PubMed Health⌕ Search

Biomedical subjects

B C Delidow

Publications and source records attributed to B C Delidow.

14 recordsLinked to original sources
PubMed1998

Co-regulation of pituitary tumor cell adhesion and prolactin gene expression by glucocorticoid.

Rat 235-1 pituitary tumor cells are lactotrophs producing high levels of prolactin (PRL). Dexamethasone (Dex, 100 nM) inhibits PRL gene expression in 235-1 cells by 50%, while simultaneously decreasing cell replication and cell-cell aggregation. To determine the time course of Dex action, we used a quantitative assay for cell-cell interaction, based on the number of single cells present before and after re-aggregation of dispersed cells. 235-1 cells were cultured in growth medium or medium plus 100 nM Dex for 1-4 days before assay. Control cells had 90% re-aggregation on all days of assay. Aggregation of Dex-treated cells decreased to 55% by day 4. Dex treatment also reduced cell numbers by 40%, but this decrease did not contribute to reduced aggregation. To determine the mechanism of Dex-inhibited cell-cell adhesion, we examined the expression of cadherins and catenins. Cadherin-related mRNAs (P- and N-cadherin probes) were detectable in 235-1 cells, but their levels were unchanged by Dex. A pancadherin antibody was unable to detect classical cadherins in these cells. Both alpha- and beta-catenins were detected by Western blotting and their levels were decreased by Dex. Unlike control aggregates, aggregates of Dex-treated cells were able to inhibit expression of PRL mRNA when added to monolayers of 235-1 cells. These data suggest that Dex influences cadherin function by inhibiting catenin expression and that this has the functional consequence of altering 235-1 cell-cell interactions. Overall the data show that Dex affects important aspects of lactotroph function other than PRL gene expression. These changes may include physical alterations in pituitary cell contacts that further support a change in functional state.

Animals↗
PubMed1997

Molecular cloning of PCR fragments with cohesive ends.

Use of the polymerase chain reaction (PCR) provides a convenient means of generating DNA fragments for insertion into plasmids. Large quantities of the desired insert, bounded by convenient restriction sites, may be synthesized. The primers are chosen to span a known region of interest, and extended at their 5'-ends to include the desired restriction sites. Amplification of the target sequence is followed by precipitation of the product with ammonium acetate and ethanol to remove the primers. A small amount of product is analyzed by gel electrophoresis to ensure correct amplification, the remainder is digested with the appropriate restriction enzyme(s). Restricted insert DNA is added to similarly restricted plasmid DNA in several ratios and incubated with DNA ligase to recircularize. Ligation products are used to transform competent bacteria. Clones containing inserts are identified by restriction digestion of plasmid minipreps from bacterial colonies.

Cloning, Molecular↗
PubMed1992

Regulation of proto-oncogene expression and deoxyribonucleic acid synthesis in granulosa cells of perifused immature rat ovaries.

The present series of experiments examined the effects of follicle-stimulating hormone (FSH) and insulin (IN) on granulosa cell (GC) proto-oncogene expression and DNA synthesis. In the first study, GCs were harvested from immature rat ovaries after 15, 30, or 60 min of perifusion and DNA synthesis (3H-thymidine incorporation) and proto-oncogene mRNA levels were determined. The presence of c-myc and c-fos proteins was localized within GCs immunocytochemically. GCs of control ovaries exhibited modest levels of DNA synthesis and proto-oncogene expression. FSH/IN not only stimulated DNA synthesis but also increased c-myc, c-fos, and c-jun mRNA levels and the percentage of cells staining for c-fos and c-myc proteins. The protein kinase inhibitor, 2-aminopurine (2-AP), inhibited the FSH/IN-induced increases in c-myc and c-fos mRNA levels, the percentage of cells staining for Myc and Fos protein, and DNA and protein synthesis. The effects of 48 h of perifusion with FSH in the presence or absence of IN were also examined. These treatments were selected because after 48 h of continuous exposure to FSH alone, estradiol-17 beta (E2) secretion is enhanced and 3H-thymidine incorporation is inhibited. Conversely, FSH/IN maintains 3H-thymidine incorporation for up to 48 h of perifusion culture without stimulating E2 (Peluso et al., Endocrinology 1991; 128:191-196). After 48 h of perifusion, both FSH and FSH/IN stimulated c-fos mRNA and protein levels. However, high levels of c-jun mRNA and protein were detected only within GCs of FSH/IN-treated ovaries.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Effects of calcium and calcium ionophores on prolactin gene expression in GH3 and 235-1 rat pituitary tumor cells.

Previous observations that extracellular calcium (Ca2+) enhanced PRL mRNA levels posttranscriptionally in GH3 rat pituitary tumor cells were made using double-stranded transcription probes. The effects of Ca2+ and the Ca2+ ionophore, ionomycin, on PRL gene expression in GH3 and 235-1 cells were investigated using site- and strand-specific probes. Treatment of GH3 and 235-1 cells with 0.5 mM Ca2+ in serum-free medium specifically increased PRL mRNA levels by severalfold. In 235-1 but not GH3 cells PRL gene transcription was comparably induced by Ca2+. Use of single-stranded 5' and 3' probes revealed no antisense transcription, nor any Ca2+ effect on transcriptional elongation. Treatment with Ca2+ plus ionomycin inhibited PRL mRNA levels and gene transcription in both cell lines. Although their PRL gene transcription rates are similar, several basic differences were noted between the cell lines. The 235-1 cells exhibit a different profile of nuclear PRL pre-RNAs than GH3 cells. Also, mRNA levels for a Ca(2+)-regulated gene (GRP78) did not change in Ca(2+)-treated GH3 cells but decreased in Ca(2+)-treated 235-1 cells. Ionomycin treatment increased GRP78 mRNA levels in both cell lines. Thus, addition of extracellular Ca2+ appears to affect [Ca2+]i in 235-1 but not GH3 cells, while ionomycin affects [Ca2+]i in both cell lines. These data suggest that changing [Ca2+]i modulates PRL gene transcription. The comparative data suggest that posttranscriptional PRL regulation is Ca(2+)-regulated in GH3 cells, but is constitutive in 235-1 cells.

Animals↗
PubMed1992

Posttranscriptional regulation of prolactin (PRL) gene expression in PRL-deficient pituitary tumor cells.

Rat pituitary acidophils consist of somatotropes (GH+/PRL-), lactotropes (GH-/PRL+), and lactosomatotropes (GH+/PRL+). Studies have indicated interconversion of these cell types in response to changing hormonal status. Representative tumor cell lines have been obtained for each acidophil cell type, and some display spontaneous interconversions. We examined whether the switch from GH3 cells (GH+/PRL+) to GH3LP and GC cells (both GH+/PRL-) involves repression of PRL gene expression at a transcriptional vs. posttranscriptional level. PRL mRNA is undetectable or barely detectable in GH3LP and GC cells. In contrast, nuclear extracts from these cells transcribe the PRL promoter in vitro, and their Pit-1 mRNA levels are comparable to those in GH3 cells. Nuclear run-on transcription assays demonstrated that the PRL gene is transcribed in GH3LP and GC cells at a rate of about 60% of that observed in GH3 cells. No evidence was obtained for a block to transcriptional elongation or for transcription in the antisense direction across the PRL gene. Northern blot analysis of nuclear RNA revealed partially degraded and undetectable PRL gene transcripts in GH3LP cells and GC cells, respectively. These findings indicate that PRL gene transcripts are specifically degraded in tumor cells which display a pure somatotrope phenotype and raise the possibility that the trans-differentiation of lactosomatotropes to somatotropes involves posttranscriptional regulation of PRL gene expression.

Animals↗
PubMed1991

Follicle-stimulating hormone and insulin regulation of 17 beta-estradiol secretion and granulosa cell proliferation within immature rat ovaries maintained in perifusion culture.

The present study examined the effects of FSH and insulin (IN) on 17 beta-estradiol (E2) secretion and granulosa cell proliferation. For these studies, immature rat ovaries were maintained in perifusion culture and continuously exposed to FSH (0-800 ng RP-1 eq/ml) and /or IN (0.2 U/ml). At specific times, the ovaries were removed from perifusion, and the perifusate was assayed for E2 by RIA. The ovaries were then incubated with [3H]thymidine ([3H]T) in order to estimate granulosa cel mitotic activity. FSH increased E2 secretion in a dose-dependent manner (P less than 0.05), but did not enhance [3H]T incorporation (P greater than 0.05) after 48 h of perifusion. Conversely, IN increased [3H]T incorporation (P less than 0.05) without stimulating E2 secretion (P greater than 0.05) after 48 h of perifusion. FSH alone or in combination with IN stimulated [3H]T incorporation at 24 h of perifusion compared to both zero hour control (P less than 0.05) and IN treatments (P less than 0.05). The inability of FSH to stimulate [3H]T incorporation after 48 h did not appear to be due to increased E2, since IN stimulated [3H]T incorporation in the presence of 100 ng E2/ml. Further, continuous exposure to FSH was required to maintain E2 secretion, demonstrating that after 24 h, FSH loses its capacity to stimulate granulosa cell [3H]T incorporation, but not E2 secretion. Finally, when ovaries are pretreated with FSH for 48 h and then exposed to FSH and/or IN, [3H]T incorporation was stimulated, and E2 secretion inhibited. These data suggest that 1) initially, FSH acts to stimulate mitosis then promotes granulosa cell E2 secretion; and 2) once granulosa cells begin to secrete E2 they are still capable of mitosis, but their mitotic activity is no longer directed by FSH.

Animals↗
PubMed1991

Inhibition of prolactin gene transcription by transforming growth factor-beta in GH3 cells.

Transforming growth factor-beta (TGF beta) is a member of a large family of growth factors, several of which regulate pituitary function. TGF beta has recently been reported to reduce PRL production by GH4 cells. We have examined the effect of TGF beta on PRL gene expression in rat pituitary tumor GH3 cells. TGF beta 1 or TGF beta 2 reduced both basal and Ca(2+)-stimulated PRL mRNA levels. This inhibition was specific, as the mRNA levels for GH, glucose-regulated protein 78, and histone-3 were unaffected by TGF beta. Inhibition of PRL gene expression by TGF beta was dose dependent in the range of 0.5-10 ng/ml. TGF beta inhibited run-on PRL gene transcription in nuclei from treated cells to the same extent that it reduced PRL mRNA levels, indicating a transcriptional mechanism of action. However, TGF beta did not affect Pit-1 mRNA levels or run-on transcription of the Pit-1 gene. Thus, TGF beta does not appear to act through modification of Pit-1 gene expression. The PRL promotor contains two regions of homology, with a consensus sequence found in the promoters of other TGF beta-inhibited genes. These findings are consistent with other studies that have demonstrated transcriptional repression by TGF beta. The potency and specificity of the effects of TGF beta on PRL gene expression suggest that it may be a physiological regulator of lactotroph function.

Animals↗
PubMed1990

Gonadotropin induction of c-fos and c-myc expression and deoxyribonucleic acid synthesis in rat granulosa cells.

Although gonadotropins stimulate ovarian granulosa cells to proliferate and differentiate into steroidogenic cells, little is known about the molecular mechanisms by which gonadotropins induce these fundamentally different responses. In this study the acute effects of PMSG on protooncogene expression, DNA synthesis, and steroid secretion were examined. The levels of c-fos, c-myc, and beta-actin mRNA were measured in total RNA samples from granulosa cells by quantitative polymerase chain reaction. PMSG increased the mRNA levels of c-fos, c-myc, and beta-actin within 15 min. Fos and myc proteins were localized within granulosa cells by immunocytochemistry. Less than 10% of granulosa cells stained for c-fos or c-myc proteins in the control samples. In contrast, approximately 40% of the cells stained for these protooncogene proteins 30 min after PMSG injection (P less than 0.05). These values declined to about 10% of the cells 60 min after PMSG injection. DNA synthesis, as estimated by [3H]thymidine incorporation, increased 30 and 60 min after PMSG (P less than 0.05). 17 beta-Estradiol and progesterone synthesis did not change within 60 min of PMSG injection. These data demonstrate that 1) c-fos and c-myc are expressed in ovarian granulosa cells; 2) the expression of the genes encoding c-fos, c-myc, and beta-actin is rapidly increased by gonadotropin; and 3) the increase in the corresponding products of the c-fos and the c-myc genes precedes an increase in DNA synthesis and steroid production. These data suggest that the expression of c-fos and c-myc may be a part of the molecular mechanism through which gonadotropins regulate granulosa cell function.

Actins↗
PubMed1989

Reevaluation of the effects of growth hormone and prolactin on anuran tadpole growth and development.

The effects of ovine prolactin (oPRL) and ovine (o) or porcine (p) growth hormone (GH) on growth of anuran larvae were compared. In grass frog tadpoles (Rana pipiens), injections of oPRL (10 micrograms/gm/day) increased tail growth and body weight but had no effect on hindlimb growth or development. The same dose of oGH did not significantly affect the tail or body weight but it caused a striking increase in limb length and development. In bullfrog tadpoles (Rana catesbeiana) given an ergot drug (bromocriptine) to induce metamorphic changes, significant tail regression and loss of body weight occurred. Treatment with oPRL reversed these effects but had no effect on hindlimb growth or development. The pGH was much less effective than the oPRL in blocking tail regression and body weight loss but it significantly increased limb growth and development. These results show that GH and PRL can differentially regulate growth and development of structures in larval anurans. The effects of GH on limb growth and development can not be explained simply by a thyrotropic effect.

Animals↗
PubMed1988

Evidence for hepatic involvement in the regulation of amphibian development by prolactin.

Hormonal control of amphibian development involves thyroid hormones (TH), which promote metamorphosis, and prolactin (PRL), which antagonizes the effects of TH and promotes larval growth. Although the liver is not considered to be a regulator of developmental processes such as metamorphosis, it secretes a PRL-synergizing factor (synlactin) in response to PRL. We explored the possibility that the liver may participate in the antimetamorphic actions of PRL in Rana catesbeiana. Bullfrog tadpoles, in which release of endogenous PRL was suppressed by injections of bromocryptine to induce metamorphic changes including tail regression, received hormone-containing implants in various sites. PRL implants in the spleen to deliver hormone directly to the liver via the hepatic portal drainage not only prevented tail regression but actually caused a substantial increase in the height of the tail fin. PRL implanted in other sites or GH implanted in the spleen was much less effective. The liver of animals with intrasplenic PRL implants secreted more synlactin in vitro than that of tadpoles with subcutaneous PRL implants. Young grass frogs were injected with ovine (o) GH or oPRL to determine effects on hepatic synlactin secretion. Although the GH stimulated body growth it did not induce the liver to secrete synlactin. By contrast, PRL treatment did stimulate hepatic secretion of synlactin without stimulating body growth. These results indicate that the liver of pre- and postmetamorphic animals can be stimulated by PRL to secrete synlactin. Furthermore, the antimetamorphic actions of PRL in tadpoles appears to be mediated, at least in part, by an action on the liver. Synlactin may mediate this hepatic effect.

Animals↗
PubMed1986

Secretion of prolactin-synergizing activity (synlactin) by the liver of ectothermic vertebrates in vitro.

Recent work in our laboratory indicated that the liver of rats and pigeons secretes a prolactin synergist (synlactin) in vitro. We have investigated the secretion of this activity by the liver of nine species of ectothermic vertebrates. Liver from three teleosts (goby, salmon, and tilapia), four amphibians (Ambystoma, Necturus, bullfrog, and grass frog) and two reptiles (turtle and anole) was diced, washed, and incubated for 3 hr in isotonic medium. After dialysis, the liver incubation media (LIM) were tested with and without prolactin (PRL) in the local pigeon crop-sac bioassay. The LIM for turtle, larval bullfrog, freshwater salmon, and 5% seawater-adapted goby significantly augmented the local crop-sac response to PRL, but the LIM from anole, adult bullfrog, grass frog, fasted larval Ambystoma, Necturus, 100% seawater-adapted goby, and tilapia did not contain synergizing activity. We conclude that synlactin is secreted by the liver of several species representing three of the major ectothermic classes of vertebrates. It is significant that in two cases, larval bullfrog and 5% seawater-adapted goby, the presence of synlactin occurs in physiological states in which PRL is active. In the opposite cases (adult frog and 100% seawater-adapted goby) the activity was not detectable. We also found that the liver of larval and adult bullfrogs and tilapia released a factor in vitro that had proliferative activity in the crop-sac. This activity appears to be distinct from synlactin.

Amphibians↗
PubMedDate not supplied

Quantitative measurement of mRNAs by polymerase chain reaction.

Although polymerase chain reaction (PCR) has been used to detect the presence of specific mRNA species, there are no reports indicating that PCR can be used as a reliable, reproducible assay to quantify the relative level of an mRNA. In this study we examined the enzymatic steps (reverse transcription and PCR) required to analyze RNA by PCR and determined the conditions under which the product obtained reproducibly reflects the relative amounts of amplified species in the starting material. Aliquots of total RNA from rat ovaries and GH3 pituitary cells were used to prepare cDNAs for PCR amplification of beta-actin and prolactin (PRL) sequences, respectively. Assay of equivalent dilutions of ovarian cDNAs made from 10, 2, and 0.4 micrograms of RNA demonstrated that the amount of PCR product obtained was proportional to both the amount of cDNA amplified and the amount of RNA transcribed, with a relatively small variability for both reactions. cDNAs were also made against RNA prepared from GH3 cells cultured in the presence or absence of Ca2+, which induces PRL gene expression. Measurement of PRL mRNA by PCR gave results comparable to those obtained by Northern blot (4.7-fold induction vs. 5.9-fold), and again was highly reproducible. Additionally, PCR analysis of cDNA against GH3 nuclear RNA allowed us to detect an apparent splice variant of the PRL nuclear RNA that is also Ca2+ regulated. These results indicate the sensitivity and reliability of PCR as a quantitative assay for specific mRNAs, and demonstrate the possibilities for obtaining data not readily available by other means.

Actins↗