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J Mazella

Publications and source records attributed to J Mazella.

At least 55 records · Page 3Linked to original sources
PubMed1994

Solubilization and purification of a high affinity neurotensin receptor from newborn human brain.

High affinity neurotensin receptors were solubilized in an active form from newborn human brain using the non-denaturing detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS). The solubilized receptor was purified in a single step by affinity chromatography. The binding properties of the purified receptor towards [125I-Tyr3]neurotensin are very similar to those of the membrane bound and of the crude CHAPS-solubilized receptor in terms of affinity and specificity. The purified receptor is a single protein chain of molecular weight 100 kDa as shown by gel filtration and by affinity labelling with [125I-Tyr3]neurotensin in the presence of the cross-linking agent disuccinimidyl suberate.

Amino Acid Sequence↗
PubMed1994

Activation of the human IGFBP-1 gene promoter by progestin and relaxin in primary culture of human endometrial stromal cells.

We have studied the activity for the insulin-like growth factor binding protein-1 (IGFBP-1) gene promoter in human endometrial stromal cells by transient transfection. The promoter activity derived from p3.6CAT or p3.6Luc (3400 bp IGFBP-1 promoter 5' to the reporter gene chloramphenicol acetyltransferase or luciferase) was minimal in unstimulated cells. A time study over 13 days of culture showed that the promoter activity increased exponentially to > 10(4) fold in cells treated with medroxyprogesterone acetate (MPA) and relaxin (RLX). Induction of the IGFBP-1 gene promoter activity by hormones was similar to the secretion pattern of IGFBP-1 in endometrial stromal cells. MPA alone caused a moderate induction, 3-40-fold increase over the control. Deletion analysis showed that two regions in the IGFBP-1 gene promoter were responsible for the activation of the IGFBP-1 gene. The basal promoter region, termed bp1-A (+68 bp to -1.205 kb), contains multiple sections of regulatory sequence including a cis-element CCAAT (-72 bp). A DNase I protection assay in the bp-1A region revealed four distinct binding regions, one of which contained the CCAAT box region. Another promoter region, termed bp1-B (-2.6 to -3.4 kb), mediated 95% of the total promoter activity in endometrial stromal cells. The bp1-B region also contains multiple regulatory sequences. Mutation and DNase I protection assay suggest that Sp1-like binding site at -2.63 kb was a regulatory site responsible for the activation of IGFBP-1 gene promoter.

Adult↗
PubMed1994

Stable expression of the cloned rat brain neurotensin receptor into fibroblasts: binding properties, photoaffinity labeling, transduction mechanisms, and internalization.

The study of the pharmacological, biochemical, and transduction properties of the cloned rat brain neurotensin receptor was carried out in thymidine kinase mutant fibroblasts stably transfected with the receptor cDNA. The interaction of neurotensin with transfected fibroblasts leads to a concentration-dependent stimulation of phosphatidylinositol hydrolysis and intracellular calcium. These effects are totally inhibited by the nonpeptide neurotensin antagonist SR48692. By contrast, this receptor remains unable to modulate intracellular levels of cyclic nucleotides. The transfected neurotensin receptor can be solubilized in an active form by digitonin with an identical pharmacological profile, whereas the detergent 3-[(3-cholamidopropyl) dimethylammonio]-1-propane-sulfonic acid is unable to solubilize the binding activity. The binding of iodinated neurotensin to transfected fibroblasts bearing the cloned receptor remains partly un-dissociated even after an acid washing step, indicating that the transfected neurotensin receptor retains the capacity to be internalized according to a temperature-dependent mechanism. Indeed, the sequestration of the neurotensin-receptor complex can be blocked by phenylarsine oxide. Finally, photoaffinity labeling experiments reveal that the cloned rat brain neurotensin receptor is expressed under two forms with molecular masses of 50 and 60 kDa. Labeling and internalization of these two proteins are totally blocked by the neurotensin antagonist SR48692.

Animals↗
PubMed1994

Identification of a distal regulatory sequence of the human IGFBP-1 gene promoter and regulation by the progesterone receptor in a human endometrial adenocarcinoma cell line.

The activity of the insulin-like growth factor binding protein-1 (IGFBP-1) gene promoter was studied in the human endometrial adenocarcinoma cell line HEC-1B. Basal promoter activity was directed by the region +68 to -207 bp, similar to observations in the hepatoma HepG2 cell line. A distal regulatory sequence approximately -2.6 kb from the transcription initiation site strongly enhanced the activity of the IGFBP-1 gene promoter in HEC-1B cells, but not in HepG2 cells. Sequence analysis revealed that this active region resides in 105 bp between -2,628 to -2,732 bp (the Rsa I-Cla I fragment). This region contains many putative active motifs homologous to known cis elements. Additional deletion and mutation in the Rsa I-Cla I fragment showed that the activity was confined to a 58-bp DNA fragment. In cells treated with progestin and co-transfected with progesterone receptor vector hPR1, the CAT activity derived from constructs containing the Rsa I-Cla I fragment was reduced in a dose-dependent manner. The active DNA fragment also stimulated the activity of the heterologous TK/CAT promoter in HEC-1B cells, while the PR complex inhibited this activity by 50%. These observations indicate that most of the regulation of the IGFBP-1 gene in HEC-1B cells is derived from the distal promoter region confined to the Rsa I-Cla I fragment and that the same region mediates an inhibitory effect from the progesterone receptor.

Adenocarcinoma↗
PubMed1994

Decidualization of human endometrial stromal cells in vitro: effects of progestin and relaxin on the ultrastructure and production of decidual secretory proteins.

Decidual cells arise by proliferation and differentiation of endometrial stromal cells of the uterus after appropriate stimulation by ovarian hormones. Previously we have shown that progestin and relaxin stimulate the secretion of several decidual-cell-specific secretory proteins in a long-term primary cell culture system. We now report the effects of progestin and relaxin on the morphology of stromal cells in association with the production rate of two major decidual secretory proteins, prolactin and insulin-like growth factor binding protein-1 (IGFBP-1). Stromal cells were cultured in RPMI 1640 and 2% fetal bovine serum for 22 days under control conditions (no hormone), with relaxin or medroxyprogesterone acetate (MPA), or MPA plus relaxin. Cells treated with MPA alone or MPA plus porcine relaxin grew to a high density with many areas of heaping while control cells and cells grown in medium containing relaxin alone formed discontinuous layers. The cytoplasm was distinguished by aggregates of rough endoplasmic reticulum and secretory granules. Surfaces of cells treated with MPA plus relaxin had clusters of short blunt processes containing secretory granules. The processes were rarely seen in cells exposed to MPA alone and completely absent in control cells or cells exposed to relaxin alone. Intercellular space was greatly widened in cells treated with MPA alone or MPA plus relaxin. There were many extended gap junctions in MPA- and relaxin-treated cells in contrast to controls.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1994

In vivo and in vitro structure-activity studies with peptide and pseudopeptide neurotensin analogs suggest the existence of distinct central neurotensin receptor subtypes.

The present study was designed to compare, with respect to structure-activity relationships, the receptors that subserve the hypothermic and analgesic effects of neurotensin (NT) to the receptor that mediates the effects of NT in mesencephalic dopamine (DA) neurons, and to compare these receptors to the cloned adult rat brain NT receptor and to newborn mouse and rat brain NT receptors. The results show that NT receptors in homogenates from newborn mouse and rat brain and from COS 7 cells transfected with the cloned high-affinity NT receptor from the adult rat brain displayed virtually identical structure-activity relationships toward a series of 12 peptide and pseudopeptide NT analogs, as assessed by the ability of the compounds to inhibit the binding of [125I]NT binding in these systems. Furthermore, when eight of these analogs were tested for their ability to inhibit [125I]NT binding and to potentiate K(+)-evoked DA release in primary cultures of rat mesencephalic neurons, it was found that they all behaved as agonists with binding and biological potencies quite similar to those observed in the other binding assays. Finally and strikingly, when seven of these analogs with checked metabolic stability were tested in vivo for their hypothermic and analgesic (tail-flick test) effects after i.c.v. injection in the mouse, they exhibited relative potencies that were completely different from those obtained in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1993

Implication of various forms of neurotensin receptors in the mechanism of internalization of neurotensin in cerebral neurons.

This report describes the kinetics and molecular aspects of neurotensin internalization in neurons. Incubation of alpha-125I-Bolton-Hunter neurotensin-(2-13) with cortical neurons at 37 degrees C was followed by a rapid internalization of the peptide bound to its receptors. This process was completed within 20 min and was inhibited either irreversibly by the general endocytosis inhibitor phenylarsine oxide or reversibly by incubation at low temperature (0-4 degrees C). The discrepancy of maximal binding capacities observed in the presence (150 fmol/mg of protein) or in the absence (250 fmol/mg of protein) of internalization inhibitors could be attributed to the appearance of a new pool of neurotensin binding sites on the cell surface rather than a recycling of internalized receptors. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis in denaturing conditions revealed that three different protein subunits of 50, 60, and 100 kDa were covalently labeled at 37 degrees C with a radioactive photoreactive analogue of neurotensin. The 50- and 60-kDa subunits remained labeled when internalization was blocked, whereas the specific labeling of the 100-kDa protein was abolished. These results suggest that neurotensin-induced internalization of the 50- and 60-kDa subunits initially present on the cell surface triggers insertion of the 100-kDa subunit into the membrane from an intracellular compartment. Subcellular fractionation experiments have shown that, in the absence of neurotensin, the 100-kDa protein is located in an intracellular vesicular fraction of neurons.

Animals↗
PubMed1993

Insulin-like growth factor binding protein-1 inhibits the mitogenic effect of insulin-like growth factors and progestins in human endometrial stromal cells.

This study describes the effects of insulin-like growth factors (IGFs), progestins, and IGF binding protein-1 (IGF-BP1) on DNA synthesis in human endometrial stromal cells. Endometrial stromal cells treated with IGF-I or IGF-II or progestins increased their rate of DNA synthesis 3-6-fold. In a time study, IGF-I or IGF-II stimulated DNA synthesis 3-fold in 24 h. An equivalent increase by medroxyprogesterone acetate (MPA) required 48 h. Progestin-stimulated DNA synthesis was inhibited completely by an equimolar concentration of the antiprogesterone RU486. IGF-I-stimulated DNA synthesis was inhibited completely by IGF-BP1. Half-maximal inhibition of IGF-I-stimulated DNA synthesis occurred when IGF-I and IGF-BP1 were at equimolar concentrations. Non-phosphorylated and phosphorylated forms of IGF-BP1 were equipotent inhibitors of IGF-I-stimulated DNA synthesis. IGF-BP1 also inhibited progestin-stimulated DNA synthesis. Half-maximal inhibition of progestin-stimulated DNA synthesis occurred at 100 ng/ml of phosphorylated IGF-BP1. Phosphorylated IGF-BP1 was a 3-4-fold more potent inhibitor of progestin-stimulated DNA synthesis than the nonphosphorylated form. The inhibition by IGF-BP1 of progestin-stimulated DNA synthesis was reversed by an equimolar concentration of IGF-II. This study suggests that progestins regulate an autocrine growth control loop in the endometrium that involves IGFs, IGF receptors, and IGF-BPs.

Adult↗
PubMed1992

Effect of progestin, antiprogestin, and relaxin on the accumulation of prolactin and insulin-like growth factor-binding protein-1 messenger ribonucleic acid in human endometrial stromal cells.

Prolactin (PRL) and insulin-like growth factor-binding protein (IGFBP-1) are two major secretory proteins of human endometrial/decidual cells. We have characterized the mRNA of PRL and IGFBP-1 and studied the effect of progestin, medroxyprogesterone acetate (MPA), anti-progestin (RU486), and relaxin (RLX) on the levels of these two mRNA transcripts in a long-term culture of human endometrial stromal cells. Northern blot analysis showed that the size of PRL mRNA was 1.15 kb and that of IGFBP-1 mRNA, 1.6 kb. Primer extension of endometrial/decidual IGFBP-1 mRNA showed two transcription initiation sites identical to those found in HepG2 human hepatoma cell line. The levels of mRNA in control samples remained low, approximately 2 pg PRL and approximately 5 pg IGFBP-1/microgram RNA at various times of culture. When stromal cells were treated with MPA for 28 days, PRL mRNA gradually increased 100-fold whereas IGFBP-1 mRNA exponentially increased approximately 1000-fold compared to control values and leveled after 25 days in culture. The timing of maximal stimulation was shortened by withdrawing MPA or by replacing MPA with RU486. After removal of MPA, levels of both mRNAs increased and each peaked after approximately 10 days, with PRL showing a 2-fold and IGFBP-1 a 20-fold increase compared to cells treated with MPA continuously. Replacing MPA by RU486 caused a rapid increase of PRL mRNA (2-3-fold) in 2-3 days followed by a gradual reduction to less than 20% of peak levels over the next 3 days. IGFBP-1 mRNA levels increased 30- and 100-fold in 1-2 days followed by a reduction to less than 20% of peak levels over the next 24 h. The reduction of mRNA levels by RU486 was reversed when cells were rechallenged with MPA. Relaxin alone caused a transient stimulation of PRL and IGFBP-1 mRNA. Maximal stimulation occurred between 10 and 20 days of culture and was 100-fold for PRL and 1000-fold for IGFBP-1 relative to control values. Cells treated with MPA and RLX in sequence had higher mRNA levels than cells treated with MPA continuously or cells subjected to MPA withdrawal. Maximal mRNA levels reached 0.4 ng PRL and approximately 8 ng IGFBP-1/microgram total RNA, approximately 0.04% and 0.8% of cellular RNA. The mRNA levels under various hormonal manipulations were similar to the previously published synthesis and secretion patterns of PRL and IGFBP-1 proteins in this system.(ABSTRACT TRUNCATED AT 400 WORDS)

Base Sequence↗
PubMed1991

Binding and internalization of iodinated neurotensin in neuronal cultures from embryonic mouse brain.

The binding and internalization of labeled neurotensin were studied by means of biochemical and light microscopic radioautography techniques in primary cultures of neurons from whole cerebral hemispheres of mouse embryos. Saturable, high affinity neurotensin binding was detected 5-7 days postplating in cells incubated with 0.1 nM 125I-Tyr3-neurotensin at 37 degrees C or 10 degrees C. The binding capacity at equilibrium was 3 times higher at 37 degrees C than at 10 degrees C. Moreover, whereas virtually all the radioactivity bound at 10 degrees C was membrane-bound (i.e. was readily washable by a hypertonic, high pH, NaCl solution), more than 70% of the radioactivity bound at 37 degrees C was intracellular (i.e. resisted the same treatment). Light microscopic radioautograms of whole cells revealed that approximately 16% of neurons were labeled with 125I-Tyr3-neurotensin at either 37 degrees C or 10 degrees C. The labeling was observed over cell bodies and processes, and the density of silver grains associated with perikarya, as compared to processes, was proportionally higher at 37 degrees C than at 10 degrees C. Semi-thin (1 micron thick) sections through cells incubated at 37 degrees C confirmed that a major fraction of the radioactivity was intracellular and showed that it was mainly confined to the cytoplasm. These results indicate that 125I-Tyr3-neurotensin binds to a distinct subset of primary cultured neurons and that a large proportion of the bound radioactivity undergoes rapid internalization in a temperature-dependent manner. It is proposed that this internalization is ligand-induced and that it may play a role in the modulation of central neurotensin receptor levels.

Animals↗
PubMed1990

Differential effects of progestin and relaxin on the synthesis and secretion of immunoreactive prolactin in long term culture of human endometrial stromal cells.

PRL secretion from human endometrium is a continuous process extending from the luteal phase of the menstrual cycle throughout the entire gestational stage. We have developed a long term primary cell culture system to elucidate the hormonal requirements for this sustained production of PRL. The effects of medroxyprogesterone acetate (MPA), progesterone, and relaxin (RLX) on the production of immunoreactive PRL were investigated. MPA stimulated cell growth and PRL production rate during days 5-20 of culture. Progesterone was 20-40% less effective in stimulating PRL than MPA. Stimulation of PRL was continued 1-2 weeks after MPA withdrawal. Relaxin did not promote cell growth. However, it induced the PRL production which fluctuated during the long term culture. The maximal response to RLX was 2- to 3-fold higher or similar to that of MPA. Only five of nine endometrial specimens examined responded to RLX alone. The effect of MPA plus RLX was significantly greater than that of MPA or RLX alone. The highest production rate was shown in cells treated with MPA and then RLX in sequence. After a month of culture, the production rates (micrograms of PRL per 0.1 mg cell DNA/day) under various culture conditions (A, control; B, MPA; C, MPA for 10-15 days and no hormone afterward; D, both MPA and RLX; and E, MPA and RLX in sequence) were: A, about 0-0.01 (n = 12); B, 2.5 +/- 0.9 (n = 8); C, 4.8 +/- 2.5 (n = 8); D, 5.7 +/- 3.0 (n = 5); and E, 11 +/- 3.7 (n = 7); mean +/- SD; n, number of specimens). Endometrial stromal cells were incubated with [35S]methionine, and [35S]immunoreactive PRL and other secretory proteins were analyzed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis to characterize the size and isoforms of immunoreactive PRL. PRL was one of the five major secretory proteins (23-25K, 32K, 42K, 78K, and 150K daltons, sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing condition) induced by MPA and RLX in endometrial stromal cells. More than 90% of immunoreactive PRL was secreted into the medium. The apparent mol wt of immunoreactive PRL were 21K, 23K (the predominant size), and 25K daltons. Results obtained from the incorporation of [14C]glucosamine into immunoreactive PRL indicated that both 23K and 25K PRL contained glycosylated PRL. A 45K-dalton glycosylated immunoreactive PRL was also present in the culture medium.(ABSTRACT TRUNCATED AT 400 WORDS)

Adult↗
PubMed1990

Colocalization of neurotensin receptors and of the neurotensin-degrading enzyme endopeptidase 24-16 in primary cultures of neurons.

This paper compares the localization of neurotensin receptors and of endopeptidase 24-16, a peptidase likely involved in the inactivation of neurotensin in primary cultures of neurons. Neurotensin binding sites were radiolabeled with 125I-Tyr3-neurotensin, whereas endopeptidase 24-16 was stained by immunohistochemical techniques using a monospecific polyclonal antibody. Endopeptidase 24-16 is present in 80-85% of the nondifferentiated neurons. The proportion of immunoreactive neurons decreased during maturation to reach 35-40% after 4-8 d of culture. By contrast, neurotensin receptors were not detectable in nondifferentiated cells and appear during maturation. Specific 125I-Tyr3-neurotensin labeling is maximal after 4 d of culture and is located on about 10% of differentiated neurons. Double-labeling experiments show that about 90% of cortical, hypothalamic, and mesencephalic neurons bearing the neurotensin receptor also contained endopeptidase 24-16, supporting the hypothesis that one of the functions of endopeptidase 24-16 is the physiological inactivation of neurotensin. However, the presence of endopeptidase 24-16 on numerous neurons that do not contain neurotensin receptors also suggests that the enzyme could be involved in the degradation and/or maturation of other neuropeptides.

Animals↗
PubMed1989

Purification of the neurotensin receptor from mouse brain by affinity chromatography.

The neurotensin receptor was purified from newborn mouse brain by affinity chromatography. Active neurotensin binding sites were solubilized from brain homogenates using the nondenaturing detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS) in the presence of cholesteryl hemisuccinate. Chromatography of the soluble extract on SP-Sephadex C-25 and hydroxylapatite eliminated 50% of proteins without loss of neurotensin binding activity. This prepurified material was loaded into an affinity column prepared by coupling neurotensin (2-13) to glutaraldehyde-activated Ultrogel AcA22. Nonspecifically adsorbed proteins were eliminated by extensive washing, and the receptor was eluted with a buffer containing 1 M NaCl, 0.1% CHAPS, and 0.02% cholesteryl hemisuccinate. After desalting, the purified receptor bound 125I-labeled neurotensin with a dissociation constant of 0.26 nM and retained its specificity towards a series of neurotensin analogues. The desalted NaCl eluate appeared on sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a major band of molecular weight 100,000 which was identified as the receptor by affinity labeling with 125I-labeled neurotensin in the presence of disuccinimidyl suberate. The purity of the mouse brain receptor eluted from the affinity column was estimated to be 78%. Electroelution of the 100-kDa protein band gave an homogenous preparation of receptor. Very similar results were obtained with CHAPS-solubilized neurotensin receptors from rat and rabbit brain.

Animals↗
PubMed1989

Long-term effects of progestin and RU 486 on prolactin production and synthesis in human endometrial stromal cells.

Previous studies have shown that the production of prolactin (PRL) is increased in human endometrial stromal cells cultured with medroxyprogesterone acetate (MPA) for 3-5 days. In the present study, we have investigated the effect of prolonged treatment of progestin and anti-progestin, RU 486, on the production and synthesis of PRL. Stromal cells were isolated from human endometrium obtained from non-pregnant women and cultured for 20-30 days in medium RPMI 1640 with 2% fetal calf serum or supplemented with MPA, RU 486, alone or in sequence. The PRL content in medium was measured by radioimmunoassay. The production rate was estimated from the PRL content in medium accumulated in 24 h. The PRL production rate was progressively increased in stromal cells continuously treated with MPA for 30 days (greater than 100-fold over the control value, i.e. 0-0.01 microgram/0.1 mg cell DNA/day). RU 486 alone had no effect on the production of PRL. However, the production of PRL was increased by MPA in stromal cells pretreated with RU 486 indicating that the effect of RU 486 is reversible. When stromal cells were treated with MPA and RU 486 sequentially, RU 486 stimulated the PRL production (approximately 2-fold over the MPA-treated cells) for 2-3 days and then reduced to basal levels over a 5-day period. The stimulatory and inhibitory effects of RU 486 on PRL production in stromal cells pretreated with progestin was also observed in the rate of synthesis of PRL estimated by incubating the stromal cells with [35S]methionine and immuno-isolating the [35S]PRL with anti-PRL.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗