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.

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.

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.

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.

Regulation of steroid sulphatase expression and activity in breast cancer.

Steroid sulphatase (STS) catalyzes the conversion of oestrone sulphate (E1S) to oestrone (E1) and its action in breast tumours makes a major contribution to in situ oestrogen production in this tissue. Although expression of STS mRNA and STS activity are increased in malignant breast tissues compared with that in non-malignant tissues, little is known about the regulation of its expression or activity. In the present study we have used a RT-PCR technique to investigate the regulation of STS mRNA expression in cultured breast tissue fibroblasts and MCF-7 cells. STS mRNA expression was readily detectable in fibroblasts derived from breast tissue proximal to tumours, breast tumour tissue and reduction mammoplasty tissue. For two pre-menopausal subjects, STS mRNA expression was similar in proximal and tumour fibroblasts whereas for a third, post-menopausal subject, expression in breast tumour fibroblasts was 2.4-fold that in proximal fibroblasts. The cytokine tumour necrosis factor alpha (TNFalpha) or the STS inhibitor, 2-methoxyoestrone-3-O-sulphamate, had no effect on STS mRNA expression in fibroblasts. STS mRNA was detectable in MCF-7 cells but neither TNFalpha nor interleukin 6 (IL-6) affected its expression. Transient transfection of COS-1 and MCF-7 cells with a STS cDNA lacking STS 5' and 3' sequences increased activity 17-fold and 2-fold, respectively. TNFalpha plus IL-6 increased STS activity in mock transfected MCF-7 cells and further increased STS activity in transfected MCF-7 cells. This indicates that activation can occur independently of STS promoter and enhancer elements. In conjunction with the lack of regulation of STS mRNA it suggest that TNFalpha and IL-6 may increase STS activity via a post-translational modification of the enzyme or by increasing substrate availability.

Uptake of dehydroepiandrosterone-3-sulfate by isolated trophoblasts from human term placenta, JEG-3, BeWo, Jar, BHK cells, and BHK cells transfected with human sterylsulfatase-cDNA.

The human placenta lacks the enzyme 17alpha-hydroxylase/17-20-lyase, and is thus unable to convert cholesterol into estrogens. Therefore estrogen synthesis of trophoblast cells depends on the supply of precursors such as dehydroepiandrosterone-3-sulfate (DHEA-S) and 16alpha-hydroxy-dehydroepiandrosterone-3-sulfate by maternal and fetal blood. To investigate the cellular internalisation of these anionic hydrophilic precursors, the uptake of [(3)H]-/[(35)S]-DHEA-S and [(3)H]-taurocholate by isolated cytotrophoblasts, cells of choriocarcinoma cell lines (JEG-3, BeWo, Jar), BHK and BHK cells transfected with human sterylsulfatase-cDNA (BHK-STS cells) was studied. Furthermore, the activity of sterylsulfatase of these cells in suspension and in corresponding cell homogenate was measured. During the first 5 min of incubation with [(3)H]-DHEA-S or [(35)S]-DHEA-S, radioactivity of cytotrophoblasts increased significantly, while radioactivity of JEG-3, Jar, BHK and BHK-STS cells did not increase. Radioactivity of BeWo cells increased slightly. For all cell types, there was no significant difference for uptake of either substrate. During incubation with [(3)H]-taurocholate, radioactivity of cytotrophoblasts did not increase. Sterylsulfatase activity of cytotrophoblast homogenate was significantly lower than that of cytotrophoblast suspension. Sterylsulfatase activity of BHK-STS, JEG-3 or BeWo cell homogenate was significantly higher than that of the corresponding cell suspension. In BHK and Jar cells sterylsulfatase activity was not detectable. Cytotrophoblasts take up DHEA-S without prior hydrolysis. BHK, BHK-STS, JEG-3, and Jar cells do not take up and BeWo cells slowly take up DHEA-S. In cytotrophoblasts extracellular DHEA-S rapidly gains access to intracellular sterylsulfatase, while in choriocarcinoma and BHK-STS cells access of DHEA-S to sterylsulfatase is limited. Our results indicate, that uptake by cytotrophoblasts is mediated by a carrier which is not expressed in choriocarcinoma or BHK cells and which is different from the known taurocholate-transporting organic anion transporting polypetides.

Glucocorticoid deficiency delays stratum corneum maturation in the fetal mouse.

The stratum corneum (SC) matures during late gestation in man and other mammals. Using the fetal rat as an experimental model, we have previously shown that glucocorticoids given in pharmacologic doses accelerate fetal SC maturation and barrier formation. To determine whether glucocorticoids are required for normal SC maturation, we examined the epidermal morphology of glucocorticoid-deficient (C-) murine pups, derived from matings of mice homozygous for null mutations of the corticotropin-releasing hormone alleles. In control pups on day 17.5 of gestation (term is 19.5 d), a multilayered SC was present and neutral lipid deposition in a membrane pattern was observed using Nile red fluorescence histochemistry. Ultrastructurally, mature lamellar unit structures predominate in the SC intercellular domains. In contrast, in C-pups only a single layer of SC was evident on day 17.5, and secreted lamellar material was not organized into mature lamellar structures. Furthermore, the expression of structural proteins necessary for cornified envelope formation, involucrin, loricrin, and filaggrin, and the activity of the lipid synthetic enzymes beta-glucocerebrosidase and steroid sulfatase, markers of barrier maturation, were reduced in day 17.5 C-pups. C-pups derived from pregnancies supplemented with physiologic amounts of cortisone, however, display normal SC ultrastructure on day 17.5 of gestation. Furthermore, at birth, both control and C-pups exhibit a multilayered SC replete with mature lamellar membrane structures. These data demonstrate that fetal glucocorticoid deficiency delays SC maturation, and suggests that normal levels of glucocorticoids are not absolutely required for SC development.

In vivo metabolism of 24R,25-dihydroxyvitamin D3: structure of its major bile metabolite.

In vivo metabolism of 24R,25-dihydroxyvitamin D3 (24,25-(OH)2D3) in female dogs has been studied thoroughly, and its major bile metabolite identified. After single oral administration of 24,25-(OH)2 [6,19,19-3H]D3 the plasma concentrations of radioactive metabolites were monitored for 504 h, and the metabolites in the bile collected and analyzed. The concentration of 24,25-(OH)2D3 in plasma reached a maximum after 6 h and decayed in two distinct phases; a fast-phase with a half-life of 17 h, followed by a slow-phase with a 17-day half-life. The area under the concentration/time curve (AUC) was 78-84% (0-504 h). The only detectable metabolite in the plasma was 25-hydroxy-24-oxovitamin D3 whose AUC was less than 5%. At 504 h, about 50% of administered radioactivity has been excreted, of which about 90% was found in the feces, indicating most of the administered 24,25-(OH)2D3 to be excreted in bile. A major metabolite, which constituted 23% of the total bile radioactivity at 504 h, was found in the bile. This metabolite was efficiently deconjugated by beta-glucuronidase to afford an aglycone which was identified as 23S,25-dihydroxy-24-oxovitamin D3 (23S,25-(OH)2-24-oxo-D3), by co-chromatography on HPLC with synthetic standards. The glucuronide was isolated from the bile of dogs given large doses of 24,25-(OH)2D3, and the structure determined being 23-(beta-glucuronide) of 23S,25-(OH)2-24-oxo-D3, by analyzing its negative ion mass spectrum and the positive ion mass spectrum of its derivatives. Thus it was concluded that, in dogs, 24,25-(OH)2D3 is a long lasting vitamin D metabolite, is mainly excreted in bile when metabolized to 23S,25-(OH)2-24-oxo-D3 and is conjugated at 23-OH as glucuronide.

Sterylsulfatase expression in normal and transformed human placental cells.

Isolated cytotrophoblast cells and choriocarcinoma cell lines are commonly applied in-vitro systems for the study of human placental endocrine function. We tested these normal and transformed placental cells for expression of the enzyme sterylsulfatase which is necessary for the production of free steroids from sulfoconjugated precursors in the placenta as well as in other human tissues, and compared the results with respective data obtained from term placental tissue. Specific sterylsulfatase activity was highest in placental homogenates but was lower by about a factor of 5 to 10 in homogenates of freshly isolated cytotrophoblast or JEG-3 cells and by about a factor of 100 in BeWo cell homogenates; the enzyme activity could not be detected in Jar cells. Sterylsulfatase mRNA levels as analyzed by Northern blotting roughly paralleled the levels of enzyme activity measured in cytotrophoblast, JEG-3, and BeWo cells; in Jar cells, RNA species complementary to the specific probe were clearly detectable but differed by size from the mRNA species found in the other cells. Our results indicate that sterylsulfatase activity is differently expressed in normal and transformed placental cells due to different rates or products of gene transcription in these cells.

Mode of action of iron on arylsulphatases under acute lead acetate treated conditions in rats.

Arylsulphatases A, B and C were found to be inhibited in liver and kidney tissues under lead acetate-treated conditions (both in vivo and in vitro) in rats. When lead acetate-treated animals (in vivo) were supplemented with ferric ammonium citrate (in vivo), a remarkable recovery was found in the activities of all arylsulphatases A, B and C whereas ferric ammonium citrate itself had no effect on the activities of arylsulphatases. When both the in vivo and in vitro lead acetate-treated arylsulphatases were supplemented with the purified ferritins (in vitro) it was observed that lead-induced inhibition of the activities of arylsulphatases was successfully reversed. It was also found that ferritins were able to bind a large quantity of lead. These results indicated that ferritins were directly involved for reactivation of arylsulphatases which were inhibited by lead. It was well established that a response to iron administration in rats was an immediate de novo stimulation of ferritin biosynthesis. Iron might therefore protect the enzymatic activities of arylsulphatases by enhancing the level of ferritin in liver and kidney tissues which is known to bind a large quantity of lead thereby ameliorating their toxic effects in the living system.