ConA-mediated binding and uptake of purified alpha-galactosidase A in Fabry fibroblasts

In most human tissues there are at least two different alpha-galactosidases, A and B. The former is deficient in patients hemizygous for Fabry disease. We have isolated it from human placenta and found that it was labile even at culture conditions, but was stabilized after binding to concanavalin A (conA). The alpha-galactosidase activity was markedly increased in Fabry fibroblasts when these were treated with conA and exposed to alpha-galA at 37 degrees C. The maximum activity was obtained after 1/2-2 h of incubation and was maintained for at least 4 h. The binding and uptake of conA into Fabry cells was followed by microscopical studies of fluorescein-labelled conA. We assume that alpha-galA is taken up by endocytosis of the enzyme-conA complex.     

Mitogenic stimulation and the redistribution of concanavalin A receptors on lymphocytes

When mouse spleen (Ig⁻) cells undergo maximal mitogenic stimulation by optimal concentrations of concanavalin A (conA), the Ig⁻ cells form caps of conA very slowly, with 50% of maximum cap formation occurring after about 10 h and maximal capping after about 24 h. Anti-conA antibody added after optimal conA accelerates the rate of cap formation and effectively blocks mitogenic stimulation (< 10%) by optimal conA concentrations when the rate of capping is increased more than about 2-fold. The effect of anti-conA antibody in accelerating cap formation by optimal conA is antagonized by cytochalasin D (CD), which substantially restores the mitogenic action of optimal conA. Thus there is an inverse relationship between rate of cap formation and extent of mitogenic stimulation. Further experiments showed that if anti-conA antibody, α-methyl mannoside or EGTA were added at increasing intervals after the addition of conA, these inhibitors block the stimulation of the cells with very similar time courses. Addition of appropriate concentrations of an inhibitor at the same time as optimal conA blocks mitogenic stimulation completely, but has negligible effects after 24 h. The extent of stimulation which occurs after the addition of inhibitor at intermediate times closely follows the extent of cap formation at the same time. The simplest interpretation of these results is that mitogenic action by optimal conA can be blocked by (i) accelerated capping of uncapped cells; or (ii) by the removal of either conA or calcium before, but not after, cap formation has occurred. These results suggest that the rate of cap formation by conA, and the presence of external calcium (>10⁻⁴ M) in the medium for some unspecified period before cap formation occurs are both significant factors in generating the primary mitogenic signals which commit the cells to DNA synthesis.     

Fabry Disease – Underestimated in the Differential Diagnosis of Multiple Sclerosis?

Objective

Fabry disease is a rare X-linked inherited lysosomal storage disorder affecting multiple organ systems. It includes central nervous system involvement via micro- and macroangiopathic cerebral changes. Due to its clinical symptoms and frequent MRI lesions, Fabry disease is commonly misdiagnosed as multiple sclerosis. We present an overview of cases from Fabry centres in Germany initially misdiagnosed with multiple sclerosis and report the clinical, MR-tomographical, and laboratory findings.

Methods

Eleven Fabry patients (one male, ten females) initially diagnosed with multiple sclerosis were identified from 187 patient records (5.9%) and analyzed for presenting symptoms, results of the initial diagnostic workup, and the clinical course of the disease.

Results

Four patients were identified as having a “possible” history of MS, and 7 patients as “definite” cases of multiple sclerosis (revised McDonald criteria). On average, Fabry disease was diagnosed 8.2 years (±9.8 years) after the MS diagnosis, and 12.8 years after onset of first symptoms (±10.3 years). All patients revealed white matter lesions on MRI. The lesion pattern and results of cerebrospinal fluid examination were inconsistent and non-specific. White matter lesion volumes ranged from 8.9 mL to 34.8 mL (mean 17.8 mL±11.4 mL). There was no association between extra-neurological manifestations or enzyme activity and lesion load.

Conclusion

There are several anamnestic and clinical hints indicating when Fabry disease should be considered a relevant differential diagnosis of multiple sclerosis, e.g. female patients with asymmetric, confluent white matter lesions on MRI, normal spinal MR imaging, ectatic vertebrobasilar arteries, proteinuria, or lack of intrathecally derived immunoglobulin synthesis.     

Expression and Characterization of Glycosylated and Catalytically Active Recombinant Human α-Galactosidase A Produced in Pichia pastoris

Fabry disease is an X-linked inborn error of glycolipid metabolism caused by deficiency of the lysosomal enzyme α-galactosidase A. This enzyme is responsible for the hydrolysis of terminal α-galactoside linkages in various glycolipids. An improved method of production of recombinant α-galactosidase A for use in humans is needed in order to develop new approaches for enzyme therapy. Human α-galactosidase A for use in enzyme therapy has previously been obtained from human sources and from recombinant clones derived from human cells, CHO cells, and insect cells. In this report we describe the construction of clones of the methylotrophic yeast Pichia pastoris that produce recombinant human α-galactosidase A. Recombinant human α-galactosidase A is secreted by these Pichia clones and the level of production is more than 30-fold greater than that of previously used methods. Production was optimized using variations in temperature, pH, cDNA copy number, and other variables using shake flasks and a bioreactor. Expression of the human enzyme increased with increasing cDNA copy number at 25°C, but not at the standard growth temperature of 30°C. The recombinant α-galactosidase A was purified to homogeneity using ion exchange (POROS 20 CM, POROS 20 HQ) and hydrophobic (Toso-ether, Toso-butyl) chromatography with a BioCAD HPLC Workstation. Purified recombinant α-galactosidase A was taken up by fibroblasts derived from Fabry disease patients and normal enzyme levels could be restored under these conditions. Analysis of the carbohydrate present on the recombinant enzyme indicated the predominant presence of N-linked high-mannose structures rather than complex carbohydrates.     

The Expression and Release of Adhesion Molecules by Human Endothelial Cell Lines and Their Consequent Binding of Lymphocytes

We report the characterization of a novel series of human endothelial cell lines (designated SGHEC) regarding the expression and release of adhesion molecules and their binding of lymphocytes. SGHEC expressed significant levels of intercellular adhesion molecule-1 (ICAM-1; CD54) which increased after stimulation with tumor necrosis factor-α (TNFα), interleukin-1β (IL-1β), or interferon-γ (IFN-γ). Vascular cell adhesion molecule-1 (VCAM-1; CD106) and E-selectin (CD62E) were not detectable on unstimulated SGHEC but substantial levels were expressed after stimulation with either TNFα or IL-1β but not with IFN-γ. The increased expression of ICAM-1 and VCAM-1 was evident after 4 h stimulation and was even higher after 24 h; E-selectin was maximal after 4 h and returned almost to basal levels by 24 h. Substantial quantities of immunoreactive ICAM-1 and VCAM-1 also accumulated as soluble material in the supernatants of TNFα-stimulated SGHEC (VCAM-1 was substantially higher than ICAM-1), but E-selectin remained below the limits of detection. Various quantitative data suggest that this is a controlled release regulated by cytokine and provide support for a physiological function for these soluble molecules. Primary human lymphocytes and lymphoblastoid cell lines expressing lymphocyte function-associated antigen-1 (LFA-1) bound to SGHEC; this binding increased substantially after activation of either cell type. The binding was inhibited by monoclonal antibodies against LFA-1 and, to a lesser extent, ICAM-1, thus demonstrating the importance of these molecules in the observed binding; neither anti-VCAM-1 nor anti-E-selectin antibodies affected the binding. From these various data, we conclude that LFA-1/ICAM-1 interactions are partially responsible for the binding of lymphocytes to endothelial cells. The SGHEC lines should prove useful in investigating leukocyte-endothelial interactions and the mechanism of release of soluble adhesion molecules.     

Efficacy of Enzyme and Substrate Reduction Therapy with a Novel Antagonist of Glucosylceramide Synthase for Fabry Disease

Fabry disease, an X-linked glycosphingolipid storage disorder, is caused by the deficient activity of α-galactosidase A (α-Gal A). This results in the lysosomal accumulation in various cell types of its glycolipid substrates, including globotriaosylceramide (GL-3) and lysoglobotriaosylceramide (globotriaosyl lysosphingolipid, lyso-GL-3), leading to kidney, heart, and cerebrovascular disease. To complement and potentially augment the current standard of care, biweekly infusions of recombinant α-Gal A, the merits of substrate reduction therapy (SRT) by selectively inhibiting glucosylceramide synthase (GCS) were examined. Here, we report the development of a novel, orally available GCS inhibitor (Genz-682452) with pharmacological and safety profiles that have potential for treating Fabry disease. Treating Fabry mice with Genz-682452 resulted in reduced tissue levels of GL-3 and lyso-GL-3 and a delayed loss of the thermal nociceptive response. Greatest improvements were realized when the therapeutic intervention was administered to younger mice before they developed overt pathology. Importantly, as the pharmacologic profiles of α-Gal A and Genz-682452 are different, treating animals with both drugs conferred the greatest efficacy. For example, because Genz-682452, but not α-Gal A, can traverse the blood–brain barrier, levels of accumulated glycosphingolipids were reduced in the brain of Genz-682452–treated but not α-Gal A–treated mice. These results suggest that combining substrate reduction and enzyme replacement may confer both complementary and additive therapeutic benefits in Fabry disease.     

No Fabry Disease in Patients Presenting with Isolated Small Fiber Neuropathy

Objective

Screening for Fabry disease in patients with small fiber neuropathy has been suggested, especially since Fabry disease is potentially treatable. However, the diagnostic yield of testing for Fabry disease in isolated small fiber neuropathy patients has never been systematically investigated. Our aim is to determine the presence of Fabry disease in patients with small fiber neuropathy.

Methods

Patients referred to our institute, who met the criteria for isolated small fiber neuropathy were tested for Fabry disease by measurement of alpha-Galactosidase A activity in blood, lysosomal globotriaosylsphingosine in urine and analysis on possible GLA gene mutations.

Results

725 patients diagnosed with small fiber neuropathy were screened for Fabry disease. No skin abnormalities were seen except for redness of the hands or feet in 30.9% of the patients. Alfa-Galactosidase A activity was tested in all 725 patients and showed diminished activity in eight patients. Lysosomal globotriaosylsphingosine was examined in 509 patients and was normal in all tested individuals. Screening of GLA for mutations was performed for 440 patients, including those with diminished α-Galactosidase A activity. Thirteen patients showed a GLA gene variant. One likely pathogenic variant was found in a female patient. The diagnosis Fabry disease could not be confirmed over time in this patient. Eventually none of the patients were diagnosed with Fabry disease.

Conclusions

In patients with isolated small fiber neuropathy, and no other signs compatible with Fabry disease, the diagnostic yield of testing for Fabry disease is extremely low. Testing for Fabry disease should be considered only in cases with additional characteristics, such as childhood onset, cardiovascular disease, renal failure, or typical skin lesions.     

Inactivation by chloroquine of α-galactosidase in cultured human skin fibroblasts

When human skin fibroblasts are cultured in the presence of chloroquine or NH₄Cl there is a decrease in the intracellular level of lysosomal hydrolases and a concomitant increase in the extracellular activity as compared with cells grown in the absence of a base (cf [18]). In a medium with 25 μM chloroquine or 5 mM NH₄Cl, the decrease in the intracellular activity of β-hexosaminidase, arylsulphatase and β-glucuronidase is 10–40% after 1 day. A similar decrease in α-galactosidase activity is observed in cells grown in the presence of 5 mM NH₄Cl. However, in the presence of 25 μM chloroquine, the intracellular activity of α-galactosidase decreases by 80–90% within 6 h. The inactivation is irreversible. After removal of the chloroquine and further culture of the cells in chloroquine-free medium, α-galactosidase activity gradually increases due to de novo synthesis. The turnover time of α-galactosidase was calculated to be 1.9 days. Inactivation of α-galactosidase also occurs when homogenates are incubated with chloroquine, but the concentration of the base required for maximum inactivation is at least three orders of magnitude higher than that which must be present in the medium of intact cells to obtain the same effect.     

Circulating Stromal Cell-Derived Factor 1α Levels in Heart Failure: A Matter of Proper Sampling

Background

The chemokine Stromal cell-derived factor 1α (SDF1α, CXCL12) is currently under investigation as a biomarker for various cardiac diseases. The correct interpretation of SDF1α levels is complicated by the occurrence of truncated forms that possess an altered biological activity.

Methodology

We studied the immunoreactivities of SDF1α forms and evaluated the effect of adding a DPP4 inhibitor in sampling tubes on measured SDF1α levels. Using optimized sampling, we measured DPP4 activity and SDF1α levels in patients with varying degrees of heart failure.

Results

The immunoreactivities of SDF1α and its degradation products were determined with three immunoassays. A one hour incubation of SDF1α with DPP4 at 37°C resulted in 2/3 loss of immunoreactivity in each of the assays. Incubation with serum gave a similar result. Using appropriate sampling, SDF1α levels were found to be significantly higher in those heart failure patients with a severe loss of left ventricular function. DPP4 activity in serum was not altered in the heart failure population. However, the DPP4 activity was found to be significantly decreased in patients with high SDF1α levels

Conclusions

We propose that all samples for SDF1α analysis should be collected in the presence of at least a DPP4 inhibitor. In doing so, we found higher SDF1α levels in subgroups of patients with heart failure. Our work supports the need for further research on the clinical relevance of SDF1α levels in cardiac disease.     

Mosaicism of Podocyte Involvement Is Related to Podocyte Injury in Females with Fabry Disease

Background

Fabry disease. an X-linked deficiency of α-galactosidase A coded by the GLA gene, leads to intracellular globotriaosylceramide (GL-3) accumulation. Although less common than in males, chronic kidney disease, occurs in ∼15% of females. Recent studies highlight the importance of podocyte injury in Fabry nephropathy development and progression. We hypothesized that the greater the % of podocytes with active wild-type GLA gene (due to X-inactivation of the mutant copy) the less is the overall podocyte injury.

Methods

Kidney biopsies from 12 treatment-naive females with Fabry disease, ages 15 (8–63), median [range], years were studied by electron microscopy and compared with 4 treatment-naive male patients.

Results

In females, 51 (13–100)% of podocytes (PC) per glomerulus had no GL-3 inclusions, this consistent with a non-Fabry podocyte phenotype (NFPC). In PC with GL-3 inclusions [Fabry podocyte phenotype (FPC)], GL-3 volume density per podocyte was virtually identical in females and males, consistent with little or no cross-correction between FPC and NFPC. %NFPC per glomerulus (%NFPC/glom) correlated with age in females (r = 0.65, p = 0.02), suggesting a survival disadvantage for FPC over time. Age-adjusted %NFPC/glom was inversely related to foot process width (FPW) (r = −0.75, p = 0.007), an indicator of PC injury. GL-3 volume density in FPC in females correlated directly with FPW.

Conclusions

These findings support important relationships between podocyte mosaicism and podocyte injury in female Fabry patients. Kidney biopsy, by providing information about podocyte mosaicism, may help to stratify females with Fabry disease for kidney disease risk and to guide treatment decisions.     

Induction and regulation of matrix metalloproteinase-12in human airway smooth muscle cells

Background

The elastolytic enzyme matrix metalloproteinase (MMP)-12 has been implicated in the development of airway inflammation and remodeling. We investigated whether human airway smooth muscle cells could express and secrete MMP-12, thereby participating in the pathogenesis of airway inflammatory diseases.

Methods

Laser capture microdissection was used to collect smooth muscle cells from human bronchial biopsy sections. MMP-12 mRNA expression was analysed by quantitative real-time RT-PCR. MMP-12 protein expression and secretion from cultured primary airway smooth muscle cells was further analysed by Western blot. MMP-12 protein localization in bronchial tissue sections was detected by immunohistochemistry. MMP-12 activity was determined by zymography. The TransAM AP-1 family kit was used to measure c-Jun activation and nuclear binding. Analysis of variance was used to determine statistical significance.

Results

We provide evidence that MMP-12 mRNA and protein are expressed by in-situ human airway smooth muscle cells obtained from bronchial biopsies of normal volunteers, and of patients with asthma, COPD and chronic cough. The pro-inflammatory cytokine, interleukin (IL)-1β, induced a >100-fold increase in MMP-12 gene expression and a >10-fold enhancement in MMP-12 activity of primary airway smooth muscle cell cultures. Selective inhibitors of extracellular signal-regulated kinase, c-Jun N-terminal kinase and phosphatidylinositol 3-kinase reduced the activity of IL-1β on MMP-12, indicating a role for these kinases in IL-1β-induced induction and release of MMP-12. IL-1β-induced MMP-12 activity and gene expression was down-regulated by the corticosteroid dexamethasone but up-regulated by the inflammatory cytokine tumour necrosis factor (TNF)-α through enhancing activator protein-1 activation by IL-1β. Transforming growth factor-β had no significant effect on MMP-12 induction.

Conclusion

Our findings indicate that human airway smooth muscle cells express and secrete MMP-12 that is up-regulated by IL-1β and TNF-α. Bronchial smooth muscle cells may be an important source of elastolytic activity, thereby participating in remodeling in airway diseases such as COPD and chronic asthma.     

Epithelial–Mesenchymal Transition in Kidney Tubular Epithelial Cells Induced by Globotriaosylsphingosine and Globotriaosylceramide

Fabry disease is a lysosomal storage disorder caused by deficiency of alpha-galactosidase A (α-gal A), which results in the deposition of globotriaosylceramide (Gb3) in the vascular endothelium. Globotriaosylsphingosine (lyso-Gb3), a deacylated Gb3, is also increased in the plasma of patients with Fabry disease. Renal fibrosis is a key feature of advanced Fabry disease patients. Therefore, we evaluated the association of Gb3 and lyso-Gb3 accumulation and the epithelial–mesenchymal transition (EMT) on tubular epithelial cells of the kidney. In HK2 cells, exogenous treatments of Gb3 and lyso-Gb3 increased the expression of TGF-β, EMT markers (N-cadherin and α-SMA), and phosphorylation of PI3K/AKT, and decreased the expression of E-cadherin. Lyso-Gb3, rather than Gb3, strongly induced EMT in HK2 cells. In the mouse renal mesangial cell line, SV40 MES 13 cells, Gb3 strongly induced phenotype changes. The EMT induced by Gb3 was inhibited by enzyme α-gal A treatment, but EMT induced by lyso-Gb3 was not abrogated by enzyme treatment. However, TGF-β receptor inhibitor (TRI, SB525334) inhibited the activation of TGF-β and EMT markers in HK2 cells with Gb3 and lyso-Gb3 treatments. This study suggested that increased plasma lyso-Gb3 has a crucial role in the development of renal fibrosis through the cell-specific induction of the EMT in Fabry disease, and that TRI treatment, alongside enzyme replacement therapy, could be a potential therapeutic option for patients with Fabry disease.     

Analysis of Alpha-2 Macroglobulin from the Long-Lived and Cancer-Resistant Naked Mole-Rat and Human Plasma

Background

The naked mole-rat (NMR) is a long-lived and cancer resistant species. Identification of potential anti-cancer and age related mechanisms is of great interest and makes this species eminent to investigate anti-cancer strategies and understand aging mechanisms. Since it is known that the NMR expresses higher liver mRNA-levels of alpha 2-macroglobulin than mice, nothing is known about its structure, functionality or expression level in the NMR compared to the human A2M.

Results

Here we show a comprehensive analysis of NMR- and human plasma-A2M, showing a different prediction in glycosylation of NMR-A2M, which results in a higher molecular weight compared to human A2M. Additionally, we found a higher concentration of A2M (8.3±0.44 mg/mL vs. and 4.4±0.20 mg/mL) and a lower total plasma protein content (38.7±1.79 mg/mL vs. 61.7±3.20 mg/mL) in NMR compared to human. NMR-A2M can be transformed by methylamine and trypsin resulting in a conformational change similar to human A2M. NMR-A2M is detectable by a polyclonal antibody against human A2M. Determination of tryptic and anti-tryptic activity of NMR and human plasma revealed a higher anti-tryptic activity of the NMR plasma. On the other hand, less proteolytic activity was found in NMR plasma compared to human plasma.

Conclusion

We found transformed NMR-A2M binding to its specific receptor LRP1. We could demonstrate lower protein expression of LRP1 in the NMR liver tissue compared to human but higher expression of A2M. This was accompanied by a higher EpCAM protein expression as central adhesion molecule in cancer progression. NMR-plasma was capable to increase the adhesion in human fibroblast in vitro most probably by increasing CD29 protein expression. This is the first report, demonstrating similarities as well as distinct differences between A2M in NMR and human plasma. This might be directly linked to the intriguing phenotype of the NMR and suggests that A2M might probably play an important role in anti-cancer and the anti-aging mechanisms in the NMR.     

Localization of the gene for a third G protein β-subunit to mouse chromosome 6 near Raf-1

The large family of signal transducing proteins known as G proteins are heterotrimers that dissociate into an independent α-subunit and βγ-subunit complex after ligand binding or other stimulation. For Gα, at least 30 distinct sequences representing 10 different classes have been identified. On the other hand, cDNAs for only three Gβ-subunit genes have been isolated so far. All three of the Gβ genes have been chromosomally mapped in the human, but only two in the mouse. Using a human retinal cDNA for the third G protein β-subunit, we have mapped the corresponding gene, termed Gnb-3, to mouse Chromosome 6 with somatic cell hybrids and have positioned it distal to but near the marker Raf-1 by analysis of the progeny of three genetic crosses.     

Katanin Severing and Binding Microtubules Are Inhibited by Tubulin Carboxy Tails

Microtubule dynamics in cells are regulated by associated proteins that can be either stabilizers or destabilizers. A class of destabilizers that is important in a large number of cellular activities is the microtubule-severing enzymes, yet little is known about how they function. Katanin p60 was the first ATPase associated with microtubule severing. Here, we investigate the activity of katanin severing using a GFP-labeled human version. We quantify the effect of katanin concentration on katanin binding and severing activity. We find that free tubulin can inhibit severing activity by interfering with katanin binding to microtubules. The inhibition is mediated by the sequence of the tubulin and specifically depends on the carboxy-terminal tails. We directly investigate the inhibition effect of tubulin carboxy-terminal tails using peptide sequences of α-, β-, or detyrosinated α-tubulin tails that have been covalently linked to bovine serum albumin. Our results show that β-tubulin tails are the most effective at inhibiting severing, and that detyrosinated α-tubulin tails are the least effective. These results are distinct from those for other severing enzymes and suggest a scheme for regulation of katanin activity in cells dependent on free tubulin concentration and the modification state of the tubulin.     

Melittin Suppresses HIF-1α/VEGF Expression through Inhibition of ERK and mTOR/p70S6K Pathway in Human Cervical Carcinoma Cells

Objective

Melittin (MEL), a major component of bee venom, has been associated with various diseases including arthritis, rheumatism and various cancers. In this study, the anti-angiogenic effects of MEL in CaSki cells that were responsive to the epidermal growth factor (EGF) were examined.

Methodology/Principal Findings

MEL decreased the EGF-induced hypoxia-inducible factor-1α (HIF-1α) protein and significantly regulated angiogenesis and tumor progression. We found that inhibition of the HIF-1α protein level is due to the shortened half-life by MEL. Mechanistically, MEL specifically inhibited the EGF-induced HIF-1α expression by suppressing the phosphorylation of ERK, mTOR and p70S6K. It also blocked the EGF-induced DNA binding activity of HIF-1α and the secretion of the vascular endothelial growth factor (VEGF). Furthermore, the chromatin immunoprecipitation (ChIP) assay revealed that MEL reduced the binding of HIF-1α to the VEGF promoter HRE region. The anti-angiogenesis effects of MEL were confirmed through a matrigel plus assay.

Conclusions

MEL specifically suppressed EGF-induced VEGF secretion and new blood vessel formation by inhibiting HIF-1α. These results suggest that MEL may inhibit human cervical cancer progression and angiogenesis by inhibiting HIF-1α and VEGF expression.     

Luteolytic action of RU486: Modulation of luteal 3β-hydroxysteroid dehydrogenase and 20α-hydroxysteroid dehydrogenase activities in late pregnant rats

The effect of the synthetic antiprogestin RU486 on luteal function in late pregnant rats was studied by evaluating the activities of the enzymes 3β-hydroxysteroid dehydrogenase (3β-HSD) and 20α-hydroxysteroid dehydrogenase (20α-HSD). RU486 (2 mg/kg) administered to rats on day 18 of pregnancy at 10.00 h induced preterm delivery 26.4 ± 0.35 h (n = 8) after treatment. Luteal 3β-HSD activity increased 24 and 34 h after RU486 injection, but a significant and progressive decrease started at 48 h with the maximal reduction 72 h after RU486 treatment, when compared with controls. Serum progesterone concentration decreased at the time of 3β-HSD activity reduction. Interestingly, 20α-HSD activity started to increase 58 h after RU486 injection. The administration of the cyclooxygenase inhibitor, diclofenac (1.3 mg/kg), on days 17–19 of pregnancy to RU486-treated rats, delayed abortion and the duration of delivery, and prevented the decrease in 3β-HSD and the increase in 20α-HSD activities observed 58 h after antiprogesterone treatment. RU486 administered intrabursally (1 μg per ovary) on day 20 (14.00–15.00 h) increased 3β-HSD and decreased 20α-HSD luteal activities at 18.00 h on day 21 of pregnancy, without modifying serum progesterone concentration, when compared with normal pregnant rats. In conclusion, the luteolytic process after preterm delivery induced by RU486 administration in late pregnant rats is characterized by a decrease in luteal 3β-HSD activity and circulating progesterone, which may trigger the increase in luteal 20α-HSD activity. Prostaglandins seems to be involved in the increase of 20α-HSD activity and therefore, in the demise of corpora lutea.