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1.
Biotechnol Prog ; 27(6): 1522-34, 2011.
Article in English | MEDLINE | ID: mdl-21916026

ABSTRACT

An experimental protocol to evaluate the structured biomass model proposed by Lavallée (Lavallée, Lessard, and Vanrolleghem, J Environ Eng Sci 2005;4:517-532) is presented. The protocol was devised to induce transient behavior and characterize the evolution of several internal biomass components. The proposed model is based on biochemical principles, and was fitted to the collected data. In these experiments, it was observed that filling the storage capacity of cells leads to special transient behavior, including a temporarily reduced metabolic activity. The model-based interpretation of the results showed that the observed transient behavior can be explained by cross-regulation of carbon and nitrogen metabolism. Hence, according to an extensive literature review, the cross-regulation of carbon and nitrogen can be used to model some observed transient behaviors and regulation of the storage process in activated sludge.


Subject(s)
Bacteria/metabolism , Carbon/metabolism , Nitrogen/metabolism , Sewage/microbiology , Bacteria/genetics , Bacteria/growth & development , Biomass , Bioreactors/microbiology , Models, Biological
2.
Water Sci Technol ; 59(4): 661-71, 2009.
Article in English | MEDLINE | ID: mdl-19237760

ABSTRACT

Models currently used have been developed to describe the storage response in the activated sludge process. In these models the distribution of the substrate flux between growth and storage is an empirical function. rRNA-structured biomass models are proposed to describe the metabolic status of cells in view of predicting the growth response (dmicro/dt) of cells in activated sludge process. The autocatalytic reaction rate of the synthesis of the PSS component (rRNA) can provide a mechanistic explanation for the growth response and the growth lag phase. The proposed models were able to describe and predict properly the growth response of the biomass in various types of reactor. Such models could be more widely applicable by using intrinsic model parameters. This would be a key improvement for as it would lead to improved models for design.


Subject(s)
Biomass , Models, Biological , RNA, Ribosomal/genetics , Kinetics
3.
Radiat Res ; 159(5): 693-7, 2003 May.
Article in English | MEDLINE | ID: mdl-12710882

ABSTRACT

The current study extends our previous investigations of 2-h radiofrequency (RF)-field exposures on genotoxicity in human blood cell cultures by examining the effect of 24-h continuous-wave (CW) and pulsed-wave (PW) 1.9 GHz RF-field exposures on both primary DNA damage and micronucleus induction in human leukocyte cultures. Mean specific absorption rates (SARs) ranged from 0 to 10 W/kg, and the temperature within the cultures was maintained at 37.0 +/- 1.0 degrees C for the duration of the 24-h exposure period. No significant differences in primary DNA damage were observed between the sham-treated controls and any of the CW or PW 1.9 GHz RF-field-exposed cultures when processed immediately after the exposure period by the alkaline comet assay. Similarly, no significant differences were observed in the incidence of micronuclei, incidence of micronucleated binucleated cells, frequency of binucleated cells, or proliferation index between the sham-treated controls and any of the CW or PW 1.9 GHz RF-field-exposed cultures. In conclusion, the current study found no evidence of 1.9 GHz RF-field-induced genotoxicity in human blood cell cultures after a 24-h exposure period.


Subject(s)
DNA Damage , Leukocytes/radiation effects , Micronuclei, Chromosome-Defective/radiation effects , Radio Waves , Adult , Cells, Cultured , Humans , Middle Aged
4.
Mol Psychiatry ; 8(1): 39-49, 2003 Jan.
Article in English | MEDLINE | ID: mdl-12556907

ABSTRACT

In recent studies that addressed the transcriptional control of steroid synthesis, a transcriptional regulating protein of 132 kDa (TReP-132) was cloned and demonstrated to regulate expression of the human P450 side chain cleavage (P450scc) gene. In the present study, we describe the cloning and characterization of the mouse orthologue of the human factor, mouse transcriptional regulating protein (mTReP-132). mTReP-132 encodes a 1216-residue protein that is 85.5% homologous to the human protein. Both factors contain characteristic motifs, namely glutamine-, proline- and acidic-rich regions. The primary structure also exhibits two zinc fingers of the C(2)H(2) subtype, suggesting that this protein has the ability to act as a DNA binding transcription factor. mTReP-132 may also be a co-regulator of nuclear receptors because of two nuclear box motifs in this protein. Northern blot analysis demonstrated the expression of two transcripts of 4.4 and 7.5 kb in several tissues, but expression was clearly highest in the brain, thymus and testis of mice. In the brain, the hybridization signal was quite localized and strong in the basal ganglia, hippocampus, piriform cortex, cerebral cortex, ventromedial nucleus of the hypothalamus, and the dorsal and superior central nuclei of the raphe. Although classical steroidogenesis pathways have yet to be firmly established in the brain, expression of both mTReP-132 and P450scc provides anatomical evidence that mTReP-132 may regulate this key steroidogenic enzyme within specific regions involved in behavioral and psychiatric disorders. Moreover, the presence of both mTReP-132 and steroidogenic factor 1 (SF-1) transcripts in the ventromedial nucleus of the hypothalamus suggests a role for mTReP-132 in brain development and function. The molecular cloning and the highly specific expression of mTReP-132 across the brain further consolidate the hypothesis that this tissue is able to synthesize de novo steroids in a region-specific manner.


Subject(s)
Brain Chemistry/genetics , DNA-Binding Proteins/genetics , Mental Disorders/genetics , Transcription Factors/genetics , Adrenal Glands/physiology , Amino Acid Sequence , Animals , Behavior, Animal/physiology , Cloning, Molecular , Gene Expression , In Situ Hybridization , Male , Mice , Molecular Sequence Data , RNA, Messenger/analysis , Steroidogenic Factor 1 , Testis/physiology , Thymus Gland/physiology , Zinc Fingers/genetics
5.
Radiat Res ; 158(4): 523-33, 2002 Oct.
Article in English | MEDLINE | ID: mdl-12236820

ABSTRACT

Human blood cultures were exposed to a 1.9 GHz continuous-wave (CW) radiofrequency (RF) field for 2 h using a series of six circularly polarized, cylindrical waveguides. Mean specific absorption rates (SARs) of 0.0, 0.1, 0.26, 0.92, 2.4 and 10 W/kg were achieved, and the temperature within the cultures during a 2-h exposure was maintained at 37.0 +/- 0.5 degrees C. Concurrent negative (incubator) and positive (1.5 Gy (137)Cs gamma radiation) control cultures were run for each experiment. DNA damage was quantified immediately after RF-field exposure using the alkaline comet assay, and four parameters (tail ratio, tail moment, comet length and tail length) were used to assess DNA damage for each comet. No evidence of increased primary DNA damage was detected by any parameter for RF-field-exposed cultures at any SAR tested. The formation of micronuclei in the RF-field-exposed blood cell cultures was assessed using the cytokinesis-block micronucleus assay. There was no significant difference in the binucleated cell frequency, incidence of micronucleated binucleated cells, or total incidence of micronuclei between any of the RF-field-exposed cultures and the sham-exposed controls at any SAR tested. These results do not support the hypothesis that acute, nonthermalizing 1.9 GHz CW RF-field exposure causes DNA damage in cultured human leukocytes.


Subject(s)
DNA Damage , Leukocytes/radiation effects , Micronuclei, Chromosome-Defective/radiation effects , Radio Waves , Adult , Cells, Cultured , Female , Humans , Leukocytes/diagnostic imaging , Male , Middle Aged , Ultrasonography
6.
Radiat Res ; 158(4): 534-7, 2002 Oct.
Article in English | MEDLINE | ID: mdl-12236821

ABSTRACT

Blood cultures from human volunteers were exposed to an acute 1.9 GHz pulse-modulated radiofrequency (RF) field for 2 h using a series of six circularly polarized, cylindrical waveguides. Mean specific absorption rates (SARs) ranged from 0 to 10 W/kg, and the temperature within the cultures during the exposure was maintained at 37.0 +/- 0.5 degrees C. DNA damage was quantified in leukocytes by the alkaline comet assay and the cytokinesis-block micronucleus assay. When compared to the sham-treated controls, no evidence of increased primary DNA damage was detected by any parameter for any of the RF-field-exposed cultures when evaluated using the alkaline comet assay. Furthermore, no significant differences in the frequency of binucleated cells, incidence of micronucleated binucleated cells, or total incidence of micronuclei were detected between any of the RF-field-exposed cultures and the sham-treated control at any SAR tested. These results do not support the hypothesis that acute, nonthermalizing 1.9 GHz pulse-modulated RF-field exposure causes DNA damage in cultured human leukocytes.


Subject(s)
DNA Damage , Leukocytes/radiation effects , Radio Waves , Cells, Cultured , Humans , Micronuclei, Chromosome-Defective/radiation effects
7.
Water Sci Technol ; 46(1-2): 423-30, 2002.
Article in English | MEDLINE | ID: mdl-12216661

ABSTRACT

Hypotheses of the ASM 1 model state that the specific respiration rate of heterotrophic active biomass is a constant. It thus becomes possible to assess the active heterotrophic biomass decay rate (bH) during starvation. The object of this study is to verify if the specific respiration rate of heterotrophic biomass remains constant throughout starvation, and if it is typical of global metabolic biomass activity, as proposed by the model hypotheses. The decay rate bH was evaluated using respirometric and enzymatic measurements. Measurement of bH through respirometry on various sludges varied between 0.28 and 0.76 d(-1). According to the enzyme and respirometric measurements carried out, a large portion of this value is related to enzymatic regulation. During starvation, when initial active biomass activity is high, down regulation of the respiratory chain results in a decrease in the specific heterotrophic active biomass respiration rate, thus creating an overevaluation of the decay rate as measured by respirometry. Thus a high decay rate used in the ASM 1 model could eventually lead to a poor forecast of biomass production.


Subject(s)
Bacteria , Bioreactors , Models, Theoretical , Biomass , Oxygen/metabolism , Population Dynamics , Waste Disposal, Fluid
8.
Am J Physiol Heart Circ Physiol ; 281(3): H1407-12, 2001 Sep.
Article in English | MEDLINE | ID: mdl-11514313

ABSTRACT

Estrogen replacement therapy reduces risk of cardiovascular events by altering coronary vasoregulation and distribution of blood flow. Vessel reactivity and blood flow distribution were assessed in anesthetized female rabbits in the following groups: 1) sham, 2) ovariectomy, 3) ovariectomy + 17beta-estradiol, and 4) ovariectomy + dehydroepiandrosterone. After a 2-wk treatment, cardiac hemodynamics, vascular reserve, and blood flow were evaluated during the following infusions: 1) NaCl, or vehicle (0.5 ml/min), 2) acetylcholine (2 mg/kg), 3) isoproterenol (2 mg. kg(-1). min(-1)), and 4) chromonar (8 mg/kg). In hearts from ovariectomized rabbits, autoregulatory blood flow was preserved despite lower diastolic perfusion pressures (55 +/- 8 vs. 64 +/- 8 mmHg in sham) and rate-pressure product (14.4 +/- 0.8 vs. 19.3 +/- 0.8 beats/min. mmHg x 10(-3)). Estrogen replacement therapy restored coronary pressure and reserve, and all drugs increased vascular conductance. In conclusion, in hearts from ovariectomized rabbits, vascular reserve declined because coronary pressure was lower; however, blood flow was preserved at a higher level than expected for oxygen demand. Estrogen replacement therapy restores myocardial oxygen supply-demand indices and returns coronary pressure-flow data to levels observed in animals with intact ovaries.


Subject(s)
Coronary Circulation/drug effects , Estradiol/pharmacology , Estrogen Replacement Therapy , Acetylcholine/pharmacology , Anesthesia , Animals , Blood Flow Velocity/drug effects , Blood Flow Velocity/physiology , Blood Pressure/drug effects , Cardiotonic Agents/pharmacology , Chromonar/pharmacology , Coronary Circulation/physiology , Dehydroepiandrosterone/pharmacology , Female , Isoproterenol/pharmacology , Ovariectomy , Oxygen Consumption/drug effects , Oxygen Consumption/physiology , Rabbits , Vasodilator Agents/pharmacology
9.
J Biol Chem ; 276(36): 33881-92, 2001 Sep 07.
Article in English | MEDLINE | ID: mdl-11349124

ABSTRACT

The human CYP11A1 gene is expressed specifically in steroidogenic tissues and encodes cytochrome P450scc, which catalyzes the first step in steroid synthesis. A region of the 5'-flanking DNA of the gene from nucleotides -155 to -131 (-155/-131) is shown to activate transcription in steroidogenic human placental JEG-3 (1) and adrenal NCI-H295 cells. Using this region of the gene as probe, a cDNA clone of 4.4 kilobase pairs was isolated by screening JEG-3 cell and human placental cDNA expression libraries. The open reading frame encodes three zinc fingers of the C(2)H(2) subtype, and separate regions rich in glutamate, proline, and glutamine, which are indicative of a DNA-binding protein involved in gene transcription. Expression of the cDNA in vitro and in HeLa cells yields a protein of 132 kDa, which concurs with the predicted size. Northern blot analysis demonstrate expression of two TReP-132 transcripts of 4.4 and 7.5 kilobase pairs in the thymus, adrenal cortex, and testis; and expression is also found in the steroidogenic JEG-3, NCI-H295, and MCF-7 cell lines. Immunocytochemistry analysis demonstrates localization of the HA-tagged TReP-132 protein in the nucleus. The expression of exogenous TReP-132 in HeLa cells was demonstrated to interact with the -155/-131 region in bandshift analysis. Transfection of the cDNA in placental JEG-3 and adrenal NCI-H295 cells increases expression of a reporter construct controlled by the P450scc gene 5'-flanking region from nucleotides -1676 to +49. Moreover, a chimeric protein generated by fusion of TReP-132 with the Gal4 DNA-binding domain was able to significantly increase promoter activity of a reporter construct via Gal4-binding sites upstream of the E1b minimal promoter. Coexpression of CREB-binding protein (CBP)/p300 with TReP-132 has an additive effect on promoter activity, and the proteins were demonstrated to interact physically. Thus, these results together indicate the isolation of a novel zinc-finger transcriptional regulating protein of 132 kDa (TReP-132) involved in the regulation of P450scc gene expression.


Subject(s)
Cholesterol Side-Chain Cleavage Enzyme/biosynthesis , Cholesterol Side-Chain Cleavage Enzyme/genetics , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/physiology , Gene Expression Regulation, Enzymologic , Nuclear Proteins/chemistry , Nuclear Proteins/metabolism , Trans-Activators/chemistry , Trans-Activators/metabolism , Transcription Factors/chemistry , Transcription Factors/physiology , Zinc Fingers , Adrenal Cortex/metabolism , Amino Acid Sequence , Base Sequence , Binding Sites , Blotting, Northern , Blotting, Western , Cell Line , Cell Nucleus/metabolism , DNA, Complementary/metabolism , Gene Library , HeLa Cells , Humans , Immunohistochemistry , Male , Microscopy, Fluorescence , Molecular Sequence Data , Placenta/metabolism , Plasmids/metabolism , Precipitin Tests , Promoter Regions, Genetic , Protein Binding , Protein Structure, Tertiary , Ribonucleases/metabolism , Steroids/metabolism , Testis/metabolism , Thymus Gland/metabolism , Tissue Distribution , Transfection , Tumor Cells, Cultured
10.
DNA Cell Biol ; 17(8): 707-15, 1998 Aug.
Article in English | MEDLINE | ID: mdl-9726253

ABSTRACT

Microsomal cytochrome P450c17 (17a-hydroxylase/17,20-Lyase) catalyzes two reactions in the delta5 and delta4 pathways leading to the production of C19 steroids. Transient expression of human, bovine, porcine, rat, and mouse P450c17 cDNAs showed that the protein has 17alpha-hydroxylase and 17,20-Lyase activities, converting pregnenolone and progesterone into delta5- and delta4-Cl9 steroids, respectively, although the rat and mouse proteins have a preferential pathway toward the delta4 steroids. The guinea pig (gp) P450c17 shares 46% to 70% amino acid identity with the corresponding proteins of other species, and further characterization indicated that the guinea pig enzyme only converts progesterone to androstenedione. In this study, we have tried to identify amino acid(s) in the gpP450c17 that governs such a steroid specificity. Among the various mutants that we have created, change of the arginine (R) residue at position 200 to an asparagine (N) (R200N) in the gpP450c17 protein increased reactivity toward pregnenolone compared with the wild-type enzyme. Pregnenolone was converted into 17alpha-hydroxypregnenolone and dehydroepiandrosterone. However, this gain occurred at the expense of the 17,20-lyase activity toward 17alpha-hydroxyprogesterone. The R200N mutation in the gpP450c17 protein introduced a potential N-linked glycosylation site (200Asn-X-Thr202); however, substitution of the Thr202 residue by an asparagine (R200N/T202N), which abolishes the site, did not change the preference of the gpP450c17 mutant for pregnenolone. Furthermore, introduction of a putative glycosylation site at amino acid 185 in the gpP450c17 enzyme did not alter substrate specificity. The properties of the amino acid were also investigated, and neither the charge nor the size of the sidechain elicited change in the substrate specificity of gpP450c17. Thus, our results demonstrate that the mutation of arginine to asparagine at position 200 changes the substrate specificity of the gpP450c17 enzyme.


Subject(s)
Multienzyme Complexes/metabolism , Steroid 17-alpha-Hydroxylase/metabolism , Steroids/metabolism , 17-alpha-Hydroxypregnenolone/metabolism , Amino Acid Sequence , Animals , Binding Sites/genetics , Dehydroepiandrosterone/metabolism , Evolution, Molecular , Guinea Pigs , Molecular Sequence Data , Multienzyme Complexes/genetics , Mutagenesis, Site-Directed , Protein Processing, Post-Translational , Recombinant Proteins/metabolism , Sequence Homology, Amino Acid , Species Specificity , Steroid 17-alpha-Hydroxylase/genetics , Substrate Specificity
11.
J Clin Endocrinol Metab ; 82(1): 182-7, 1997 Jan.
Article in English | MEDLINE | ID: mdl-8989256

ABSTRACT

Dehydroepiandrosterone (DHEA)- and pregnenolone (PREG)-fatty acid esters (FA) are formed in plasma high density lipoproteins (HDL), whereas they accumulate in very low density lipoproteins (VLDL), low density lipoproteins (LDL), and HDL. We have hypothesized that these lipoidal steroids could be transferred from HDL to VLDL and LDL by the cholesteryl ester (CE) transfer protein (CETP), which mediates CE transfer activity in human plasma. In this study, we further investigated this hypothesis. Lipoproteins and lipoprotein-deficient plasma (LPDP) were purified and analyzed by Western blots. LPDP was rich in CETP, in contrast to lipoprotein preparations, which contained very low amounts. Using these preparations in in vitro transfer assays, CE transfer from radiolabeled steroid ester-HDL to VLDL or LDL was only observed in the presence of LPDP. In contrast, time- and temperature-dependent transfer of DHEA-FA and PREG-FA were observed in the absence of LPDP. The addition of LPDP had no effect on the DHEA-FA transfer rate, whereas the PREG-FA transfer rate was increased. Moreover, in the absence of LPDP, no decrease in the transfer levels of DHEA-FA and PREG-FA was observed after the removal of CETP from lipoprotein preparations by immunoaffinity column chromatography. The PREG-FA transfer activity of LPDP was studied using the anti-CETP monoclonal antibody TP-2, which is known to block the CE transfer activity of CETP. In the presence of LPDP, this antibody led to a dose-dependent decrease in CE transfer activity, whereas PREG-FA transfer activity was unaffected. In conclusion, we have shown that DHEA-FA and PREG-FA are transferred from HDL to VLDL and LDL by a CETP-independent mechanism. There is a major difference in the transport of lipoidal steroids by human lipoproteins compared to that of CE.


Subject(s)
Carrier Proteins/blood , Dehydroepiandrosterone/blood , Esters/blood , Fatty Acids/blood , Glycoproteins , Lipoproteins/blood , Pregnenolone/blood , Adult , Biological Transport , Blotting, Western , Cholesterol Ester Transfer Proteins , Humans , Kinetics , Lipoproteins, HDL/blood , Lipoproteins, LDL/blood , Lipoproteins, VLDL/blood , Male
12.
Clin Endocrinol (Oxf) ; 46(1): 93-100, 1997 Jan.
Article in English | MEDLINE | ID: mdl-9059564

ABSTRACT

OBJECTIVE: Insulin was found to decrease the concentration of serum dehydroepiandrosterone (DHEA) and DHEA-sulphate (DHEAS) and recent data suggest that an increase in the metabolic clearance rate of DHEA (MCRDHEA) may be involved. In this study, we have investigated the effects of insulin on DHEA metabolism in men. PATIENTS: A total of 10 men were enrolled into the study, and all subjects completed the study. Subjects were healthy, non-obese, and 20-30 years old. DESIGN: DHEA was administered intravenously to subjects, alone or in combination with insulin. A hyperinsulinaemic-euglycaemic clamp was initiated for subjects with the insulin infusion and euglycaemia was maintained by checking blood glucose and adjusting the rate of a 25% dextrose infusion as needed. Serum was collected before DHEA infusion, during DHEA infusion after attaining steady state (3.5-4 h), and during DHEA plus insulin infusion (6-6.5 h) (steady state) and then assayed for DHEA, DHEA metabolites, and DHEA acylation by LCAT. RESULTS: Results showed rapid transformation of DHEA into androst-5-ene-3 beta, 17 beta-diol, DHEA fatty-acid esters (DHEA-FA), androstenedione and 5 alpha-androstan-3 alpha-ol-17-one glucuronide (androsterone glucuronide) whereas DHEAS, testosterone, androstane-3 alpha, 17 beta-diol glucuronide and oestradiol serum levels were not affected. When insulin was simultaneously infused once steady-state DHEA levels had been attained, we observed a decline only in DHEA, DHEA-FA and DHEAS levels, with no effect on other steroids examined. Although serum DHEA esterification was not affected by DHEA, a stimulation by insulin was observed. CONCLUSIONS: These results suggest that insulin increases the DHEA metabolic clearance rate by stimulating its conversion to DHEA-FA and by enhancing uptake of DHEA-FA by peripheral tissues.


Subject(s)
Dehydroepiandrosterone/metabolism , Insulin/pharmacology , Adult , Androstenediols/metabolism , Androstenedione/metabolism , Androsterone/analogs & derivatives , Androsterone/metabolism , Dehydroepiandrosterone/pharmacology , Dehydroepiandrosterone Sulfate/blood , Esters , Fatty Acids/metabolism , Humans , Male , Metabolic Clearance Rate/drug effects , Stimulation, Chemical
13.
J Endocrinol ; 150 Suppl: S119-24, 1996 Sep.
Article in English | MEDLINE | ID: mdl-8943795

ABSTRACT

In addition to dehydroepiandrosterone (DHEA) sulfate (S), the human plasma also contains a second form of DHEA ester: DHEA-fatty acid esters (DHEA-FA). In the human adult, the plasma concentrations of DHEA-FA, DHEA and DHEAS are in the range of 6, 12 and 2000 nM respectively. Although the adrenal is responsible for almost all production of DHEAS in the circulation, DHEA-FA is formed from DHEA by an enzyme present in the circulation. Our work has clearly demonstrated that lecithin-cholesterol acyltransferase, localized on high density lipoprotein, is responsible for DHEA-FA production. Once DHEA-FA is formed, it is subsequently transferred to very low density lipoprotein (VLDL) and low density lipoprotein (LDL), like cholesteryl esters. Plasma lipoproteins contain at least 90% of circulating DHEA-FA of which 40% are found in the LDL fraction. Analysis of the fatty acid composition of tritiated DHEA-FA-labelled LDL ([3H]DHEA-FA-LDL) indicated the prevalence of DHEA-linoleate/palmitoleate and DHEA-oleate. Treatment of [3H]steroid-FA-LDL with charcoal does not remove radioactivity, thus suggesting that the non-polar steroid is incorporated into the central non-polar core of the lipoproteins. Incubation of [3H]DHEA-FA-LDL with ZR-75-1 breast cancer cells produced a time-dependent increase in labeled non-conjugated steroids in the cell culture medium, whereas the levels of tritiated DHEA-FA decreased. Lipoidal radioactivity in cells increased with time, but non-conjugated radioactivity associated with the cells showed no such increase. HPLC analysis of the culture medium indicated the presence of tritiated DHEA and androst-5-one-3 beta, 17 beta-diol. Our study indicates that circulating DHEA-FA incorporated into lipoproteins may indeed act as a substrate for potent steroid formation following their entry into steroid target cells.


Subject(s)
Dehydroepiandrosterone/metabolism , Fatty Acids/metabolism , Adrenal Glands/metabolism , Dehydroepiandrosterone/blood , Esters , Fatty Acids/blood , Female , Gonads/metabolism , Humans , Lipoproteins, VLDL/metabolism , Male , Phosphatidylcholine-Sterol O-Acyltransferase/metabolism , Placenta/metabolism
14.
Biochim Biophys Acta ; 1299(3): 306-12, 1996 Feb 16.
Article in English | MEDLINE | ID: mdl-8597585

ABSTRACT

Pregnenolone- (PREG-), and dehydroepiandrosterone- (DHEA-) fatty acid esters (FA) are present in human plasma, where they are associated with lipoproteins. Because plasma has the ability to form PREG-FA and DHEA-FA in vitro from their unconjugated steroid counterparts, we postulated that the LCAT enzyme might be responsible for their formation. Here we show that lecithin-cholesterol acyltransferase (LCAT) has PREG and DHEA esterifying activities. First, VLDL, IDL, LDL, and HDL were isolated by the sequential ultracentrifugation micromethod from the plasma of fasting men and women and tested for their ability to form PREG-FA, DHEA-FA, and cholesteryl esters in vitro from their respective unconjugated counterparts. The results showed that the three steroids were esterified only in HDL subfractions. The rate of tritiated PREG esterification was clearly higher than that of tritiated cholesterol and DHEA, both in total plasma and isolated HDL, and no gender difference was observed. Second, human and guinea pig LCAT were purified and used in phosphatidylcholine-reconstituted vesicles containing human apoAI to show their ability to esterify tritiated cholesterol, PREG, and DHEA in the absence of unlabeled steroid. The amount of cholesteryl ester, PREG-FA, and DHEA-FA increased after incubation as a function of time and amount of purified LCAT, showing that PREG is preferentially acylated by LCAT compared to cholesterol and DHEA. The PREG and DHEA esterifying activities of LCAT were cofactor-dependent, as shown by the absence of acylation without apoAI. Finally, we determined by HPLC the fatty acid moiety of PREG-FA and DHEA-FA formed in human plasma and guinea pig and rat sera in vitro after incubation with unconjugated tritiated PREG and DHEA. We showed that the fatty acid moieties of newly formed tritiated PREG-FA and DHEA-FA were similar to that reported for cholesteryl esters in the plasma of the three species. We conclude that LCAT has a lecithin-steroid acyltransferase activity and that PREG is probably the preferential substrate of this enzyme. In addition, the fact that the differences in the fatty acid moieties of cholesteryl esters of human, guinea pig, and rat plasmas are also observed for PREG-FA and DHEA-FA suggests that the LCAT is the sole circulating enzyme that has PREG and DHEA esterifying activities.


Subject(s)
Dehydroepiandrosterone/metabolism , Fatty Acids/metabolism , Lipoproteins, HDL/metabolism , Phosphatidylcholine-Sterol O-Acyltransferase/blood , Pregnenolone/metabolism , Adult , Animals , Apolipoprotein A-I/pharmacology , Cholesterol/metabolism , Esterification/drug effects , Esters/chemistry , Female , Guinea Pigs , Humans , Male , Phosphatidylcholine-Sterol O-Acyltransferase/isolation & purification , Rats , Species Specificity
17.
Appl Ther ; 10(5): 331-3, 1968 May.
Article in English | MEDLINE | ID: mdl-5649877
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