrate with microsomes prepared from a granulosa cell line, the K(m) of STS activity was approximately 72 microM, a value in agreement with previously published data for rat STS. Therefore, ovarian cells possess STS and can remove the sulfate from adrenal androgens such as dehydroepiandrosterone sulfate (DHEA-S). Using DHEA-S as a steroidogenic substrate represents an alternative model for the production of ovarian steroids versus the "two cell, two gonadotropin" model of ovarian estrogen synthesis, whereby thecal cells produce androgens from substrate cholesterol and granulosa cells convert the androgens into estrogens. The relative contribution of STS activity to ovarian steroidogenesis remains unclear but may have important physiological and pathophysiological implications.