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ABSTRACT: Estrogen signaling plays an important role in breast carcinogenesis. An increased understanding of estrogen gene targets and their effects will allow for more directed and effective therapies for individuals with breast cancer, particularly those with estrogen receptor positive tumors resistant to tamoxifen therapy. Here, we identify YPEL3 as a growth suppressive protein downregulated by estrogen in estrogen receptor positive breast cancer cell lines. Estrogen repression of YPEL3 expression was found to be independent of p53 but dependent on estrogen receptor alpha expression. Importantly, YPEL3 expression, which is induced by the removal of estrogen or treatment with tamoxifen triggers cellular senescence in MCF-7 cells while loss of YPEL3 increases the growth rate of MCF-7 cells. Taken together these findings suggest that YPEL3 may represent a potential target for directed hormonal therapy for estrogen receptor positive breast cancer patients.
International Journal of Cancer 06/2011; 130(10):2291-9. · 5.44 Impact Factor
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ABSTRACT: Sarcoplasmic calcium binding protein (SCP) is an invertebrate EF-hand calcium buffering protein that has been proposed to fulfill a similar function in muscle relaxation as vertebrate parvalbumin. We have identified three SCP variants in the freshwater crayfish Procambarus clarkii. The variants (pcSCP1a, pcSCP1b, and pcSCP1c) differ across a 37 amino acid region that lies mainly between the second and third EF-hand calcium binding domains. We evaluated tissue distribution and response of the variants to cold exposure, a stress known to affect expression of parvalbumin. Expression patterns of the variants were not different and therefore do not provide a functional rationale for the polymorphism of pcSCP1. Compared to hepatopancreas, expression of pcSCP1 variants was 100,000-fold greater in axial abdominal muscle and 10-fold greater in cardiac muscle. Expression was 10-100 greater in fast-twitch deep flexor and extensor muscles compared to slow-twitch superficial flexor and extensors. In axial muscle, no significant changes of pcSCP1, calmodulin (CaM), or sarcoplasmic/endoplasmic reticulum Ca-ATPase (SERCA) expression were measured after one week of 4°C exposure. In contrast, large decreases of pcSCP1 were measured in cardiac muscle, with no changes in CaM or SERCA. Knockdown of pcSCP1 by dsRNA led to reduced muscle activity and decreased expression of SERCA. In summary, the pattern of pcSCP1 tissue expression is similar to parvalbumin, supporting a role in muscle contraction. However, the response of pcSCP1 to cold exposure differs from parvalbumin, suggesting possible functional divergence between the two proteins.
Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology 04/2011; 160(1):8-14. · 1.61 Impact Factor
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ABSTRACT: Since the classical studies of Ussing employing a nonmammalian isolated epithelium (frog skin) to explore the basic principles
of ion transport, physiologists have adopted increasingly reductionist approaches to dissect the biophysical mechanisms undergirding
biological transport. In vitro characterization has employed isolated perfused organs, isolated epithelia, and reconstituted
vesicle studies. Depth of resolution has been further enhanced by the emerging molecular revolution. Following years of deconstruction,
physiologists are now engaging in reconstruction, namely putting the genes back into the organism. This contribution attempts
such an integrative approach for a single electrolyte, calcium (Ca2+), in a nonmammalian epithelium, the crayfish antennal gland (kidney). Two collaborating laboratories have archived an inventory
of Ca2+ associated proteins believed to play a role in transcellular Ca2+ movement. Using the basic building blocks (expression profiles of key Ca2+ associated proteins and their regulators), the authors attempt to reconstruct a whole cell model for Ca2+ regulation in transporting epithelium (compared with a nonepithelial tissue) under stressors that perturb Ca2+ homeostasis which originate either intrinsically (the postmolt stage of the molting cycle) or extrinsically (unanticipated
cold acclimation). Through horizontal integration of expression profiles of seven target Ca2+ associated proteins in epithelial and nonepithelial tissue under two contrasting experimental conditions, emergent themes
inform the physiological complexity of Ca2+ homeostasis. Integration at the next level will require placing the epithelium in the context of organismic Ca2+ balance. The unique Ca2+ handling capabilities of the freshwater crayfish make it an excellent nonmammalian model for those studies.
KeywordsCrayfish-Calcium-Antennal gland-Muscle-Posmolt-Cold-ECaC-PMCA-NCX-SERCA-SCP-CaM-eEF1Bγ
03/2010: pages 73-90;
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ABSTRACT: This study describes the cloning, sequencing and functional characterization of an epithelial Ca(2+) channel (ECaC)-like gene isolated from antennal gland (kidney) of the freshwater crayfish Procambarus clarkii. The full-length cDNA consisted of 2687 bp with an open reading frame of 2169 bp encoding a protein of 722 amino acids with a predicted molecular mass of 81.7 kDa. Crayfish ECaC had 76-78% identity at the mRNA level (80-82% amino acid identity) with published fish sequences and 56-62% identity at the mRNA level (52-60% amino acid identity) with mammalian ECaCs. Secondary structure of the crayfish ECaC closely resembled that of cloned ECaCs. Postmolt ECaC expression was exclusively restricted to epithelia associated with Ca(2+) influx and was virtually undetectable in non-epithelial tissues (eggs, muscle). Compared with expression levels in hepatopancreas, expression in gill was 10-fold greater and expression was highest in antennal gland (15-fold greater than in hepatopancreas). Compared with baseline expression levels in intermolt stage, expression of ECaC in antennal gland increased 7.4- and 23.8-fold, respectively, in pre- and postmolt stages of the molting cycle. This increase was localized primarily in the labyrinth and nephridial canal, regions of the antennal gland associated with renal Ca(2+) reabsorption. The ECaC in crayfish appears to be expressed in epithelia associated with unidirectional Ca(2+) influx and relative expression is correlated with rate of Ca(2+) influx.
Journal of Experimental Biology 06/2007; 210(Pt 10):1813-24. · 3.00 Impact Factor
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ABSTRACT: Sarcoplasmic Calcium-binding Protein (SCP) is believed to function as the invertebrate equivalent of vertebrate parvalbumin, namely to "buffer" cytosolic Ca2+. We have cloned and characterized a novel SCP from axial abdominal muscle of crayfish Procambarus clarkii (referred to as pcSCP1), and have examined tissue specific distribution and expression as a function of molting stage in non-epithelial and epithelial tissues. The complete sequence of pcSCP1 consists of 1,052 bp with a 579 bp open reading frame, coding for 193 amino acid residues (molecular mass of 21.8 kDa). There is a 387 bp 3' terminal non-coding region with a poly (A) tail. The deduced pcSCP1 protein sequence matched most closely with published SCP sequences from another crayfish Astacus leptodactylus (92.8%) and from shrimp (78.6-81.2%) and fruit fly (53%). Real-time PCR analysis confirmed that pcSCP1 is ubiquitously expressed in all tissues tested (gill, hepatopancreas, intestine, antennal gland, muscle); however it is most abundant in muscle particularly in the axial abdominal muscle. The real-time PCR analysis revealed that pcSCP1 expression is downregulated in pre- and postmolt stages compared with intermolt. Epithelial (hepatopancreas and antennal gland) SCP expression exhibited a more dramatic decrease than that observed in muscle. Expression trends for pcSCP1 paralleled published trends for sarco/endoplasmic reticular calcium ATPase (SERCA), suggesting that their cellular function in regulating intracellular Ca2+ is linked.
Comparative Biochemistry and Physiology Part B Biochemistry and Molecular Biology 09/2006; 144(4):478-87. · 1.92 Impact Factor
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ABSTRACT: This article will review the utility of comparative animal models in understanding the molecular biology of ion transport. Due to the breadth of this field some 'disclaimers' need to be established up front. 'Comparative' will be defined as non-mammalian. 'Genetic species' will be defined as organisms that have been selected as models for genetic studies and for which the genome has been largely sequenced. 'Non-genetic species' will include other non-mammalian organisms. The review will be limited to ions that play a major role in extracellular (EC) ionoregulation (Na/K/Ca/Cl) and not to micronutrients (Fe) or heavy metals (Cd, Zn). The review will focus only on ion motive proteins that have been associated with vectorial transfer at epithelial tissues. The review is therefore intended as a guidepost to researchers new to the field as well as to inform biologists of the power of comparative genomics.
Journal of Experimental Biology 10/2004; 207(Pt 19):3253-63. · 3.00 Impact Factor
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ABSTRACT: The discontinuous pattern of crustacean cuticular mineralization (the molting cycle) has emerged as a model system to study the spatial and temporal regulation of genes that code for Ca2+-transporting proteins including pumps, channels and exchangers. The plasma membrane Ca2+-ATPase (PMCA) is potentially of significant interest due to its role in the active transport of Ca2+ across the basolateral membrane, which is required for routine maintenance of intracellular Ca2+ as well as unidirectional Ca2+ influx. Prior research has suggested that PMCA expression is upregulated during periods of elevated Ca2+ influx associated with postmolt cuticular mineralization. This paper describes the cloning, sequencing and functional characterization of a novel PMCA3 gene from the antennal gland (kidney) of the crayfish Procambarus clarkii. The complete sequence, the first obtained from a non-genetic invertebrate species, was obtained through reverse transcription-polymerase chain reaction (RTPCR) and rapid amplification of cDNA ends (RACE) techniques. Crayfish PMCA3 consists of 4148 bp with a 3546 bp open reading frame coding for 1182 amino acid residues with a molecular mass of 130 kDa. It exhibits 77.5-80.9% identity at the mRNA level and 85.3-86.9% identity at the protein level with PMCA3 from human, mouse and rat. Membrane topography was typical of published mammalian PMCAs. Northern blot analysis of total RNA from crayfish gill, antennal gland, cardiac muscle and axial abdominal muscle revealed that a 7.5 kb species was ubiquitous. The level of PMCA3 mRNA expression in all tissues (transporting epithelia and muscle) increased significantly in pre/postmolt stages compared with relatively low abundance in intermolt. Western analysis confirmed corresponding changes in PMCA protein expression (130 kDa).
Journal of Experimental Biology 09/2004; 207(Pt 17):2991-3002. · 3.00 Impact Factor
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ABSTRACT: This paper describes the cloning and functional characterization of the heart muscle isoform of Sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA) from crayfish Procambarus clarkii. The complete crayfish heart SERCA, identified by reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE), consists of 4495 bp with a 3060 bp open reading frame, coding for 1020 amino acids. This isoform differs from the previously identified axial abdominal (tail) muscle SERCA solely in its C-terminal amino acids. The last nine amino acids of the tail muscle isoform are replaced by 27 hydrophobic amino acids in the heart isoform that have the potential to form an additional transmembrane domain. Consistent with other invertebrate studies, Southern blot analysis suggested that the heart and tail muscle isoforms are encoded from the same gene that is equally related to SERCA-1, -2 and -3 of vertebrates. The tissue distributions of these two isoforms have been assessed using isoform-specific probes and northern analysis. A cardiac-specific probe bound only to a 5.8 kb species in heart and had minimal cross-hybridization with 7.6 and 5.8 kb species in eggs and no hybridization with tail muscle. A tail-isoform-specific probe hybridized with a 4.5 kb species in tail muscle and cross-hybridized with a 4.5 kb species in eggs and 8.8 kb in heart muscle. Both isoforms are expressed in eggs suggesting that transcripts are formed early in development and are subsequently broadly expressed in all tissue types. Expression of the cardiac muscle SERCA isoform varied with the stage of moulting. Expression was high in intermoult and decreased in premoult. However, expression was restored rapidly in postmoult (within 2 days) unlike expression of tail muscle SERCA, which remained downregulated for weeks. Differences in contractility between the two muscle types in the postmoult period may explain these expression patterns.
Journal of Experimental Biology 10/2002; 205(Pt 17):2677-86. · 3.00 Impact Factor
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ABSTRACT: The molting cycle of the freshwater crayfish, Procambarus clarkii, has been used as a model to study the cellular physiology and molecular biology of Ca "supply" proteins that effect transcellular vectorial Ca(2+) movement to achieve organismal Ca homeostasis. Specifically, periods of net Ca(2+) influx (postmolt) have been compared with periods of net Ca(2+) balance (intermolt). The broader goal is to understand the paradox facing epithelial cells of maintaining low cytosolic Ca(2+)in the face of mass Ca(2+)transit across epithelial cells. This mini-review compares mRNA and protein expression profiles for a series of proteins that are of strategic importance in effecting transcellular Ca(2+) flux in a selected epithelium, the antennal gland (kidney analog) specifically during apical to basolateral Ca(2+) conveyance. Target proteins were selected as representative of key "stages" in the transcellular transfer of Ca(2+): import (epithelial Ca(2+) channel, ECaC); storage (sarco/endoplasmic reticulum Ca(2+) ATPase, SERCA); buffering (sarcoplasmic Ca(2+) binding protein, SCP); and export (plasma membrane Ca(2+) ATPase, PMCA and Na(+)/Ca(2+) exchanger, NCX). The purpose of this review is to assess coordination of expression of these target proteins at times of high Ca(2+) demand (premolt and postmolt) compared to low Ca demand (intermolt) as a function of cellular location (apical vs. basolateral; endomembranes vs. plasma membranes) and relative abundance within different regions of the antennal gland. Understanding the spatiotemporal regulation of Ca(2+) handling proteins involved in transcellular transport is fundamental to investigating their endocrine regulation.
General and Comparative Endocrinology 152(2-3):267-72. · 3.27 Impact Factor
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ABSTRACT: 1.This study examined expression of two primary transmembrane Ca2+ export proteins (plasma membrane Ca2+ ATPase, (PMCA); Na+/Ca2+ exchanger, sodium/calcium exchanger (NCX)) in epithelial (antennal gland, kidney) and non-epithelial (axial abdominal muscle) tissues of the freshwater crayfish Procambarus clarkii following exposure (28 days) to 4 °C (compared with control 23 °C).2.Crayfish tissues responded to cold exposure within 5–7 days by upregulating the mRNA (determined through real-time polymerase chain reaction (PCR)) for both PMCA and NCX. After 28 days, levels were 4–5× higher in antennal gland and 2–3× higher in muscle. In situ hybridization localized the mRNA increase to the nephridial canal in antennal gland sections and to the extensor muscles in the tail. Western blotting confirmed corresponding increases in PMCA protein expression; however, expression of NCX protein was unchanged.
Journal of Thermal Biology.
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ABSTRACT: Eukaryotic elongation factor 1Bγ (eEF1Bγ) is a subunit of elongation factor 1 (EF1), which regulates the recruitment of amino acyl-tRNAs to the ribosome during protein synthesis in eukaryotes. In addition to structural roles within eEF1, eEF1Bγ has properties which suggest sensory or regulatory activities. We have cloned eEF1Bγ from axial abdominal muscle of freshwater crayfish, Procambarus clarkii. The predicted amino acid sequence has 66% identity to Locusta migratoria eEF1Bγ and 65% identity to Artemia salina eEF1Bγ. We measured eEF1Bγ expression by real-time PCR, using the relative quantification method with 18s ribosomal RNA as an internal calibrator. eEF1Bγ expression was lowest in gill, axial abdominal muscle, and hepatopancreas, and was highest in the antennal gland (5.7-fold above hepatopancreas) and cardiac muscle (7.8-fold above hepatopancreas). In axial abdominal muscle, eEF1Bγ expression was 4.4-fold higher in premolt and 11.9 higher in postmolt compared to intermolt. In contrast, eEF1Bγ was decreased or unchanged in epithelial tissues during pre- and postmolt. eEF1Bγ expression in the hepatopancreas was 3.5-fold higher during intermolt compared to premolt and was unchanged in gill and antennal gland. No significant differences in eEF1Bγ were found after 1 week of acclimation to 4 °C. These results show that eEF1Bγ is regulated at the mRNA level with tissue-specific differences in expression patterns.
Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology.
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ABSTRACT: The molting cycle of the freshwater (FW) crayfish, Procambarus clarkii, has been used as a model to study the cellular physiology and molecular biology of proteins (channels, exchangers and pumps) that effect epithelial Ca transport (ECT). Specifically, periods of net Ca flux (typically postmolt influx or premolt efflux) have been compared with periods of net Ca balance (intermolt). In the present study, we further explore the spatial and temporal regulation of plasma membrane Ca ATPase (PMCA) in the antennal gland (kidney). Crayfish are uniquely adapted to produce dilute urine through reabsorption of filtered Ca at the nephridial canal; reabsorption increases in pre/postmolt to compensate for the freshwater absorbed at ecdysis to elicit shedding. Prior work has suggested that PMCA mRNA and protein increase in pre- and postmolt stages compared with intermolt at the antennal gland. In the present paper, we used state-of-the-art techniques to increase the spatial and temporal resolution of this observation. Real-time PCR indicated that the PMCA mRNA expression increased 10-fold in premolt with a further doubling in postmolt. In situ hybridization confirmed that the PMCA mRNA was expressed primarily within the nephridial canal and labyrinth of the antennal gland, and that labelling increased in pre- and postmolt. Immunolocalization with confocal visualization confirmed that the PMCA protein is primarily membrane associated in the nephridial canal cells (basolateral but also apical).
International Congress Series.
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ABSTRACT: Calmodulin (CaM) is a highly conserved calcium (Ca2+) binding protein that transduces Ca2+ signals into downstream effects influencing a range of cellular processes, including Ca2+ homeostasis. The present study explores CaM expression when Ca2+ homeostasis is challenged during the mineralization cycle of the freshwater crayfish (Procambarus clarkii). In this paper we report the cloning of a CaM gene from axial abdominal crayfish muscle (referred to as pcCaM). The pcCaM mRNA is ubiquitously expressed but is far more abundant in excitable tissue (muscle, nerve) than in any epithelia (gill, antennal gland, digestive) suggesting that it plays a greater role in the biology of excitation than in epithelial ion transport. In muscle cells the pcCaM was colocalized on the plasma membrane with the Ca2+ ATPase (PMCA) known to regulate intracellular Ca2+ through basolateral efflux. While PMCA exhibits a greater upregulation in epithelia (than in non-epithelial tissues) during molting stages requiring transcellular Ca2+ flux (pre- and postmolt compared with intermolt), expression of pcCaM exhibited a uniform increase in epithelial and non-epithelial tissues alike. The common increase in expression of CaM in all tissues during pre- and postmolt stages (compared with intermolt) suggests that the upregulation is systemically (hormonally) mediated. Colocalization of CaM with PMCA confirms physiological findings that their regulation is linked.
Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology.
