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Biomedical subjects

G A Garcia

Publications and source records attributed to G A Garcia.

At least 19 recordsLinked to original sources
PubMed2002

Effect of norethisterone and its A-ring reduced metabolites on the acrosome reaction in porcine spermatozoa.

The synthetic progestin, norethisterone (NET), has been reported as a contragestational postcoital agent in humans, rodents and rabbits. The effect and molecular mechanisms of NET and its A-ring reduced metabolites, 5alpha-NET and 3beta5alpha-NET, on the acrosome reaction (AR) are unknown. The aim of this study was to assess the effect of these compounds on an in vitro progesterone-induced AR in porcine spermatozoa. The spermatozoa were obtained from semen ejaculated by proven fertile adult pigs. Seminal plasma removed and incubated under capacitating conditions was performed in TALP-Hepes medium for 4 h. Progesterone (P4) and three different progestins: norethisterone (NET), 5alpha-norethisterone (5alpha-NET) and 3beta5alpha-NET were then added at equimolar doses, and the spermatozoa were incubated for 15 min. Double-staining with PSA-FITC and Hoechst-33258 assessed the AR and sperm viability. Both P4 and NET induced the AR, while 5alpha-NET not only did not induce this process, but was able to block the effect of P4 on the spermatozoa. 3beta5alpha-NET was not able to inhibit P4 action. These results suggest that NET and its A-ring reduced metabolites act in different ways on the progesterone-induced AR in porcine spermatozoa.

Acrosome Reaction↗
PubMed2001

tRNA-guanine transglycosylase from Escherichia coli: molecular mechanism and role of aspartate 89.

The enzyme tRNA-guanine transglycosylase (TGT, EC 2.4.2.29) catalyzes a posttranscriptional transglycosylation reaction involved in the incorporation of the modified base queuine [Q, 7-(4,5-cis-dihydroxy-2-cyclopenten-1-ylaminomethyl)-7-deazaguanine] into tRNA. Previously, the crystal structure of the TGT from Zymomonas mobilis was solved in complex with preQ(1) (the substrate for the eubacterial TGT) [Romier et al. (1996) EMBO J. 15, 2850-2857]. An aspartate residue at position 102 (position 89 in the Escherichia coli TGT) was proposed to play a nucleophilic role in an associative catalytic mechanism. Although this is an attractive and precedented mechanism, a dissociative mechanism is equally plausible. In a dissociative mechanism, aspartate 89 would provide electrostatic stabilization of an oxocarbenium ion intermediate that is formed by dissociation of guanine. To clarify the nature of the catalytic mechanism of TGT, we have generated and characterized four mutations of aspartate 89 in the E. coli TGT (alanine, asparagine, cysteine, and glutamate). All four mutant TGTs were able to noncovalently bind tRNA, but only the glutamate mutant was able to form a stable complex with the RNA substrate under denaturing conditions that was comparable to wild type. Furthermore, the glutamate mutant was the only mutant TGT that demonstrated significant activity. Kinetic parameters were determined for this enzyme and shown to be comparable to wild type, revealing that the enzyme is considerably tolerant of the positioning of the carboxylate. Under conditions of high enzyme concentrations and long time courses, the alanine, asparagine, and cysteine mutants showed very low levels (ca. 10(3)-fold lower than wild type) of activity that were linear with respect to enzyme concentration and dependent upon pH in a fashion similar to that of the wild type. However, the observed initial velocities were too low to accurately determine k(cat) and K(m) values. We hypothesize that the activity observed for these mutants is most likely derived from host strain TGT (wt) contamination. These results are most consistent with aspartate 89 acting as a nucleophile in an associative catalytic mechanism.

Aspartic Acid↗
PubMed2001

A new target for shigellosis: rational design and crystallographic studies of inhibitors of tRNA-guanine transglycosylase.

Eubacterial tRNA-guanine transglycosylase (TGT) is involved in the hyper-modification of cognate tRNAs leading to the exchange of G34 at the wobble position in the anticodon loop by preQ1 (2-amino-5-(aminomethyl)pyrrolo[2,3-d]pyrimidin-4(3H)-one) as part of the biosynthesis of queuine (Q). Mutation of the tgt gene in Shigella flexneri results in a significant loss of pathogenicity of the bacterium, revealing TGT as a new target for the design of potent drugs against Shigellosis. The X-ray structure of Zymomonas mobilis TGT in complex with preQ1 was used to search for new putative inhibitors with the computer program LUDI. An initial screen of the Available Chemical Directory, a database compiled from commercially available compounds, suggested several hits. Of these, 4-aminophthalhydrazide (APH) showed an inhibition constant in the low micromolar range. The 1.95 A crystal structure of APH in complex with Z. mobilis TGT served as a starting point for further modification of this initial lead.

Binding Sites↗
PubMed2001

tRNA recognition by tRNA-guanine transglycosylase from Escherichia coli: the role of U33 in U-G-U sequence recognition.

In eubacteria, the biosynthesis of queuine, a modified base found in the wobble position (#34) of tRNAs coding for Tyr, His, Asp, and Asn, occurs via a multistep pathway. One of the key enzymes in this pathway, tRNA-guanine transglycosylase (TGT), exchanges the genetically encoded guanine at position 34 with a queuine precursor, preQ1. Previous studies have identified a minimal positive RNA recognition motif for Escherichia coli TGT consisting of a stable minihelix that contains a U-G-U sequence starting at the second position of its seven base anticodon loop. Recently, we reported that TGT was capable of recognizing the U-G-U sequence outside of this limited structural context. To further characterize the ability of TGT to recognize the U-G-U sequence in alternate contexts, we constructed mutants of the previously characterized E. coli tRNA(Tyr) minihelix. The U-G-U sequence was shifted to various positions within the anticodon loop of these mutants. Characterization of these analogs demonstrates that in addition to the normal U33G34U35 position, TGT can also recognize the U34G35U36 analog (UGU(+1)). The other analogs were not active. This indicates that the recognition of the U-G-U sequence is not strictly dependent upon its position relative to the stem. In E. coli, the full-length tRNA with a U34G35U36 anticodon sequence is one of the isoacceptors that codes for threonine. We found that TGT is able to recognize tRNA(Thr(UGU)) but only in the absence of a uridine at position 33. U33, an invariant base present in all tRNAs, has been shown to strongly influence the conformation of the anticodon loop of certain tRNAs. We find that mutation of this base confers on TGT the ability to recognize U34G35U36, and suggests that loop conformation affects recognition. The fact that the other analogs were not active indicates that although TGT is capable of recognizing the U-G-U sequence in additional contexts, this recognition is not indiscriminate.

Anticodon↗
PubMed2000

tRNA-guanine transglycosylase from Escherichia coli: recognition of noncognate-cognate chimeric tRNA and discovery of a novel recognition site within the TpsiC arm of tRNA(Phe).

tRNA-guanine transglycosylase (TGT) is a key enzyme involved in the posttranscriptional modification of tRNA across the three kingdoms of life. In eukaryotes and eubacteria, TGT is involved in the introduction of queuine into the anticodon of the cognate tRNAs. In archaebacteria, TGT is responsible for the introduction of archaeosine into the D-loop of the appropriate tRNAs. The tRNA recognition patterns for the eubacterial (Escherichia coli) TGT have been studied. These studies are all consistent with a restricted recognition motif involving a U-G-U sequence in a seven-base loop at the end of a helix. While attempting to investigate the potential of negative recognition elements in noncognate tRNAs via the use of chimeric tRNAs, we have discovered a second recognition site for the E. coli TGT in the TpsiC arm of in vitro-transcribed yeast tRNA(Phe). Kinetic analyses of synthetic mutant oligoribonucleotides corresponding to the TpsiC arm of the yeast tRNA(Phe) indicate that the specific site of TGT action is G53 (within a U-G-U sequence at the transition of the TpsiC stem into the loop). Posttranscriptional base modifications in tRNA(Phe) block recognition by TGT, most likely due to a stabilization of the tRNA structure such that G53 is inaccessible to TGT. These results demonstrate that TGT can recognize the U-G-U sequence within a structural context that is different than the canonical U-G-U in the anticodon loop of tRNA(Asp). Although it is unclear if this second recognition site is physiologically relevant, this does suggest that other RNA species could serve as substrates for TGT in vivo.

Base Sequence↗
PubMed1999

Mutagenesis and crystallographic studies of Zymomonas mobilis tRNA-guanine transglycosylase to elucidate the role of serine 103 for enzymatic activity.

The tRNA modifying enzyme tRNA-guanine transglycosylase (TGT) is involved in the exchange of guanine in the first position of the anticodon with preQ1 as part of the biosynthesis of the hypermodified base queuine (Q). Mutation of Ser90 to an alanine in Escherichia coli TGT leads to a dramatic reduction of enzymatic activity (Reuter, K. et al. (1994) Biochemistry 33, 7041-7046). To further clarify the role of this residue in the catalytic center, we have mutated the corresponding Ser103 of the crystallizable Zymomonas mobilis TGT into alanine. The crystal structure of a TGT(S103A)/preQ1 complex combined with biochemical data presented in this paper suggest that Ser103 is essential for substrate orientation in the TGT reaction.

Crystallography↗
PubMed1999

The androgenic effect of norethisterone and 5alpha-norethisterone on the contractile response of the rat vas deferens to methoxamine and serotonin.

Norethisterone (NET) and its metabolite 5alpha-norethisterone (5alpha-NET) are competitors for the androgen receptor. The sensitivity of the rat vas deferens to the contractile action of methoxamine and serotonin is regulated by hormonal and anatomical factors. The aim of this study was to evaluate the ability of NET and 5alpha-NET to induce the androgen-regulated contractile response to methoxamine and serotonin in the epididymal and prostatic portions of rat vas deferens. Adult male rats either intact, castrated or steroid-treated castrated were used. The contractility was recorded isometrically, and non-cumulative concentration-response curves to either methoxamine or serotonin were obtained. NET and 5alpha-NET partially restored the sensitivity to methoxamine and serotonin in the epididymal portion of castrated rats. The maximal responses to both agonists were significantly higher than those observed in castrated rats, and significantly lower than the responses observed in either intact or androgen-treated castrated rats. The prostatic portion was less responsive to both agonists than the epididymal portion, in all groups but castrated rats, as castration induced sensitivity to both agonists. NET and 5alpha-NET displayed a partial though similar androgenic activity in the rat vas deferens. These results contrast with previous reports where a decrease of androgenic effect due to the 5alpha-reduction of NET has been found.

Animals↗
PubMed1998

tRNA-guanine transglycosylase from Escherichia coli: recognition of full-length 'queuine-cognate' tRNAs.

A key enzyme involved in the incorporation of the modified base queuine into tRNA (position 34) is tRNA-guanine transglycosylase (TGT). Studies of the recognition of truncated tRNAs by the Escherichia coli TGT have established a minimal recognition motif involving a minihelix with a 7 base loop containing a U-G-U sequence (where G is replaced with queuine) [Curnow, A.W. and Garcia, G.A. (1995) J. Biol. Chem. 270, 17264-17267; Nakanishi, S. et al. (1994) J. Biol. Chem. 269, 32221-32225]. Still, a clearer understanding of the recognition of full-length 'queuine-cognate' tRNAs by TGT remains lacking. In this paper, we report the in vitro transcription and enzymological characterization (Km, and kcat) of all four 'queuine-cognate' tRNAs from E. coli and from Saccharomyces cerevisiae with the TGT from E. coli. No primary or secondary structures emerge as important recognition elements from this study. The modest differences in substrate specificity (relative kcat/Km values vary from 0.5 to 8.4) seen among these 'queuine-cognate' tRNAs most likely result from the accumulated effects of many subtle factors. Interestingly, the yeast tRNAs are essentially equivalent to the E. coli tRNAs as substrates for TGT, indicating that there is nothing intrinsic to the yeast tRNAs that accounts for the absence of queuine in yeast.

Base Sequence↗
PubMed1997

Cysteine 265 is in the active site of, but is not essential for catalysis by tRNA-guanine transglycosylase (TGT) from Escherichia coli.

Site-directed mutagenesis and X-ray absorption spectroscopy studies have previously shown that the tRNA-guanine transglycosylase (TGT) from Escherichia coli is a zinc metalloprotein and identified the enzymic ligands to the zinc [Chong et al. (1995), Biochemistry 34, 3694-3701; Garcia et al. (1966), Biochemistry 35, 3133-3139]. During these studies one mutant, TGT (C265A), was found to exhibit a significantly lower specific activity, but was not found to be involved in the zinc site. The present report demonstrates that TGT is inactivated by treatment with thiol reagents (e.g., DTNB, MMTS, and N-ethylmaleimide). Further, this inactivation is shown to be due to modification of cysteine 265. The kinetic parameters for the mutants TGT (C265A) and TGT (C265S), however, suggest that this residue is not performing a critical role in the TGT reaction. We conclude that cysteine 265 is in the active site of TGT, but is not performing a critical catalytic function. This conclusion is supported by the recent determination of the X-ray crystal structure of the TGT from Zymomonas mobilis [Romier et al. (1966), EMBO J. 15, 2850-2857], which reveals that the residue corresponding to cysteine 265 is distant from the putative catalytic site, but is in the middle of a region of the enzyme surface proposed to bind tRNA.

Binding Sites↗
PubMed1997

Vitreous body glutamate concentration in dogs with glaucoma.

OBJECTIVE: To analyze the vitreal amino acid concentrations in dogs with breed-related primary glaucoma to determine whether excitotoxic amino acids associated with retinal genglion cell death in other species were present in affected dogs. SAMPLES: 11 normal control and 10 glaucomatous canine eyes. PROCEDURE: Amino acid analyses were performed by high-pressure liquid chromatography in masked manner. RESULTS: Eyes from dogs with primary glaucoma had significantly high vitreal glutamate concentration, compared with values for eyes of clinically normal control dogs. Mean (+/-SD) glutamate concentrations were 31.7 +/- 12.4 and 6.9 +/- 6.3 microM in glaucomatous and normal eyes, respectively (P < 0.0001). Eyes from dogs with glaucoma also had lower vitreal glycine (37.0 +/- 17.0 vs 59.4 +/- 28.2 microM; P < 0.043) and higher of vitreal tryptophan (39.0 +/- 22.8 vs 17.5 +/- 11.2 microM; P < 0.012) concentrations, compared with values for normal eyes. CONCLUSION: Glutamate concentration potentially toxic to retinal ganglion cells is associated with the pathogenesis of primary glaucoma in dogs. Increased glutamate concentration provides evidence of an ischemic mechanism for retinal ganglion cell death and optic nerve atrophy in dogs with glaucoma. CLINICAL RELEVANCE: The emphasis on reduction and normalization of high intraocular pressure as the primary focus of treatment for glaucoma in dogs should be augmented by other therapeutic approaches.

Amino Acids↗
PubMed1996

X-ray absorption spectroscopy of the zinc site in tRNA-guanine transglycosylase from Escherichia coli.

A key step in the post-transcriptional modification of tRNA with queuine in Escherichia coli is the exchange of the queuine precursor, preQ1 into tRNA. This reaction is catalyzed by tRNA-guanine transglycosylase (TGT). We have previously shown that the E. coli TGT is a zinc metalloprotein [Chong et al. (1995) Biochemistry 34, 3694-3701]. Site-directed mutagenesis studies indicated that cysteines 302, 304, 307 and histidine 317 constitute the four ligands to the zinc. The involvement of histidine 317 is somewhat confounded by the presence of histidine 316. We have examined the zinc site in TGT (wt) and TGT (H317C) by X-ray absorption spectroscopy. The TGT (wt) data are most consistent with a tetracoordinate zinc with one nitrogen and three sulfur ligands. Interestingly, the data for TGT (H317C) are also consistent with a tetracoordinate zinc with one nitrogen and three sulfur ligands. The outer shell imidazole scattering for TGT (H317C) appears to be somewhat more ordered than that for TGT (wt), consistent with our previous suggestion that the wild-type enzyme may exist in two conformations the predominant one involving histidine 317 liganding to the zinc and the minor conformer involving histidine 316 liganding to the zinc. The minor conformer, with histidine 316 coordinating the zinc, appears to have an overall conformation that is subtly different from that of the wild-type enzyme. While TGT (H317C) has kinetic parameters very similar to the wild-type, it does not form the homotrimer quaternary structure of the wild-type. TGT (H317A) has previously [Chong et al. (1995) Biochemistry 34, 3694-3701] been found to contain a significant amount of zinc, but is essentially inactive. This suggests that careful analysis of EXAFS data can reveal subtle conformational changes in metal binding sites that are not observed in more common probes of protein conformation such as CD spectroscopy.

Absorptiometry, Photon↗
PubMed1995

Mechanism-based inactivation of tRNA-guanine transglycosylase from Escherichia coli by 2-amino-5-(fluoromethyl)pyrrolo[2,3-d]pyrimidin-4 (3H)-one.

In Escherichia coli, tRNA-guanine transglycosylase (TGT) catalyzes the incorporation of the queuine precursor preQ1 [2-amino-5-(aminomethyl)pyrrolo[2,3-d]pyrimidin-4(3H)-one] into tRNA. This precursor is further elaborated to queuine by two subsequent enzymic reactions [Slany, R. K., & Kersten, H. (1994) Biochimie 76, 1178-1182]. Our previous studies [Hoops, G. C., Townsend, L. B., & Garcia, G. A., (1995) Biochemistry (in press)] on a series of synthetic 5- and 6-substituted 2-aminopyrrolo[2,3-d]pyrimidin-4(3H) -ones have revealed that the E. coli TGT tolerates a wide diversity of substituents (isosteric, or nearly so, to the aminomethyl group of preQ1) at the 5 position. We report here that 2-amino-5-(fluoromethyl)pyrrolo[2,3-d]pyrimidin-4 (3H)-one (FMPP) inactivates TGT in a time- and concentration-dependent manner with k(inact) = 0.074 min-1 and KI = 136 microM. A competitive inhibitor (7-methyl-preQ1), with respect to preQ1, of TGT [Hoops, G.C., Townsend, L.B., & Garcia, G.A. (1995) Biochemistry (in press)] protects the enzyme from inactivation by FMPP. FMPP also acts as a competitive inhibitor (KI = 114 microM) of TGT under initial velocity conditions. The rate of fluoride release from FMPP is slightly faster (0.064 min-1) than the k(inact) (0.053 min-1) at 300 microM FMPP, consistent with fluoride release preceding inactivation. FMPP appears to partition between "normal" turnover (kcat = 0.461 min-1 and Km = 152 microM), inactivation, and an alternative processing to an unidentified, fluoride-released product.(ABSTRACT TRUNCATED AT 250 WORDS)

Enzyme Inhibitors↗
PubMed1995

tRNA-guanine transglycosylase from Escherichia coli: structure-activity studies investigating the role of the aminomethyl substituent of the heterocyclic substrate PreQ1.

A series of 5-substituted 2-aminopyrrolo[2,3-d]pyrimidin-4(3H)-ones have been synthesized in order to study the substrate specificity of the tRNA-guanine transglycosylase (TGT) from Escherichia coli. A number of these compounds were initially examined as inhibitors of radiolabeled guanine incorporation into tRNA catalyzed by TGT [Hoops, G. C., Garcia, G. A., & Townsend, L. B. (1992) 204th National Meeting of the American Chemical Society, Washington, DC, August 23-28, 1992, Division of Medicinal Chemistry, Abstract 113]. The kinetic parameters of these analogues as substrates in the TGT reaction have been determined by monitoring the loss of radiolabeled guanine from 8-[14C]G34-tRNA. This study reveals that the tRNA-guanine transglycosylase from E. coli will tolerate a wide variety of substituents at the 5-position. The role of the 5-substituent appears to be entirely in binding/recognition with no apparent effects upon catalysis. A correlation between N7 pKa and Vmax suggests the deprotonation of N7 during the reaction, which must occur prior to subsequent glycosidic bond formation, appears to be partially rate-determining for the natural substrate. Comparison of the Kis of 7-methyl-substituted competitive inhibitors to the Kms of their corresponding substrates suggests that some substrates (including preQ1) are kinetically "sticky" (i.e., Km is equivalent to Kd) and other substrates have Kms that reflect catalytic rates as well as binding.

Binding, Competitive↗
PubMed1995

tRNA-guanine transglycosylase from Escherichia coli. Minimal tRNA structure and sequence requirements for recognition.

Previously, we have demonstrated that the tRNA-guanine transglycosylase (TGT) from Escherichia coli is capable of utilizing an in vitro generated minihelix consisting of the anticodon stem and loop sequence of E. coli tRNA(Tyr) (Curnow, A. W., Kung, F. L., Koch, K. A., and Garcia, G. A. (1993) Biochemistry 32, 5239-5246). This suggests that the tRNA structural motifs necessary for recognition comprise a loop at the end of a short helix. To gain further insight into the structural requirements for TGT recognition, we have investigated the conformation of this minimal substrate. Thermal denaturation studies and kinetic analyses at 20 and 37 degrees C indicate that this minihelix is predominantly melted at 37 degrees C and that the melted conformation is not a substrate for TGT. This is confirmed by the determination that a non-helical analogue of the minihelix is not a substrate for TGT at either temperature. Two additional minihelices designed to be stable at 37 degrees C, ECYMH (a 4-base pair extension of the previous minihelix) and SCDMH (a yeast tRNA(Asp) analogue of ECYMH), were generated and characterized. Finally, several sequence mutants of SCDMH, focusing on the G30U40 base pair and U33G34U35 loop sequence, have been produced, and kinetic parameter determinations have been performed at 37 degrees C. Our results are consistent with a recent report (Nakanishi, S., Ueda, T., Hori, H., Yamazaki, N., Okada, N., and Watanabe, K. (1994) J. Biol. Chem. 269, 32221-32225) indicating that a UGU sequence in a 7-base loop is the minimal requirement for TGT recognition.

Anticodon↗
PubMed1995

tRNA-guanine transglycosylase from Escherichia coli is a zinc metalloprotein. Site-directed mutagenesis studies to identify the zinc ligands.

tRNA-guanine transglycosylase (TGT) from Escherichia coli catalyzes the exchange of the queuine precursor, preQ1, into tRNA as part of the biosynthetic pathway for the posttranscriptionally modified base, queuine. No significant sequence homologies exist between TGT and any of the proteins in the GenBank database. However, an unusual arrangement of cysteine residues was observed upon manual examination of the TGT sequence. Comparison of this sequence (residues 302-321) revealed similarities to structural zinc-binding motifs in proteins of known structure [Jaffe (1993) Comments Inorg. Chem. 15, 67-93]. Within this region of the TGT sequence, there are six residues (four cysteines and two histidines), any four of which could serve as the ligands to the zinc. We report here that wild-type TGT contains ca. 0.8 mol of zinc/mol of subunit, determined by atomic emission spectrometry. In order to determine which enzyme residues are serving as the ligands to the zinc, site-directed mutagenesis studies have been performed. Gross structural probes (native PAGE and CD spectra), enzyme activity assays, and tRNA-binding assays indicate that cysteines 302, 304, and 307 and histidine 317 are the ligands to the zinc. These results also suggest that the zinc site is necessary for TGT homotrimer formation and for tRNA binding.

Amino Acid Sequence↗
PubMed1995

Molecular mechanisms of the antihormonal and antiimplantation effects of norethisterone and its A-ring reduced metabolites.

Norethisterone (NET) has been used as a contragestational postcoital agent. It is biotransformed to 5 alpha dihydro-NET (5 alpha-NET) and 3 beta,5 alpha tetrahydro-NET (3 beta,5 alpha-NET) in target tissues. The participation of these metabolites in NET effects is unknown. We have examined the antiimplantation and antiprogestational effects of NET and its metabolites, in adult mated female rabbits, by assessing the number of implantation sites and the expression products of the uteroglobin (UTG) gene in the uterus, and by comparing them with those of RU-486 and estradiol. Steroids were daily administered s.c. at several doses for 7 consecutive days, starting 24 hr after coitus. To assure that fertilization occurred in all animals, the presence of early pregnancy factor was determined. The results demonstrated that high doses (5 mg/kg) of NET reduced both implantation and the expression of the UTG gene. On the other hand, lower doses (1.5 mg/kg) of 5 alpha-NET produced an antiimplantation effect and suppressed UTG synthesis and its mRNA. These effects were similar to those of RU-486. At lower doses (1 mg/kg), both estradiol and the estrogenic metabolite 3 beta,5 alpha-NET were also effective in inhibiting implantation and UTG gene expression. The overall results suggest that NET metabolites exert antiimplantation and antiprogestational effects through their interaction with progesterone and estrogen receptors, and provide an explanation for the molecular mechanisms involved in the postcoital contraceptive action of NET.

Animals↗
PubMed1995

Norethisterone metabolites modulate the uteroglobin and progesterone receptor gene expression in prepubertal rabbits.

Norethisterone (NET) is a synthetic progestin, used as a contraceptive agent, that is biotransformed at target tissues into 5 alpha-NET and 3 beta,5 alpha-NET, which possess different pharmacological properties. The effects of these metabolites on the expression of uteroglobin (UG) and progesterone receptor (PR) genes, both regulated by progesterone (P4), were evaluated in the uterus of prepubertal female rabbits that were simultaneously treated with P4 (1.0 mg) for 5 consecutive days. As determined by Western and Northern blot analyses, 5 alpha-NET inhibited the P4-induced UG gene expression in a dose-dependent manner. A similar inhibition was observed with the administration of RU-486. The estrogenic agent 3 beta,5 alpha-NET and estradiol at a dose of 1.0 mg also inhibited the UG gene expression induced by P4. Both 5 alpha-NET and 3 beta,5 alpha-NET blocked the PR down-regulation induced by P4 as assessed by Western and Northern blot methods. The inhibition of UG synthesis and PR down-regulation by 5 alpha-NET and 3 beta,5 alpha-NET indicates that these NET metabolites possess antiprogestational properties.

Animals↗