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Papers by Isabel Neuman

Copyright © 2012 Roxana N. Peroni et al. This is an open access article distributed under the Cre... more Copyright © 2012 Roxana N. Peroni et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. In rat isolated mesenteric beds that were contracted with NA as an in vitro model of the vascular adrenergic hyperactivity that usually precedes the onset of primary hypertension, the oral administration (3 daily doses) of either 10 mg/kg genistein or 20 mg/kg daidzein potentiated the anandamide-induced reduction of contractility to NA in female but not in male rats. Oral treatment with phytoestrogens also restored the vascular effects of anandamide as well as the mesenteric content of calcitonin gene-related peptide (CGRP) that were reduced after ovariectomy. The enhancement of anandamide effects caused by phytoestrogens was prevented by the concomitant administration of the estrogen receptor antagonist fulvestrant (2.5 mg/kg, s.c...

European Journal of Biochemistry, 1994

In previous reports we have demonstrated the presence of a soluble factor that responds to cAMP s... more In previous reports we have demonstrated the presence of a soluble factor that responds to cAMP signals to induce steroid synthesis in adrenocortical tissue. Here, we describe the purification of this factor from adrenal zona fasciculata cells by using a five-step procedure that includes DEAEcellulose, gel filtration, Mono Q HPLC and Superose HPLC, and elution of the protein from SDS/ PAGE. This procedure results in the purification to homogeneity of a protein of 43-kDa that retains the capacity to stimulate steroid synthesis in an in v i m recombination assay. This activity is inhibited by the use of phospholipase A, inhibitors. Antipeptide antibodies against the N-terminal region recognize p43 as a double band on SDSPAGE that resolves in different spots on two-dimensional gel electrophoresis. Adrenocorticotropin treatment of adrenal glands results in the appearence of multiple spots that migrated towards a lower pH compared to controls, suggesting the presence of phosphorylated and dephosphorylated forms of p43. Sequencing of the N-terminal region and internal peptides reveals no significant similarities with other proteins, suggesting that p43 is a novel protein. We conclude from our data that the isolated protein (p43) is a novel, soluble protein that acts as intermediary in adrenocorticotropin-induced stimulation of arachidonic acid release and steroid synthesis. Although the mechanism of hormonal stimulation of steroidogenesis in Leydig and adrenal cells is not completely understood, it is generally accepted that lutropin or adrenocorticotropin, respectively, bind to a specific receptor on the cell surface activating adenylate cyclase which results in a rapid increase of intracellular cAMP levels and an increase of cAMP bound to the regulatory subunit of the CAMP-dependent protein kinase (PKA) [l-51. This causes activation of PKA which leads to protein phosphorylation and ultimately to increased biosynthesis of steroids [3, 6-81. The initial and apparently rate-limiting step in the production of steroid hormones in both tissues is the conversion of cholesterol to pregnenolone catalyzed by the side-chain-cleavage enzyme complex, taking place in the inner mitochondrial membrane. This reaction is, in turn, limited by the transport of cholesterol from the outer to the inner mitochondrial membrane [9-121. In Leydig and adrenal cells, arachidonic acid and the lipoxygenase products have been shown to play an obligatory role as intermediaries in the mechanism of action of lutropin

Endocrine Research, 1997

Evidence has been introduced linking the lipoxygenase products and steroidogenesis in Leydig cell... more Evidence has been introduced linking the lipoxygenase products and steroidogenesis in Leydig cells, thereby supporting that this pathway may be a common event in the hormonal control of steroid synthesis. On the other hand, it has also been reported that lipoxygenase products of arachidonic acid (AA) may not be involved in Leydig cells steroidogenesis. In this paper, we investigated the effects of PLA2 and lipoxygenase pathway inhibitors on steroidogenesis in rat testis Leydig cells. The effects of two structurally unrelated PLA2 inhibitors (4-bromophenacyl bromide (BPB) and quinacrine) were determined. BPB blocked the LH- and Bt2cAMP-stimulated testosterone production but had no effect on 22(4)-OH-cholesterol conversion to testosterone. Quinacrine caused a dose-dependent inhibition of LH- and Bt2cAMP-induced steroidogenesis. The effects of different lipoxygenase pathway inhibitors (nordihydroguaiaretic acid (NDGA), 5,8,11,14-eicosatetraynoic acid (ETYA), caffeic acid and esculetin) have also been determined. Both NDGA and ETYA inhibited LH- and Bt2cAMP-stimulated steroid synthesis in a dose-related manner. Furthermore caffeic acid and esculetin also blocked the LH-stimulated testosterone production. Moreover, exogenous AA induced a dose-dependent increase of testosterone secretion which was inhibited by NDGA. Our results strongly support the previous concept that the lipoxygenase pathway is involved in the mechanism of action of LH on testis Leydig cells.

Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, 1999

Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, 1996

European Journal of Biochemistry, 1998

We have previously reported the purification of a phosphoprotein (p43) intermediary in steroid sy... more We have previously reported the purification of a phosphoprotein (p43) intermediary in steroid synthesis from adrenal zona fasciculata [

An arachidonic acid-preferring acyl-CoA synthetase is a hormone-dependent and obligatory protein ... more An arachidonic acid-preferring acyl-CoA synthetase is a hormone-dependent and obligatory protein in the signal

Prostaglandins & Other Lipid Mediators

It is well accepted that protein(s) with a short half-life are required in the pathway leading to... more It is well accepted that protein(s) with a short half-life are required in the pathway leading to steroid synthesis following stimulation by trophic hormones. A correlation between the disappearance of several proteins in different subcellular compartments and the inhibition of steroid synthesis produced by cycloheximide (CHx) has also been shown, In the present report we describe the effect of CHx in the stimulation of steroid synthesis using a cell-free assay. Mitochondrial progesterone (P4) production was studied by recombination of the different subcellular fractions of adrenal zona fasciculate and determined by radioimmunoassay. Soluble factors from ACTH-treated adrenals produced a four-fold stimulation of mitochondrial steroidogenesis (3.0 ± 0.6 vs. 13.3 ± 0.5 ng P4/tube for control and ACTH-treated adrenals respectively). Mitochondria obtained from CHx-ACTH-treated adrenals fail to respond to soluble ACTH-dependent factors. A permeable analogue of cholesterol (22(R)-OH choles...

International journal of hypertension, 2012

In rat isolated mesenteric beds that were contracted with NA as an in vitro model of the vascular... more In rat isolated mesenteric beds that were contracted with NA as an in vitro model of the vascular adrenergic hyperactivity that usually precedes the onset of primary hypertension, the oral administration (3 daily doses) of either 10 mg/kg genistein or 20 mg/kg daidzein potentiated the anandamide-induced reduction of contractility to NA in female but not in male rats. Oral treatment with phytoestrogens also restored the vascular effects of anandamide as well as the mesenteric content of calcitonin gene-related peptide (CGRP) that were reduced after ovariectomy. The enhancement of anandamide effects caused by phytoestrogens was prevented by the concomitant administration of the estrogen receptor antagonist fulvestrant (2.5 mg/kg, s.c., 3 daily doses). It is concluded that, in the vasculature of female rats, phytoestrogens produced an estrogen-receptor-dependent enhancement of the anandamide-vascular actions that involves the modulation of CGRP levels and appears to be relevant wheneve...

Endocrine research, 1996

It is well accepted that protein(s) with a short half-life are required in the pathway leading to... more It is well accepted that protein(s) with a short half-life are required in the pathway leading to steroid synthesis following stimulation by trophic hormones. A correlation between the disappearance of several proteins in different subcellular compartments and the inhibition of steroid synthesis produced by cycloheximide (CHx) has also been shown. In the present report we describe the effect of CHx in the stimulation of steroid synthesis using a cell-free assay. Mitochondrial progesterone (P4) production was studied by recombination of the different subcellular fractions of adrenal zona fasciculata and determined by radioimmunoassay. Soluble factors from ACTH-treated adrenals produced a four-fold stimulation of mitochondrial steroidogenesis (3.0 +/- 0.6 vs. 13.3 +/- 0.5 ng P4/tube for control and ACTH-treated adrenals respectively). Mitochondria obtained from CHx-ACTH-treated adrenals fail to respond to soluble ACTH-dependent factors. A permeable analogue of cholesterol (22(R)-OH ch...

Endocrine research, 1996

We have previously isolated and partially-sequenced a soluble phosphoprotein (p43) that acts as i... more We have previously isolated and partially-sequenced a soluble phosphoprotein (p43) that acts as intermediary in the stimulation of steroid synthesis. In this report we have used synthetic peptides whose sequences match those obtained from p43 to generate antipeptide antibodies and show that these antibodies bind to purified p43 protein as determined by immunoblot analysis. The presence of p43 was detected by Western blot in both steroidogenic and non-steroidogenic tissues. One of the antibodies was also used to purify p43 on immunoaffinity chromatography columns. Proteins eluting from affinity columns produce a twelve-fold stimulation of progesterone synthesis. This effect was blocked by the use of an inhibitor of phospholipase A2. These results suggest the involvement of p43 in transducing the adrenocorticotropin signal to mitochondria in zona fasciculata cells. We also describe a partial cDNA clone with a predicted amino acid sequence that matches the sequences of the internal pep...

Biochemical and Biophysical Research Communications, 2002

Arachidonic acid is not freely stored in the cells. A number of different pathways for the mobili... more Arachidonic acid is not freely stored in the cells. A number of different pathways for the mobilization of this compound have been proposed, including a novel mechanism that involves the release of arachidonic acid from arachidonoyl-CoA by a thioesterase with substrate specificity for very-long-chain fatty acids. In rat heart, the acyl-CoA thioesterase activity can be regulated by a mechanism that

Molecular and Cellular Endocrinology, 2015

Hormone-regulated steroidogenesis and StAR protein induction involve the action of lipoxygenated ... more Hormone-regulated steroidogenesis and StAR protein induction involve the action of lipoxygenated products. The products of 5-lipoxygenase act on inflammation and immunity by stimulation of a membrane receptor called OxeR1. The presence of OxeR1 in other systems has not been described up to date and little is known about its mechanism of action and other functions. In this context, the aim of this study was the identification and characterization of OxeR1 as a mediator of cAMP-dependent and independent pathways. Overexpression of OxeR1 in MA-10 Leydig cells increased cAMP-dependent progesterone production. Angiotensin II and cAMP stimulation of adrenocortical human H295R cells produced an increase in StAR protein induction and steroidogenesis in cells overexpressing OxeR1 as compared to mock-transfected cells. Additionally, activation of OxeR1 caused a time-dependent increase in ERK1/2 phosphorylation. In summary, membrane receptor OxeR1 is involved in StAR protein induction and activation of steroidogenesis triggered by cAMP or angiotensin II, acting, at least in part, through ERK1/2 activation.

SpringerPlus, 2012

Background: The spectrum of BRCA1/2 genetic variation in breast-ovarian cancer patients has been ... more Background: The spectrum of BRCA1/2 genetic variation in breast-ovarian cancer patients has been scarcely investigated outside Europe and North America, with few reports for South America, where Amerindian founder effects and recent multiracial immigration are predicted to result in high genetic diversity. We describe here the results of BRCA1/BRCA2 germline analysis in an Argentinean series of breast/ovarian cancer patients selected for young age at diagnosis or breast/ovarian cancer family history.

European Journal of Biochemistry, 1998

1. Eur. J. Biochem. 224, 709Ϫ716]. Here, we describe the cloning and sequencing of a cDNA enco... more 1994) Eur. J. Biochem. 224, 709Ϫ716]. Here, we describe the cloning and sequencing of a cDNA encoding p43 as well as the hormonal regulation of the p43 transcript. The protein resulted homologous to a very recently described mitochondrial peroxisome-proliferator-induced very-long-chain acyl-CoA thioesterase (MTE-I). The deduced amino acid sequence of the protein shows consensus sites for phosphorylation by different protein kinases, and a lipase serine motif. Antibodies raised against a synthetic peptide that includes the lipase serine motif and against the N-terminal region of p43 block the action of the protein. The transcript of p43 was detected in ovary of pseudopregnant rats, rat adrenal zona fasciculata and glomerulosa, mouse Leydig tumor cell line (MA-10), rat brain and human placenta. Inhibition of adrenocorticotropin hormone (ACTH) release and steroid synthesis by dexamethasone produced a dose-dependent decrease in the abundance of the adrenal transcript. The transcript was induced by in vivo stimulation of the adrenals with ACTH. The effect had a rapid onset (5 min), reached maximal stimulation (62%) at 15 min, and returned to basal levels at 30 min. The effect of ACTH on the p43 transcript was inhibited by actinomycin D and enhanced by cycloheximide. Our results provide the first evidence linking acyl-CoA thioesterases with very-long-chain specificities, and a protein intermediary in steroid synthesis, thereby supporting a regulatory role for acyl-CoA thioesterases in steroidogenic tissues. Abbreviations. ARTISt, arachidonic-acid-related thioesterase involved in steroidogenesis; ACTH, adrenocorticotropin hormone; dexamethasone, 9-fluoro-11β,17,21-trihydroxy-16A-methylpregna-1,4-diene-3,20-dione; AA, arachidonic acid; RT-PCR, reverse-transcription PCR ; CTE-I, cytosolic acyl-CoA thioesterase I; MTE-I, mitochondrial acyl-CoA thioesterase.

The Journal of Steroid Biochemistry and Molecular Biology, 1991

Luteinizing hormone (LH) and human chorionic gonadotrophin (hCG) receptors are coupled to intrace... more Luteinizing hormone (LH) and human chorionic gonadotrophin (hCG) receptors are coupled to intracellular effector systems, most notably adenylate cyclase, through guanyl nucleotide-binding proteins or G-proteins. The molecular mechanism involved in the dynamic coupling of the LH/hCG receptor however, are not known. It has been postulated that receptor aggregation at the molecular level plays a critical role in this process. There have been attempts to understand the receptor association and dissociation phenomena at the molecular level. One of them involves the participation of the major histocompatibility complex (MHC) class I antigen in the mechanism of receptor activation and/or expression. One molecular basis for these mechanisms consists of a physical interaction between MHC proteins and receptors to form "compound receptors" able to transfer a hormonal signal to the cell. Using a photo-reactive probe we demonstrated that the LH/hCG receptors and the class I antigens are closely associated in the membrane. Thus, it is possible to form covalent complexes of hCG and class I antigens through the binding of the hormone to specific receptors. These findings imply that LH/hCG receptors and the MHC class I antigens may interact at the level of the plasma membrane in the mechanism of LH action. We also performed experiments using a single cell and limiting stimulation to a patch of membrane. The results stimulating the cell in a localized area suggested that even if all components are entirely free to float there is a constraint in the localization of the receptor, G-protein, and/or the effector, supporting the constraint dissociation model. Within a limited area subunits could dissociate, but they would not be free to diffuse throughout the membrane. Moreover the concept of compartmentalization that has been utilized to explain some inconsistencies in second-messenger action now can be proved by experimental design.

The Journal of Steroid Biochemistry and Molecular Biology, 2006

The activation of the rate-limiting step in steroid biosynthesis, that is the transport of choles... more The activation of the rate-limiting step in steroid biosynthesis, that is the transport of cholesterol into the mitochondria, is dependent on PKA-mediated events triggered by hormones like ACTH and LH. Two of such events are the protein tyrosine dephosphorylation mediated by protein tyrosine phosphatases (PTPs) and the release of arachidonic acid (AA) mediated by two enzymes, ACS4 (acyl-CoA synthetase 4) and Acot2 (mitochondrial thioesterase). ACTH and LH regulate the activity of PTPs and Acot2 and promote the induction of ACS4. Here we analyzed the involvement of PTPs on the expression of ACS4. We found that two PTP inhibitors, acting through different mechanisms, are both able to abrogate the hormonal effect on ACS4 induction. PTP inhibitors also reduce the effect of cAMP on steroidogenesis and on the level of StAR protein, which facilitates the access of cholesterol into the mitochondria. Moreover, our results indicate that exogenous AA is able to overcome the inhibition produced by PTP inhibitors on StAR protein level and steroidogenesis. Then, here we describe a link between PTP activity and AA release, since ACS4 induction is under the control of PTP activity, being a key event for AA release, StAR induction and steroidogenesis.

European Journal of Biochemistry, 1994

In previous reports we have demonstrated the presence of a soluble factor that responds to cAMP s... more In previous reports we have demonstrated the presence of a soluble factor that responds to cAMP signals to induce steroid synthesis in adrenocortical tissue. Here, we describe the purification of this factor from adrenal zona fasciculata cells by using a five-step procedure that includes DEAEcellulose, gel filtration, Mono Q HPLC and Superose HPLC, and elution of the protein from SDS/ PAGE. This procedure results in the purification to homogeneity of a protein of 43-kDa that retains the capacity to stimulate steroid synthesis in an in v i m recombination assay. This activity is inhibited by the use of phospholipase A, inhibitors. Antipeptide antibodies against the N-terminal region recognize p43 as a double band on SDSPAGE that resolves in different spots on two-dimensional gel electrophoresis. Adrenocorticotropin treatment of adrenal glands results in the appearence of multiple spots that migrated towards a lower pH compared to controls, suggesting the presence of phosphorylated and dephosphorylated forms of p43. Sequencing of the N-terminal region and internal peptides reveals no significant similarities with other proteins, suggesting that p43 is a novel protein.

Copyright © 2012 Roxana N. Peroni et al. This is an open access article distributed under the Cre... more Copyright © 2012 Roxana N. Peroni et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. In rat isolated mesenteric beds that were contracted with NA as an in vitro model of the vascular adrenergic hyperactivity that usually precedes the onset of primary hypertension, the oral administration (3 daily doses) of either 10 mg/kg genistein or 20 mg/kg daidzein potentiated the anandamide-induced reduction of contractility to NA in female but not in male rats. Oral treatment with phytoestrogens also restored the vascular effects of anandamide as well as the mesenteric content of calcitonin gene-related peptide (CGRP) that were reduced after ovariectomy. The enhancement of anandamide effects caused by phytoestrogens was prevented by the concomitant administration of the estrogen receptor antagonist fulvestrant (2.5 mg/kg, s.c...

European Journal of Biochemistry, 1994

In previous reports we have demonstrated the presence of a soluble factor that responds to cAMP s... more In previous reports we have demonstrated the presence of a soluble factor that responds to cAMP signals to induce steroid synthesis in adrenocortical tissue. Here, we describe the purification of this factor from adrenal zona fasciculata cells by using a five-step procedure that includes DEAEcellulose, gel filtration, Mono Q HPLC and Superose HPLC, and elution of the protein from SDS/ PAGE. This procedure results in the purification to homogeneity of a protein of 43-kDa that retains the capacity to stimulate steroid synthesis in an in v i m recombination assay. This activity is inhibited by the use of phospholipase A, inhibitors. Antipeptide antibodies against the N-terminal region recognize p43 as a double band on SDSPAGE that resolves in different spots on two-dimensional gel electrophoresis. Adrenocorticotropin treatment of adrenal glands results in the appearence of multiple spots that migrated towards a lower pH compared to controls, suggesting the presence of phosphorylated and dephosphorylated forms of p43. Sequencing of the N-terminal region and internal peptides reveals no significant similarities with other proteins, suggesting that p43 is a novel protein. We conclude from our data that the isolated protein (p43) is a novel, soluble protein that acts as intermediary in adrenocorticotropin-induced stimulation of arachidonic acid release and steroid synthesis. Although the mechanism of hormonal stimulation of steroidogenesis in Leydig and adrenal cells is not completely understood, it is generally accepted that lutropin or adrenocorticotropin, respectively, bind to a specific receptor on the cell surface activating adenylate cyclase which results in a rapid increase of intracellular cAMP levels and an increase of cAMP bound to the regulatory subunit of the CAMP-dependent protein kinase (PKA) [l-51. This causes activation of PKA which leads to protein phosphorylation and ultimately to increased biosynthesis of steroids [3, 6-81. The initial and apparently rate-limiting step in the production of steroid hormones in both tissues is the conversion of cholesterol to pregnenolone catalyzed by the side-chain-cleavage enzyme complex, taking place in the inner mitochondrial membrane. This reaction is, in turn, limited by the transport of cholesterol from the outer to the inner mitochondrial membrane [9-121. In Leydig and adrenal cells, arachidonic acid and the lipoxygenase products have been shown to play an obligatory role as intermediaries in the mechanism of action of lutropin

Endocrine Research, 1997

Evidence has been introduced linking the lipoxygenase products and steroidogenesis in Leydig cell... more Evidence has been introduced linking the lipoxygenase products and steroidogenesis in Leydig cells, thereby supporting that this pathway may be a common event in the hormonal control of steroid synthesis. On the other hand, it has also been reported that lipoxygenase products of arachidonic acid (AA) may not be involved in Leydig cells steroidogenesis. In this paper, we investigated the effects of PLA2 and lipoxygenase pathway inhibitors on steroidogenesis in rat testis Leydig cells. The effects of two structurally unrelated PLA2 inhibitors (4-bromophenacyl bromide (BPB) and quinacrine) were determined. BPB blocked the LH- and Bt2cAMP-stimulated testosterone production but had no effect on 22(4)-OH-cholesterol conversion to testosterone. Quinacrine caused a dose-dependent inhibition of LH- and Bt2cAMP-induced steroidogenesis. The effects of different lipoxygenase pathway inhibitors (nordihydroguaiaretic acid (NDGA), 5,8,11,14-eicosatetraynoic acid (ETYA), caffeic acid and esculetin) have also been determined. Both NDGA and ETYA inhibited LH- and Bt2cAMP-stimulated steroid synthesis in a dose-related manner. Furthermore caffeic acid and esculetin also blocked the LH-stimulated testosterone production. Moreover, exogenous AA induced a dose-dependent increase of testosterone secretion which was inhibited by NDGA. Our results strongly support the previous concept that the lipoxygenase pathway is involved in the mechanism of action of LH on testis Leydig cells.

Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, 1999

Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, 1996

European Journal of Biochemistry, 1998

We have previously reported the purification of a phosphoprotein (p43) intermediary in steroid sy... more We have previously reported the purification of a phosphoprotein (p43) intermediary in steroid synthesis from adrenal zona fasciculata [

An arachidonic acid-preferring acyl-CoA synthetase is a hormone-dependent and obligatory protein ... more An arachidonic acid-preferring acyl-CoA synthetase is a hormone-dependent and obligatory protein in the signal

Prostaglandins & Other Lipid Mediators

It is well accepted that protein(s) with a short half-life are required in the pathway leading to... more It is well accepted that protein(s) with a short half-life are required in the pathway leading to steroid synthesis following stimulation by trophic hormones. A correlation between the disappearance of several proteins in different subcellular compartments and the inhibition of steroid synthesis produced by cycloheximide (CHx) has also been shown, In the present report we describe the effect of CHx in the stimulation of steroid synthesis using a cell-free assay. Mitochondrial progesterone (P4) production was studied by recombination of the different subcellular fractions of adrenal zona fasciculate and determined by radioimmunoassay. Soluble factors from ACTH-treated adrenals produced a four-fold stimulation of mitochondrial steroidogenesis (3.0 ± 0.6 vs. 13.3 ± 0.5 ng P4/tube for control and ACTH-treated adrenals respectively). Mitochondria obtained from CHx-ACTH-treated adrenals fail to respond to soluble ACTH-dependent factors. A permeable analogue of cholesterol (22(R)-OH choles...

International journal of hypertension, 2012

In rat isolated mesenteric beds that were contracted with NA as an in vitro model of the vascular... more In rat isolated mesenteric beds that were contracted with NA as an in vitro model of the vascular adrenergic hyperactivity that usually precedes the onset of primary hypertension, the oral administration (3 daily doses) of either 10 mg/kg genistein or 20 mg/kg daidzein potentiated the anandamide-induced reduction of contractility to NA in female but not in male rats. Oral treatment with phytoestrogens also restored the vascular effects of anandamide as well as the mesenteric content of calcitonin gene-related peptide (CGRP) that were reduced after ovariectomy. The enhancement of anandamide effects caused by phytoestrogens was prevented by the concomitant administration of the estrogen receptor antagonist fulvestrant (2.5 mg/kg, s.c., 3 daily doses). It is concluded that, in the vasculature of female rats, phytoestrogens produced an estrogen-receptor-dependent enhancement of the anandamide-vascular actions that involves the modulation of CGRP levels and appears to be relevant wheneve...

Endocrine research, 1996

It is well accepted that protein(s) with a short half-life are required in the pathway leading to... more It is well accepted that protein(s) with a short half-life are required in the pathway leading to steroid synthesis following stimulation by trophic hormones. A correlation between the disappearance of several proteins in different subcellular compartments and the inhibition of steroid synthesis produced by cycloheximide (CHx) has also been shown. In the present report we describe the effect of CHx in the stimulation of steroid synthesis using a cell-free assay. Mitochondrial progesterone (P4) production was studied by recombination of the different subcellular fractions of adrenal zona fasciculata and determined by radioimmunoassay. Soluble factors from ACTH-treated adrenals produced a four-fold stimulation of mitochondrial steroidogenesis (3.0 +/- 0.6 vs. 13.3 +/- 0.5 ng P4/tube for control and ACTH-treated adrenals respectively). Mitochondria obtained from CHx-ACTH-treated adrenals fail to respond to soluble ACTH-dependent factors. A permeable analogue of cholesterol (22(R)-OH ch...

Endocrine research, 1996

We have previously isolated and partially-sequenced a soluble phosphoprotein (p43) that acts as i... more We have previously isolated and partially-sequenced a soluble phosphoprotein (p43) that acts as intermediary in the stimulation of steroid synthesis. In this report we have used synthetic peptides whose sequences match those obtained from p43 to generate antipeptide antibodies and show that these antibodies bind to purified p43 protein as determined by immunoblot analysis. The presence of p43 was detected by Western blot in both steroidogenic and non-steroidogenic tissues. One of the antibodies was also used to purify p43 on immunoaffinity chromatography columns. Proteins eluting from affinity columns produce a twelve-fold stimulation of progesterone synthesis. This effect was blocked by the use of an inhibitor of phospholipase A2. These results suggest the involvement of p43 in transducing the adrenocorticotropin signal to mitochondria in zona fasciculata cells. We also describe a partial cDNA clone with a predicted amino acid sequence that matches the sequences of the internal pep...

Biochemical and Biophysical Research Communications, 2002

Arachidonic acid is not freely stored in the cells. A number of different pathways for the mobili... more Arachidonic acid is not freely stored in the cells. A number of different pathways for the mobilization of this compound have been proposed, including a novel mechanism that involves the release of arachidonic acid from arachidonoyl-CoA by a thioesterase with substrate specificity for very-long-chain fatty acids. In rat heart, the acyl-CoA thioesterase activity can be regulated by a mechanism that

Molecular and Cellular Endocrinology, 2015

Hormone-regulated steroidogenesis and StAR protein induction involve the action of lipoxygenated ... more Hormone-regulated steroidogenesis and StAR protein induction involve the action of lipoxygenated products. The products of 5-lipoxygenase act on inflammation and immunity by stimulation of a membrane receptor called OxeR1. The presence of OxeR1 in other systems has not been described up to date and little is known about its mechanism of action and other functions. In this context, the aim of this study was the identification and characterization of OxeR1 as a mediator of cAMP-dependent and independent pathways. Overexpression of OxeR1 in MA-10 Leydig cells increased cAMP-dependent progesterone production. Angiotensin II and cAMP stimulation of adrenocortical human H295R cells produced an increase in StAR protein induction and steroidogenesis in cells overexpressing OxeR1 as compared to mock-transfected cells. Additionally, activation of OxeR1 caused a time-dependent increase in ERK1/2 phosphorylation. In summary, membrane receptor OxeR1 is involved in StAR protein induction and activation of steroidogenesis triggered by cAMP or angiotensin II, acting, at least in part, through ERK1/2 activation.

SpringerPlus, 2012

Background: The spectrum of BRCA1/2 genetic variation in breast-ovarian cancer patients has been ... more Background: The spectrum of BRCA1/2 genetic variation in breast-ovarian cancer patients has been scarcely investigated outside Europe and North America, with few reports for South America, where Amerindian founder effects and recent multiracial immigration are predicted to result in high genetic diversity. We describe here the results of BRCA1/BRCA2 germline analysis in an Argentinean series of breast/ovarian cancer patients selected for young age at diagnosis or breast/ovarian cancer family history.

European Journal of Biochemistry, 1998

1. Eur. J. Biochem. 224, 709Ϫ716]. Here, we describe the cloning and sequencing of a cDNA enco... more 1994) Eur. J. Biochem. 224, 709Ϫ716]. Here, we describe the cloning and sequencing of a cDNA encoding p43 as well as the hormonal regulation of the p43 transcript. The protein resulted homologous to a very recently described mitochondrial peroxisome-proliferator-induced very-long-chain acyl-CoA thioesterase (MTE-I). The deduced amino acid sequence of the protein shows consensus sites for phosphorylation by different protein kinases, and a lipase serine motif. Antibodies raised against a synthetic peptide that includes the lipase serine motif and against the N-terminal region of p43 block the action of the protein. The transcript of p43 was detected in ovary of pseudopregnant rats, rat adrenal zona fasciculata and glomerulosa, mouse Leydig tumor cell line (MA-10), rat brain and human placenta. Inhibition of adrenocorticotropin hormone (ACTH) release and steroid synthesis by dexamethasone produced a dose-dependent decrease in the abundance of the adrenal transcript. The transcript was induced by in vivo stimulation of the adrenals with ACTH. The effect had a rapid onset (5 min), reached maximal stimulation (62%) at 15 min, and returned to basal levels at 30 min. The effect of ACTH on the p43 transcript was inhibited by actinomycin D and enhanced by cycloheximide. Our results provide the first evidence linking acyl-CoA thioesterases with very-long-chain specificities, and a protein intermediary in steroid synthesis, thereby supporting a regulatory role for acyl-CoA thioesterases in steroidogenic tissues. Abbreviations. ARTISt, arachidonic-acid-related thioesterase involved in steroidogenesis; ACTH, adrenocorticotropin hormone; dexamethasone, 9-fluoro-11β,17,21-trihydroxy-16A-methylpregna-1,4-diene-3,20-dione; AA, arachidonic acid; RT-PCR, reverse-transcription PCR ; CTE-I, cytosolic acyl-CoA thioesterase I; MTE-I, mitochondrial acyl-CoA thioesterase.

The Journal of Steroid Biochemistry and Molecular Biology, 1991

Luteinizing hormone (LH) and human chorionic gonadotrophin (hCG) receptors are coupled to intrace... more Luteinizing hormone (LH) and human chorionic gonadotrophin (hCG) receptors are coupled to intracellular effector systems, most notably adenylate cyclase, through guanyl nucleotide-binding proteins or G-proteins. The molecular mechanism involved in the dynamic coupling of the LH/hCG receptor however, are not known. It has been postulated that receptor aggregation at the molecular level plays a critical role in this process. There have been attempts to understand the receptor association and dissociation phenomena at the molecular level. One of them involves the participation of the major histocompatibility complex (MHC) class I antigen in the mechanism of receptor activation and/or expression. One molecular basis for these mechanisms consists of a physical interaction between MHC proteins and receptors to form "compound receptors" able to transfer a hormonal signal to the cell. Using a photo-reactive probe we demonstrated that the LH/hCG receptors and the class I antigens are closely associated in the membrane. Thus, it is possible to form covalent complexes of hCG and class I antigens through the binding of the hormone to specific receptors. These findings imply that LH/hCG receptors and the MHC class I antigens may interact at the level of the plasma membrane in the mechanism of LH action. We also performed experiments using a single cell and limiting stimulation to a patch of membrane. The results stimulating the cell in a localized area suggested that even if all components are entirely free to float there is a constraint in the localization of the receptor, G-protein, and/or the effector, supporting the constraint dissociation model. Within a limited area subunits could dissociate, but they would not be free to diffuse throughout the membrane. Moreover the concept of compartmentalization that has been utilized to explain some inconsistencies in second-messenger action now can be proved by experimental design.

The Journal of Steroid Biochemistry and Molecular Biology, 2006

The activation of the rate-limiting step in steroid biosynthesis, that is the transport of choles... more The activation of the rate-limiting step in steroid biosynthesis, that is the transport of cholesterol into the mitochondria, is dependent on PKA-mediated events triggered by hormones like ACTH and LH. Two of such events are the protein tyrosine dephosphorylation mediated by protein tyrosine phosphatases (PTPs) and the release of arachidonic acid (AA) mediated by two enzymes, ACS4 (acyl-CoA synthetase 4) and Acot2 (mitochondrial thioesterase). ACTH and LH regulate the activity of PTPs and Acot2 and promote the induction of ACS4. Here we analyzed the involvement of PTPs on the expression of ACS4. We found that two PTP inhibitors, acting through different mechanisms, are both able to abrogate the hormonal effect on ACS4 induction. PTP inhibitors also reduce the effect of cAMP on steroidogenesis and on the level of StAR protein, which facilitates the access of cholesterol into the mitochondria. Moreover, our results indicate that exogenous AA is able to overcome the inhibition produced by PTP inhibitors on StAR protein level and steroidogenesis. Then, here we describe a link between PTP activity and AA release, since ACS4 induction is under the control of PTP activity, being a key event for AA release, StAR induction and steroidogenesis.

European Journal of Biochemistry, 1994

In previous reports we have demonstrated the presence of a soluble factor that responds to cAMP s... more In previous reports we have demonstrated the presence of a soluble factor that responds to cAMP signals to induce steroid synthesis in adrenocortical tissue. Here, we describe the purification of this factor from adrenal zona fasciculata cells by using a five-step procedure that includes DEAEcellulose, gel filtration, Mono Q HPLC and Superose HPLC, and elution of the protein from SDS/ PAGE. This procedure results in the purification to homogeneity of a protein of 43-kDa that retains the capacity to stimulate steroid synthesis in an in v i m recombination assay. This activity is inhibited by the use of phospholipase A, inhibitors. Antipeptide antibodies against the N-terminal region recognize p43 as a double band on SDSPAGE that resolves in different spots on two-dimensional gel electrophoresis. Adrenocorticotropin treatment of adrenal glands results in the appearence of multiple spots that migrated towards a lower pH compared to controls, suggesting the presence of phosphorylated and dephosphorylated forms of p43. Sequencing of the N-terminal region and internal peptides reveals no significant similarities with other proteins, suggesting that p43 is a novel protein.