Biomedical subjects

J Sirois

Publications and source records attributed to J Sirois.

At least 37 records · Page 2Linked to original sources

PubMed2000

Interferon-tau modulates phorbol ester-induced production of prostaglandin and expression of cyclooxygenase-2 and phospholipase-A(2) from bovine endometrial cells.

Antiluteolytic actions of bovine interferon-tau (bIFN-tau) require suppression of prostaglandin F(2 alpha) (PGF(2 alpha)) production. Our objective was to test whether bIFN-tau could block PGF(2 alpha) production and synthesis of phospholipase A(2) (PLA(2)) and cyclooxygenase-2 (COX-2) enzymes induced by a protein kinase C (PKC) stimulator (phorbol 12,13 dibutyrate; PDBu). Bovine endometrial epithelial (BEND) cells were treated with PDBu in the presence or absence of bIFN-tau. Medium samples were analyzed for concentrations of PGF(2 alpha), whole-cell extracts were analyzed for abundance of PLA(2) and COX-2 by immunoblotting, and RNA extracts were examined for steady-state levels of COX-2 mRNA by Northern blotting. The PDBu stimulated production of PGF(2 alpha) between 3 and 12 h, levels of COX-2 mRNA by 3 h and protein expression of COX-2 and PLA(2) by 6 and 12 h, respectively. Added concomitantly with PDBu, bIFN-tau suppressed PGF(2 alpha) production, steady-state levels of COX-2 mRNA, and expression of COX-2 and PLA(2) proteins. Added after a 3-h stimulation with PDBu alone, bIFN-tau suppressed PGF(2 alpha) production after 1 h. Bovine IFN-tau inhibited intracellular mechanisms responsible for PGF(2 alpha) production in BEND cells, and this could be through both cytosolic and nuclear actions.

Animals↗

PubMed2000

Expression and regulation of transcripts encoding two members of the NR5A nuclear receptor subfamily of orphan nuclear receptors, steroidogenic factor-1 and NR5A2, in equine ovarian cells during the ovulatory process.

Steroidogenic factor-1 (SF-1, NR5A1a) is a member of the NR5A nuclear receptor subfamily and has been implicated as a key transcriptional regulator of all ovarian steroidogenic genes in vitro. To establish links between the expression of SF-1 and that of the steroidogenic genes in vivo, the objectives of this study were to clone equine SF-1 and examine the regulation of its messenger RNA (mRNA) in follicular cells during human CG (hCG)-induced ovulation. The equine SF-1 primary transcript was cloned by a combination of RT-PCR techniques. Results showed that the transcript was composed of a 5'-untranslated region (UTR) of 161 bp, an open reading frame (ORF) of 1386 bp that encodes a highly-conserved 461-amino acid protein, and a 3'-UTR of 518 bp. The cloning of SF-1 also led to the unexpected and serendipitous isolation of the highly-related orphan nuclear receptor NR5A2, which was shown to include a 5'-UTR of 243 bp, an ORF of 1488 bp, and a 3'-UTR of 1358 bp. The NR5A2 ORF encodes a 495-amino acid protein that is 60% identical to SF-1, including 99%-similar DNA-binding domains. Northern blot analysis revealed that SF-1 and NR5A2 were expressed in all major steroidogenic tissues, with the exception that NR5A2 was not present in the adrenal. Interestingly, NR5A2 was found to be, by far, the major NR5A subfamily member expressed in the preovulatory follicle and the corpus luteum. Using a semiquantitative RT-PCR/Southern blotting approach, the regulation of SF-1 and NR5A2 mRNAs in vivo was studied in equine follicular cells obtained from preovulatory follicles isolated between 0 and 39 h post hCG. Results showed that the theca interna was the predominant site of SF-1 mRNA expression in the follicle, and that hCG caused a significant decrease in SF-1 levels between 12-39 h in theca interna and between 24-39 h post hCG in granulosa cells (P < 0.05). In contrast, the granulosa cell layer was the predominant, if not the sole, site of NR5A2 mRNA expression in the follicle. Importantly, NR5A2 was much more highly expressed in granulosa cells than SF-1. The administration of hCG caused a significant decrease in NR5A2 transcripts in granulosa cells at 30, 36, and 39 h post hCG (P < 0.05). Thus, this study is the first to report the concomitant regulation of SF-1 in theca interna and granulosa cells throughout the ovulation/luteinization process, and to demonstrate the novel expression and hormonal regulation of NR5A2 in ovarian cells. Based on the marked expression of NR5A2 in equine granulosa and luteal cells and on mounting evidence of a functional redundancy between SF-1 and NR5A2 in other species, it is proposed that NR5A2 may play a key role in the regulation of gonadal steroidogenic gene expression.

Amino Acid Sequence↗

PubMed1999

The delayed activation of the prostaglandin G/H synthase-2 promoter in bovine granulosa cells is associated with down-regulation of truncated upstream stimulatory factor-2.

To elucidate the molecular mechanisms involved in the delayed induction of PGHS-2 in species with a long ovulatory process, a 1. 6-kilobase fragment of the bovine PGHS-2 promoter was isolated, and its activity was characterized in primary cultures of bovine granulosa cells. Promoter activity assays performed with a series of deletion mutants revealed that the promoter region from -149 to -2 (+1 = transcription start site) confers full-length promoter activity in response to forskolin (10 microM). Four consensus cis-elements were identified within this region, including an E-box, ATF/CRE, C/EBP, and AP2 site. Site-directed mutagenesis showed that the E-box was required for PGHS-2 promoter activity, that disruption of the C/EBP element decreased forskolin inducible activity by 29%, whereas point mutation within the ATF/CRE and AP2 element had no inhibitory effect. Electrophoretic mobility shift assays (EMSAs) performed with the -149/-2 fragment and granulosa cell nuclear extracts obtained before (0 h) and after (18 and 20 h) human chorionic gonadotropin (hCG) revealed the regulation of multiple DNA-protein complexes. The 0-h extract generated four complexes at the E-box, whereas only one complex was produced at this site with the 18-h extract. Supershift EMSAs identified that upstream stimulatory factor-1 and -2 (USF-1 and -2) were part of these complexes. Interestingly, the presence of the amino-terminal truncated USF-2, which lacks the transcription activation domain, was detected in the 0-h extract, but not in extracts prepared post-hCG. Supershift EMSAs also indicated high levels of C/EBPbeta binding to its cis-element in the 0-h extract, which contrasts with results previously reported in rats. Thus, high levels of amino-terminal truncated USF-2 and C/EBPbeta in bovine granulosa cells prior to hCG treatment could repress gene expression, and be involved in the delayed induction of PGHS-2 in species with a long ovulatory process.

Amino Acid Sequence↗

PubMed1999

Non-steroidal anti-inflammatory drugs (NSAIDs) block the late, prostanoid-dependent/ceramide-independent component of ovarian IL-1 action: implications for the ovulatory process.

The therapeutic efficacy and antiovulatory properties of non-steroidal anti-inflammatory drugs (NSAIDs) is attributed to their ability to suppress prostaglandin endoperoxide synthase (PGS) activity. Given the likely role of interleukin (IL)-1 in the inflammatory (and probably the ovulatory) process, we set out to evaluate whether the antiovulatory property of NSAIDs is attributable, in part, to the inhibition of ovarian IL-1 action. Whole ovarian dispersates from immature rats were cultured under serum-free conditions in the absence or presence of the indicated agents. At the conclusion of the culture period, total RNA was extracted and probed for transcripts corresponding to PGS-1, PGS-2, IL-1beta, IL-1 receptor antagonist (IL-1RA) or type I IL-1 receptor (IL-1R) by a solution hybridization/ribonuclease protection assay. Treatment with indomethacin was without significant effect on the early (1 h) response to IL-1beta; however, it led to complete and highly significant dose-dependent blockade of the late (48 h) response to IL-1beta as assessed in terms of PGS-2 transcripts, proteins and activity. The addition of PGE2 to cells augmented the ability of IL-1beta to upregulate PGS-2 transcripts. Moreover, the addition of PGE2 to indomethacin-treated cells all but reversed the ability of indomethacin to suppress the IL-1beta effect at both the PGS-2 transcript and protein levels. The upregulation by IL-1 of IL-1beta, IL-1R and IL-1RA transcripts was similarly inhibited by indomethacin. Taken together, these observations suggest that the anti-ovulatory property of NSAIDs may be due, in part, to blockade of the late, prostanoid-dependent component of ovarian IL-1 action.

Animals↗

PubMed1999

Induction of prostaglandin G/H synthase-2 in a canine model of spontaneous prostatic adenocarcinoma.

BACKGROUND: Prostate cancer is the most frequently occurring cancer in men in the United States, with an estimated 179 300 new cases in 1999. The induction of prostaglandin G/H synthase (PGHS), a key rate-limiting enzyme in prostaglandin biosynthesis, has been implicated in various cancers, most notably in colorectal cancers; however, the induction of PGHS expression in prostate cancer in vivo has not been reported for any species. The dog is the only nonhuman species that frequently develops spontaneous cancer of the prostate with increasing age, and the objective of this study was to determine whether PGHS isoenzymes were expressed in canine prostatic adenocarcinomas. METHODS: Four normal canine prostatic tissues and 24 canine prostatic adenocarcinomas were studied by means of immunohistochemistry and immunoblot analysis, using polyclonal antibodies specific for each of the two PGHS isoenzymes, PGHS-1 and PGHS-2. All P values were obtained by use of two-sided Fisher's exact tests. RESULTS: PGHS-1 immunostaining was localized to stromal fibroblasts and vascular endothelium in normal and cancerous prostates. PGHS-2 was not detected in normal prostates, but it was expressed by epithelial tumor cells in 18 (75%) of the 24 adenocarcinomas (P =.01). Immunoblot analysis confirmed the presence of PGHS-1 (69 000 molecular weight) in normal and cancerous tissues and the expression of PGHS-2 (72 000- to 74 000-molecular-weight doublet) only in prostatic adenocarcinomas. CONCLUSION: To our knowledge, these results demonstrate for the first time that PGHS-2 is induced in the majority of canine spontaneous prostatic adenocarcinomas and suggest that its expression may be involved in prostate cancer.

Adenocarcinoma↗

PubMed1999

Down-regulation of oxytocin-induced cyclooxygenase-2 and prostaglandin F synthase expression by interferon-tau in bovine endometrial cells.

Oxytocin (OT) is responsible for the episodic release of luteolytic prostaglandin (PG) F2alpha from the uterus in ruminants. The attenuation of OT-stimulated uterine PGF2alpha secretion by interferon-tau (IFN-tau) is essential for prevention of luteolysis during pregnancy in cows. To better understand the mechanisms involved, the effect of recombinant bovine IFN-tau (rbIFN-tau) on OT-induced PG production and cyclooxygenase-2 (COX-2) and PGF synthase (PGFS) expression in cultured endometrial epithelial cells was investigated. Cells were obtained from cows at Days 1-3 of the estrous cycle and cultured to confluence in RPMI medium supplemented with 5% steroid-free fetal calf serum. The cells were then incubated in the presence or absence of either 100 ng/ml OT or OT+100 ng/ml rbIFN-tau for 3, 6, 12, and 24 h. OT significantly increased PGF2alpha and PGE2 secretion at all time points (p < 0.01), while rbIFN-tau inhibited the OT-induced PG production and reduced OT receptor binding in a time-dependent manner. OT increased the steady-state level of COX-2 mRNA, measured by Northern blot, which was maximal at 3 h (9-fold increase) and then decreased with time (p < 0.01). OT also caused an increase in COX-2 protein, which peaked at 12 h (11-fold increase), as measured by Western blot. Addition of rbIFN-tau suppressed the induction of COX-2 mRNA (89%, p < 0.01) and COX-2 protein (50%, p < 0.01) by OT. OT also increased PGFS mRNA, and this stimulation was attenuated by rbIFN-tau (p < 0.01). To ensure that the decrease in COX-2 was not solely due to down-regulation of the OT receptor, cells were stimulated with a phorbol ester (phorbol 12-myristate 13-acetate; PMA) in the presence and absence of rbIFN-tau. The results showed that rbIFN-tau also decreased PMA-stimulated PG production and COX-2 protein. It can be concluded that rbIFN-tau inhibition of OT-stimulated PG production is due to down-regulation of OT receptor, COX-2, and PGFS.

Animals↗

PubMed1999

Glucocorticoid receptor-mediated post-ceramide inhibition of the interleukin-1beta-dependent induction of ovarian prostaglandin endoperoxide synthase-2 in rats.

Ovulation may constitute a cyclic, inflammatory-like process, wherein interleukin (IL)-1 induction and increased biosynthesis of prostanoids may feature prominently. In excess, glucocorticoids, potent anti-inflammatory agents, may exert an antiovulatory effect. This paper addresses the possibility that the antiovulatory action of glucocorticoids may be partly due to interference with ovarian prostanoid biosynthesis. Specifically, we examined the effect of treatment with dexamethasone, a synthetic glucocorticoid, on the IL-1-induced expression and activity of ovarian prostaglandin endoperoxide synthase (PGS)-2, the inducible variety of the rate-limiting enzyme in the prostaglandin cascade. Treatment of cultured whole ovarian dispersates from immature rats with dexamethasone for 48 h produced a significant decrease (98.9% inhibition) in the IL-1-supported expression of PGS-2 transcripts. Comparably marked inhibition was also noted for the corresponding immunoreactive protein. The dexamethasone effect was not limited to the IL-1-mediated induction of PGS-2 transcripts, comparable suppression being noted for the IL-1-mediated up-regulation of ovarian transcripts corresponding to IL-1beta, the IL-1 receptor antagonist, and the type I IL-1 receptor. The order of potency of the glucocorticoids studied was dexamethasone > prednisolone = cortisol. Dexamethasone proved equally effective in suppressing the induction of PGS-2 transcripts by congeners of the sphingomyelin-ceramide cycle (e.g., C-2 ceramide, sphingomyelinase, and sphingosine). The dexamethasone effect proved glucocorticoid-specific, as synthetic agonists representative of the progestin (R-5020), androgen (R-1881), and estrogen (diethylstilbestrol) steroid series proved to be without effect. Cotreatment with RU-486 resulted in reversal of the ability of dexamethasone to suppress PGS-2 activity or expression. Taken together, these observations suggest that dexamethasone is capable of glucocorticoid receptor-mediated/post-ceramide suppression of IL-1-supported ovarian PGS-2 transcript, protein, and activity. These findings are compatible with the view that the chronic anovulatory state associated with adrenal hyperactivity or glucocorticoid excess may be due in part to inhibition of ovarian prostaglandin biosynthesis.

Animals↗

PubMed1999

Human chorionic gonadotropin induces an inverse regulation of steroidogenic acute regulatory protein messenger ribonucleic acid in theca interna and granulosa cells of equine preovulatory follicles.

The time- and gonadotropin-dependent regulation of steroidogenic acute regulatory protein (StAR) has not been characterized in vivo in preovulatory follicles of large monoovulatory species or sexually mature animals. The objectives of this study were to clone equine StAR and describe the regulation of its messenger RNA (mRNA) in equine follicles after the administration of an ovulatory dose of hCG. The screening of an equine follicle complementary DNA (cDNA) library with a mouse StAR cDNA probe revealed two forms of equine StAR that differ only in the length of their 3'-untranslated region (3'-UTR); a long form of 2918 bp and a short form of 1599 bp. The StAR long form cDNA contains a 5'-UTR of 117 bp, an open reading frame (ORF) of 855 bp, and a 3'-UTR of 1946 bp. Primer extension analysis showed that the cDNA clone lacked the first 10 bp of the primary transcript, giving a total of 127 bp for the complete StAR 5'-UTR. The ORF encodes a 285-amino acid protein that is 86-90% identical to StAR of other species characterized to date. The regulation of StAR mRNA in vivo was studied in equine preovulatory follicles isolated during estrus at 0, 12, 24, 30, 33, 36, and 39 h (n = 4-5 follicles/time point) after an ovulatory dose of hCG. Results from Northern blots showed no significant changes in StAR mRNA levels after hCG treatment when analyses were performed on intact follicle wall (theca interna with attached granulosa cells). However, Northern blots performed on isolated follicle cells revealed an unexpected regulation of StAR mRNA. In granulosa cells, StAR transcripts were undetectable at 0 h but were significantly increased at 30 h post-hCG, and this induction was associated with a rise in follicular fluid concentrations of progesterone (P < 0.05). In contrast, StAR mRNA levels were high in theca interna at 0 h, remained unchanged until 33 h post-hCG, and dropped dramatically thereafter (P < 0.05). Thus, this study describes the primary structure of equine StAR, documents the regulation of StAR mRNA in vivo in preovulatory follicles of a large monoovulatory species, and identifies a novel inverse regulation of StAR transcripts in theca interna and granulosa cells of equine follicles before ovulation.

5' Untranslated Regions↗

PubMed1999

High expression of bovine alpha glutathione S-transferase (GSTA1, GSTA2) subunits is mainly associated with steroidogenically active cells and regulated by gonadotropins in bovine ovarian follicles.

We have previously shown that a major group of 28-30 kDa proteins decreases after the LH surge in bovine granulosa cells (GC). In the present study, we have characterized two proteins in this group in search of factors that may intervene in folliculogenesis and oocyte maturation. Polyclonal antibodies raised against 28 kDa or 29 kDa bovine GC proteins were used to screen a complementary DNA (cDNA) expression library. This resulted in the characterization of two isoenzyme subunits for alpha class glutathione S-transferase, named bGSTA1 and bGSTA2. Both bGSTA1 (25.4 kDa, pI 8.9; 791 bp cDNA; GenBank Accession No. BTU49179) and bGSTA2 (25.6 kDa, pI 7.2; 959 bp cDNA; GenBank Accession No. AF027386) have 222 amino acids. The deduced amino acid sequences were compared and showed 82% (bGSTA1) and 74% (bGSTA2) identity to human GSTA1, whereas bGSTA1 and bGSTA2 are 81% identical to each other. The bGSTA2 represents a novel GSTA subunit because it harbors a specific 16 amino acid sequence not found in any other species and GST classes. Northern blots showed that bGSTA1 and bGSTA2 are coexpressed and are tissue specific with single transcripts of 1.2 kb and 1.4 kb, respectively for bGSTA1 and bGSTA2. The messenger RNA (mRNA) were detected in GC, corpus luteum, adrenal gland, testis, liver, lung, thyroid, kidney and cotyledon, and the relative abundance of their mRNA varied. Ratios of bGSTA1/bGSTA2 mRNA vary between tisssues, indicating that expression of these genes is controlled differently. Immunohistochemistry observations revealed that expression of GSTA is cell specific, being associated with GC and theca cells, small luteal cells, Leydig cells, hepatocytes, adrenal cortex, specific chromaffin cells in the adrenal medulla, renal proximal convoluted tubular cells, and Clara cells in the bronchioles. Studies in vivo showed that levels of mRNA for bGSTA1 were elevated in follicular wall of preovulatory follicles before hCG treatment, but decreased by 77% 12 h after hCG injection. However, in FSH stimulated preovulatory follicles, the decrease in mRNA for both GSTAs was only 21% at 24 h following hCG injection. We concluded that bGSTA1 and bGSTA2 expression is tissue- and cell-specific, is associated with steroidogenically active cells, and is hormonally regulated by gonadotropins in the bovine ovarian follicle.

Amino Acid Sequence↗

PubMed1999

Dual regulation of promoter II- and promoter 1f-derived cytochrome P450 aromatase transcripts in equine granulosa cells during human chorionic gonadotropin-induced ovulation: a novel model for the study of aromatase promoter switching.

Estradiol biosynthesis is a key biochemical trait of developing follicles. To study its regulation in equine follicles, the objectives of this study were to clone and determine the structure of equine cytochrome P450 aromatase (P450AROM), and characterize the regulation of P450AROM and P450 17alpha-hydroxylase/C17-20 lyase (P45017alpha) messenger RNAs (mRNAs) in vivo in equine preovulatory follicles isolated during hCG-induced ovulation. Two distinct P450AROM complementary DNAs (cDNAs) were isolated from an equine preovulatory follicle cDNA library. One clone was 2682 bp in length and included 115 bp of 5'-untranslated region (UTR), 1509 bp of open reading frame encoding a well conserved 503-amino acid protein, and 1058 bp of 3'-UTR. Its 5'-most region represented the equine homolog of exon 1f, previously designated brain specific. The other cDNA clone encoded a truncated protein and contained a distinct 5'-UTR characteristic of transcripts derived from promoter II, previously identified as the predominant ovarian mRNA. Northern blot analyses were performed using preovulatory follicles obtained during estrus between 0-39 h after the administration of hCG and with corpora lutea isolated on day 8 of the estrous cycle (day 0 = day of ovulation). The results showed a biphasic regulation of P450AROM mRNA expression: levels were highest in follicles at 0 h post-hCG, decreased significantly during the ovulatory process at 12 and 24 h (P < 0.05), and increased again between 30-39 h post-hCG and in corpora lutea. When oligonucleotides specific for P450AROM mRNA variants were used as probes, a novel switching phenomenon was observed. Promoter II-derived transcripts accounted for the message present in follicles at 0 h post-hCG and in corpora lutea, whereas promoter 1f-derived mRNA was expressed exclusively during the ovulatory process (30-39 h post-hCG). Levels of P45017alpha mRNA were high in follicles at 0 h, but significantly decreased after hCG treatment (P < 0.05), with lowest levels in follicles at 36 and 39 h post-hCG and in corpora lutea. Northern blots performed on isolated cellular preparations revealed that P450AROM and P45017alpha transcripts were localized exclusively in granulosa cells and theca interna, respectively. Equine aromatase promoters II and 1f were cloned from a genomic library, and putative transcription start sites were characterized by primer extension assays. Sequence analyses identified distinct potential regulatory elements in each promoter. Thus, this study identifies a novel aromatase promoter-switching phenomenon in equine granulosa cells during follicular luteinization and provides a new model in which aromatase promoter switching is induced in vivo.

Amino Acid Sequence↗

PubMed1999

Characterization of cellular and vascular changes in equine follicles during hCG-induced ovulation.

In contrast to other species, the histology of the equine follicle during ovulation has not been described. Preovulatory follicles were isolated during oestrus at 0, 12, 24, 30, 33, 36 and 39 h (n = 5-6 follicles per time point) after an ovulatory dose of hCG to characterize the cellular and vascular changes associated with ovulation in mares. Pieces of follicle wall were formalin-fixed and processed for light microscopy to evaluate the general follicular morphology and quantify selected parameters. Marked changes were observed in the histology of equine follicles in the hours before ovulation. The thickness of the granulosa cell layer doubled between 0 and 39 h after hCG (77.8 +/- 4.8 versus 158.8 +/- 4.8 microns, respectively; P < 0.01). This expansion was caused primarily by a pronounced accumulation of acid mucosubstances between granulosa cells, which was first detected at 12 h after hCG and peaked at 36-39 h. In contrast, a significant thinning of the theca interna was observed after hCG treatment. Fewer cell layers were present; theca interna cells appeared smaller than before hCG; and the presence of occasional pyknotic cells was noted at 36 and 39 h after hCG. In addition, the theca layers were invaded by numerous eosinophils. No eosinophils were observed in preovulatory follicles isolated between 0 and 24 h after hCG, but the number increased to 14.0 +/- 0.8 and 5.6 +/- 0.3 eosinophils per field (x 400) in theca interna and theca externa, respectively, 39 h after hCG treatment (P < 0.01). Severe oedema, hyperaemia and haemorrhages, and significant increases in the number of blood vessels in theca interna and externa were observed at 33, 36 and 39 h after hCG. This study provides the first in-depth characterization of the sequential cellular and vascular changes that occur in equine follicles before ovulation.

Analysis of Variance↗

PubMed1998

Extrapore residues of the S5-S6 loop of domain 2 of the voltage-gated skeletal muscle sodium channel (rSkM1) contribute to the mu-conotoxin GIIIA binding site.

The tetradomain voltage-gated sodium channels from rat skeletal muscle (rSkM1) and from human heart (hH1) possess different sensitivities to the 22-amino-acid peptide toxin, mu-conotoxin GIIIA (mu-CTX). rSkM1 is sensitive (IC50 = 51.4 nM) whereas hH1 is relatively resistant (IC50 = 5700 nM) to the action of the toxin, a difference in sensitivity of >100-fold. The affinity of the mu-CTX for a chimera formed from domain 1 (D1), D2, and D3 from rSkM1and D4 from hH1 (SSSH; S indicates origin of domain is skeletal muscle and H indicates origin of domain is heart) was paradoxically increased approximately fourfold relative to that of rSkM1. The source of D3 is unimportant regarding the difference in the relative affinity of rSkM1 and hH1 for mu-CTX. Binding of mu-CTX to HSSS was substantially decreased (IC50 = 1145 nM). Another chimera with a major portion of D2 deriving form hH1 showed no detectable binding of mu-CTX (IC50 > 10 microM). These data indicate that D1 and, especially, D2 play crucial roles in forming the mu-CTX receptor. Charge-neutralizing mutations in D1 and D2 (Asp384, Asp762, and Glu765) had no effect on toxin binding. However, mutations at a neutral and an anionic site (residues 728 and 730) in S5-S6/D2 of rSkM1, which are not in the putative pore region, were found to decrease significantly the mu-CTX affinity with little effect on tetrodotoxin binding (</=1.3-fold increase in affinity). Furthermore, substitution at Asp730 with cysteine and exposure to Cd2+ or methanethiosulfonate reagents had no significant effect on sodium currents, consistent with this residue not contributing to the pore.

Animals↗

PubMed1998

Statistical Considerations in Microanalysis by Energy-Dispersive Spectrometry.

: X-ray counting statistics plays a key role in establishing confidence limits in composition determination by X-ray microanalysis. The process starts with measurements of intensity on one or more samples and standards as well as related background determinations. Since each individual measurement is subject to variability associated with counting statistics, it is necessary to combine all of the counting variability according to established mathematical procedures. The next step is to apply propagation of error calculations to equations for quantitative analysis and determine confidence limits in reported composition. Similar concepts can also be applied to trace element determination. This approach can then be combined with spectral simulation modeling, making it possible to predict detectability limits without additional measurements.

Journal Article↗

PubMed1998

Follicle size-dependent induction of prostaglandin G/H synthase-2 during superovulation in cattle.

Under physiological conditions, prostaglandin G/H synthase-2 (PGHS-2) is induced in bovine preovulatory follicles by the endogenous surge of gonadotropins. To characterize the pattern of follicular PGHS-2 expression during superovulation in cattle, heifers were treated with exogenous FSH and ovulation was induced with hCG. Animals were ovariectomized 0, 18, and 24 h post-hCG, and extracts of follicles > or = 6 mm were analyzed by Western blotting. Follicular fluid concentrations of prostaglandin (PG) E2, PGF2alpha, progesterone, and estradiol-17beta were determined by RIAs, and the morphology of the cumulus oocyte complex was examined. Results showed that PGHS-2 protein was absent in all follicles isolated at 0 h post-hCG (n = 119) and in small follicles (6 to < 8 mm) isolated between 0 and 24 h post-hCG (n = 27 follicles). In contrast, 12.3% of medium (8 to < 10 mm) and 43.7% of large (> or = 10 mm) follicles were PGHS-2-positive at 18 h post-hCG, and these percentages rose at 24 h to 45.9% and 91.0% in medium and large follicles, respectively (p < 0.05). Follicular fluid concentrations of PGE2 and PGF2alpha were low in follicles isolated at 0 h and increased only in PGHS-2-positive follicles isolated 24 h post-hCG (p < 0.05). Concentrations of progesterone and estradiol-17beta at 0 h were 28.2 +/- 5.8 and 291.8 +/- 13.0 ng/ml, respectively, and a shift from estradiol-17beta to progesterone dominance (luteinization) occurred at 24 h post-hCG only in PGHS-2-positive follicles. Also, expansion of the cumulus oocyte complex was detected at 24 h post-hCG only in PGHS-2-positive follicles. Lack of PGHS-2 induction in follicles of ovulatory size (> 8 mm) was associated with an apparent failure to respond to hCG (absence of luteinization and cumulus expansion). Collectively, these results demonstrate the presence of a time- and follicle size-dependent induction of PGHS-2 in bovine follicles during superovulatory treatment and suggest that PGHS-2 expression can be used as a marker for follicular commitment to ovulation during ovarian hyperstimulation protocols.

Animals↗

PubMed1998

Expression of prostaglandin G/H synthase type 1, but not type 2, in human ovarian adenocarcinomas.

Prostaglandin endoperoxide synthase (PGHS) is a key rate-limiting enzyme in prostaglandin biosynthesis. PGHS has recently been shown to be expressed in human colorectal cancers and in experimental cutaneous papillomas and carcinomas. However, PGHS expression has not been investigated in ovarian cancers. The objectives of this study were to determine whether PGHS isoenzymes are expressed in human ovarian cancer and, if so, to identify which isoform is involved (PGHS-1 and/or PGHS-2) and to characterize its cellular localization. Sixteen human ovarian adenocarcinomas were studied by immunohistochemistry using specific antibodies recognizing PGHS-1 or PGHS-2. Results showed that all adenocarcinomas demonstrated the presence of tumor cells expressing PGHS-1 but not PGHS-2. Patterns of staining of tumor cells varied among different types of adenocarcinomas, with cells presenting either a mostly diffuse cytoplasmic immunoreactivity or, alternatively, a staining mainly concentrated around the nucleus. No correlation between the intensity of the immunostaining and the degree of malignancy of tumors could be established (r 5 20.03; p>0.05). Immunoblot analysis with PGHS-1-selective antibodies of cell extracts from adenocarcinomas revealed the presence of a characteristic 72,000 Mr immunoreactive band. Therefore, these results show for the first time that PGHS-1 is expressed in human ovarian adenocarcinomas. (J Histochem Cytochem 46:77-84, 1998)

Adenocarcinoma, Mucinous↗

PubMed1998

Molecular characterization of equine prostaglandin G/H synthase-2 and regulation of its messenger ribonucleic acid in preovulatory follicles.

To increase our understanding of the molecular control of PG synthesis in equine preovulatory follicles, the specific objectives of this study were to clone and determine the primary structure of equine prostaglandin G/H synthase-2 (PGHS-2) and to characterize the regulation of PGHS-2 messenger RNA (mRNA) in follicles before ovulation. A complementary DNA (cDNA) library prepared from follicular mRNA and a genomic library were screened with a mouse PGHS-2 cDNA probe to isolate the equine PGHS-2 cDNA and gene, respectively. The expression library yielded three nearly full-length clones that differed only in their 5'-ends; clones 3, 5, and 6 were 2946, 3138, and 3398 bp in length, respectively. The longest clone was shown to start 9 bp downstream of the transcription initiation site, as determined by primer extension analysis, and to contain 120 bp of 5'-untranslated region (UTR), 1812 bp of open reading frame, and 1466 bp of 3'-UTR. The open reading frame encodes a 604-amino acid protein that is more than 80% identical to PGHS-2 homologs in other species. Numerous repeats (n = 11) of the Shaw-Kamen's sequence (ATTTA) are present in the 3'-UTR, a motif typically indicative of mRNAs with a short half-life. The complete equine PGHS-2 gene was isolated and sequenced from a approximately 17-kilobase clone obtained from the genomic library. The equine PGHS-2 gene structure (10 exons and 9 introns; total length of 6991 bp) is similar to its human homolog except for lacking sequence elements in introns 4, 8, and 9 and in the 3'-UTR region of exon 10. To characterize the regulation of PGHS-2 mRNA in equine follicles before ovulation, preovulatory follicles were isolated during estrus, 0, 12, 24, 30, 33, 36, and 39 h (n = 4-5 follicles/time point) after an ovulatory dose of hCG. Results from Northern blots showed significant changes in steady state levels of PGHS-2 mRNA in preovulatory follicles after hCG treatment (P < 0.05). The transcript remained undetectable between 0-24 h post-hCG, first appeared (approximately 4 kilobases) only at 30 h, and reached maximal levels 33 h post-hCG. PGHS-2 mRNA was selectively induced in granulosa cells and not in theca interna. Thus, this study provides for the first time the primary structure of the equine PGHS-2 gene, transcript, and protein. It also demonstrates that the induction of PGHS-2 gene expression in equine granulosa cells is a long molecular process (30 h post-hCG), thereby providing a model to study the molecular basis for the late transcriptional activation of PGHS-2 in species with a long ovulatory process.

Amino Acid Sequence↗

PubMed1998

Regulation of cyclooxygenase-2 and prostaglandin F synthase gene expression by steroid hormones and interferon-tau in bovine endometrial cells.

Estradiol (E2) and progesterone are responsible for regulating PG synthesis in the endometrium during the estrous cycle and interferon-tau (IFN-tau) alters PG synthesis during early pregnancy in ruminants. In this study, we examined the effects of these steroid hormones and recombinant bovine IFN-tau (rbIFN-tau) on PG production and on cyclooxygenase-2 (COX-2) and PG F (PGF) synthase (PGFS) gene expression in isolated endometrial cells. E2 decreased both PGF2alpha and PG E2 (PGE2) whereas progesterone increased PGF2alpha secretion in epithelial cells. Steroid hormones had no effect on PG production in stromal cells. rbIFN-tau attenuated both PGF2alpha and PGE2 production in epithelial cells and enhanced their production, and the ratio of PGE2 to PGF2alpha, in stromal cells. Northern blot analysis showed that E2 and rbIFN-tau decreased COX-2 messenger RNA (mRNA) levels in epithelial cells. Conversely, rbIFN-tau increased COX-2 mRNA in stromal cells. Furthermore, rbIFN-tau decreased PGFS mRNA in both cell types and this was associated with the increase in PGE2/PGF2alpha ratio. These results show that the regulation of PG synthesis by steroid hormones is different in endometrial epithelial and stromal cells in vitro. The attenuation of PGF2alpha secretion from epithelial cells and increased PGE2 production in stromal cells by rbIFN-tau are modulated by steroid hormones.

Animals↗

PubMed1998

Rat ovarian prostaglandin endoperoxide synthase-1 and -2: periovulatory expression of granulosa cell-based interleukin-1-dependent enzymes.

This laboratory has previously shown that interleukin-1 (IL-1), a putative intermediary in the ovulatory process, is capable of up-regulating PG biosynthesis by cultured whole ovarian dispersates from immature rats. In part, this phenomenon was attributable to the stimulation of ovarian phospholipase A2 activity. In this communication we examine the possibility that the PG-promoting property of IL-1 is also due to the up-regulation of PG endoperoxide synthase (PGS), the rate-limiting step in prostanoid biosynthesis. The in vivo expression of ovarian PGS-2 transcripts in the course of a simulated estrous cycle rose abruptly to a peak (35-fold increase over the control value; P < 0.05) 8-12 h after hCG administration (i.e. before or during projected ovulation). PGS-1 transcripts, in turn, were not significantly altered during the periovulatory period. Treatment of cultured whole ovarian dispersates with IL-1beta resulted in dose- and time-dependent up-regulation of PGS-2 transcripts (as well as of immunoreactive PGS-2 protein and PGE2 accumulation), characterized by an ED50 of 2 ng/ml and a maximal (72-fold) increase at 10 ng/ml. Although treatment with IL-1beta also led to an increase in PGS-1 transcripts and immunoreactive PGS-1 protein, the relative magnitude of the effect was markedly reduced compared with that of PGS-2. Cotreatment with an IL-1 receptor antagonist completely reversed the IL-1 effects, thereby suggesting mediation via the IL-1 receptor. The ability of IL-1 to up-regulate PGS-2 transcripts proved relatively specific, in that other cellular regulators (insulin-like growth factor I, activin A, endothelin-1, transforming growth factor-alpha, tumor necrosis factor-alpha, vascular endothelial growth factor, leukemia inhibitor factor, hepatocyte growth factor, or keratinocyte growth factor) were not effective. The optimal IL-1 effect required heterologous contact-dependent coculturing of granulosa and thecal-interstitial cells. Taken together, these observations 1) reaffirm (by molecular probing) the granulosa cell as the primary site of ovarian PGS-1 and PGS-2 expression, 2) document an increase in ovarian PGS-2 transcripts before ovulation, and 3) reveal a marked dependence of ovarian PGS (2 >> 1) transcripts, proteins, and activity on IL-1. The effects of IL-1 proved relatively specific, contingent upon somatic cell-cell cooperation, dose and time dependent, and IL-1 receptor mediated. These results are compatible with the proposition that the PG-promoting property of IL-1 is due, in large measure, to the activation of ovarian PGS transcription and translation. The ability of IL-1 to up-regulate ovarian PGS, an obligatory component of ovulation, is in keeping with the idea that IL-1 may constitute an intermediary in the ovulatory process.

Animals↗