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NASA NTRS1976

Nonlinear Curvature Expressions for Combined Flapwise Bending, Chordwise Bending, Torsion and Extension of Twisted Rotor Blades

The nonlinear curvature expressions for a twisted rotor blade or a beam undergoing transverse bending in two planes, torsion, and extension were developed. The curvature expressions were obtained using simple geometric considerations. The expressions were first developed in a general manner using the geometrical nonlinear theory of elasticity. These general nonlinear expressions were then systematically reduced to four levels of approximation by imposing various simplifying assumptions, and in each of these levels the second degree nonlinear expressions were given. The assumptions were carefully stated and their implications with respect to the nonlinear theory of elasticity as applied to beams were pointed out. The transformation matrices between the deformed and undeformed blade-fixed coordinates, which were needed in the development of the curvature expressions, were also given for three of the levels of approximation. The present curvature expressions and transformation matrices were compared with corresponding expressions existing in the literature.

Kvaternik, R. G.↗

NASA NTRS2003

Expression of Prox1 defines regions of the avian otocyst that give rise to sensory or neural cells

The simple primordium of the inner ear (otocyst) differentiates into many cell types, including sensory neurons and hair cells. We examined expression of the divergent homeobox transcription factor, cProx1, during otocyst development in chickens. Nuclear cProx1 protein is not evident in the otic placode but emerges in the otic cup by stage 12. At stage 16, cProx1-positive nuclei are scattered continuously throughout the neuroepithelium, from anteroventral to posteromedial. These labeled cells are neural precursors; they express betaIII-tubulin and migrate to the cochleovestibular ganglion between stages 13 and 21. By stage 18, two areas develop a dense pattern of cProx1 expression in which every nucleus is labeled. These areas emerge at the anterior and posterior extremes of the band of scattered cProx1 expression and express the sensory markers cSerrate1 and Cath1 by stage 23. Four discrete patches of dense cProx1 expression appear by stage 23 that correspond to the future superior crista, lateral crista, saccular macula, and posterior crista, as confirmed by immunolabeling for hair cell antigen (HCA) by stage 29. The remaining sensory epithelia display a dense pattern of cProx1 expression and label for HCA by stage 29. In the basilar papilla, nuclear cProx1 expression is down-regulated in most hair cells by stage 37 and in many supporting cells by stage 40. Our findings show that regions of the otocyst that give rise to neurons or hair cells are distinguished by their relative density of cProx1-positive nuclei, and suggest a role for cProx1 in the genesis of these cell types.

NASA Discipline Developmental Biology↗

NASA NTRS2000

Enhanced expression of extracellular calcium sensing receptor in monocyte-differentiated versus undifferentiated HL-60 cells: potential role in regulation of a nonselective cation channel

Human promyelocytic leukemia cells (HL-60) have been used widely as a model for studying the differentiation of hematopoietic progenitor cells in vitro. After treatment with phorbol-12-myristate-13-acetate (PMA) or 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)], HL-60 cells differentiate into cells with the phenotype of monocytes/macrophages. We previously showed that peripheral blood monocytes and the murine J774 monocytic cell line express the CaR, and myeloid progenitors in the bone marrow and myeloid cells in peripheral blood other than monocytes express lower levels of the CaR. Therefore, we investigated whether undifferentiated HL-60 cells express a functional G protein-coupled, extracellular calcium (Ca(2+)(o))-sensing receptor (CaR) and if the expression of the CaR increases as these cells differentiate along the monocytic lineage. The use of reverse transcription-polymerase chain reaction (RT-PCR) with CaR-specific primers, followed by sequencing of the amplified products, identified an authentic CaR transcript in undifferentiated HL-60 cells. Both immunocytochemistry and Western blot analysis using a CaR-specific antiserum detected low levels of CaR protein expression in undifferentiated HL-60 cells. The levels of CaR protein increased considerably following treatment of the cells with PMA (50 nM) or 1,25(OH)(2)D(3) (100 nM) for 5 days. Northern analysis using a CaR-specific riboprobe identified CaR transcripts in undifferentiated HL-60 cells, but CaR mRNA levels did not change appreciably after treatment with either agent, suggesting that upregulation of CaR protein occurs at a translational level. PMA-treated HL-60 cells expressed a nonselective cation channel (NCC), and the calcimimetic CaR activator, NPS R-467, but not its less active stereoisomer, NPS S-467, as well as the polycationic CaR agonist, neomycin, activated this NCC, demonstrating that the CaR expressed in these cells is functionally active. Therefore, HL-60 cells exhibit an increase in CaR protein expression, occurring at a translational level during their differentiation into cells with a monocyte/macrophage phenotype in response to treatment with PMA or 1, 25(OH)(2)D(3), which is functionally linked to activation of a nonselective cation channel.

NASA Discipline Musculoskeletal↗

NASA NTRS1996

Cyclooxygenases in human and mouse skin and cultured human keratinocytes: association of COX-2 expression with human keratinocyte differentiation

Epidermal expression of the two isoforms of the prostaglandin H-generating cyclooxygenase (COX-1 and COX-2) was evaluated both by immunohistochemistry performed on human and mouse skin biopsy sections and by Western blotting of protein extracts from cultured human neonatal foreskin keratinocytes. In normal human skin, COX-1 immunostaining is observed throughout the epidermis whereas COX-2 immunostaining increases in the more differentiated, suprabasilar keratinocytes. Basal cell carcinomas express little if any COX-1 or COX-2 immunostaining whereas both isozymes are strongly expressed in squamous cell carcinomas deriving from a more differentiated layer of the epidermis. In human keratinocyte cultures, raising the extracellular calcium concentration, a recognized stimulus for keratinocyte differentiation, leads to an increased expression of both COX-2 protein and mRNA; expression of COX-1 protein, however, shows no significant alteration in response to calcium. Because of a recent report that failed to show COX-2 in normal mouse epidermis, we also looked for COX-1 and COX-2 immunostaining in sections of normal and acetone-treated mouse skin. In agreement with a previous report, some COX-1, but no COX-2, immunostaining is seen in normal murine epidermis. However, following acetone treatment, there is a marked increase in COX-1 expression as well as the appearance of significant COX-2 immunostaining in the basal layer. These data suggest that in human epidermis as well as in human keratinocyte cultures, the expression of COX-2 occurs as a part of normal keratinocyte differentiation whereas in murine epidermis, its constitutive expression is absent, but inducible as previously published.

NASA Discipline Cell Biology↗

NASA NTRS1995

Expression of S100 beta in sensory and secretory cells of the vertebrate inner ear

We evaluated anti-S100 beta expression in the chick (Gallus domesticus) inner ear and determined that: 1) the monomer anti-S100 beta is expressed differentially in the vestibular and auditory perikarya; 2) expression of S100 beta in the afferent nerve terminals is time-related to synapse and myelin formation; 3) the expression of the dimer anti-S100 alpha alpha beta beta and monomer anti-S100 beta overlaps in most inner ear cell types. Most S100 alpha alpha beta beta positive cells express S100 beta, but S100 beta positive cells do not always express S100 alpha alpha beta beta. 4) the expression of S100 beta is diffused over the perikaryal cytoplasm and nuclei of the acoustic ganglia but is concentrated over the nuclei of the vestibular perikarya. 6) S100 beta is expressed in secretory cells, and it is co-localized with GABA in sensory cells. 7) Color thresholding objective quantitation indicates that the amount of S100 beta was higher (mean 22, SD +/- 4) at E19 than at E9 (mean 34, SD +/- 3) in afferent axons. 8) Moreover, S100 beta was unchanged between E11-E19 in the perikaryal cytoplasm, but did change over the nuclei. At E9, 74%, and at E21, 5% of vestibular perikarya were positive. The data suggest that S100 beta may be physically associated with neuronal and ionic controlling cells of the vertebrate inner ear, where it could provide a dual ionic and neurotrophic modulatory function.

NASA Discipline Number 40-10↗

NASA NTRS2004

Environmental impacts on the developing CNS: CD15, NCAM-L1, and GFAP expression in rat neonates exposed to hypergravity

We have previously reported that the developing rat cerebellum is affected by hypergravity exposure. The effect is observed during a period of both granule and glial cell proliferation and neuronal migration in the cerebellum and coincides with changes in thyroid hormone levels. The present study begins to address the molecular mechanisms involved in the cerebellar response to hypergravity. Specifically, the study focuses on the expression of cerebellar proteins that are known to be directly involved in cell-cell interactions [protein expressing 3-fucosyl-N-acetyl-lactosamine antigen (CD15), neuronal cell adhesion molecule (NCAM-L1)] and those that affect cell-cell interactions indirectly [glial fibrillary acidic protein (GFAP)] in rat neonates exposed to centrifuge-produced hypergravity. Cerebellar mass and protein expression in rat neonates exposed to hypergravity (1.5 G) from gestational day (G) 11 to postnatal day (P) 30 were compared at one of six time points between P6 and P30 against rat neonates developing under normal gravity. Proteins were analyzed by quantitative western blots of cerebellar homogenates prepared from male or female neonates. Cerebellar size was most clearly reduced in male neonates on P6 and in female neonates on P9, with a significant gender difference; differences in cerebellar mass remained significant even when change in total body mass was factored in. Densitometric analysis of western blots revealed both quantitative and temporal changes in the expression of selected cerebellar proteins that coincided with changes in cerebellar mass and were gender-specific. In fact, our data indicated certain significant differences even between male and female control animals. A maximal decrease in expression of CD15 was observed in HG females on P9, coinciding with maximal change in their cerebellar mass. A shift in the time-course of NCAM-L1 expression resulted in a significant increase in NCAM-L1 in HG males on P18, an isolated time at which cerebellar mass does not significantly differ between HG and SC neonates. A maximal decrease in expression of GFAP was observed in HG males on P6, coinciding with maximal change in their cerebellar mass. Altered expression of cerebellar proteins is likely to affect a number of developmental processes and contribute to the structural and functional alterations seen in the CNS developing under altered gravity. Our data suggest that both cerebellar development and its response to gravitational manipulations differ in males and females. c2004 COSPAR. Published by Elsevier Ltd. All rights reserved.

NASA Program Fundamental Space Biology↗

NASA NTRS2013

Conducting Research on the International Space Station Using the EXPRESS Rack Facilities

Conducting Research on the International Space Station using the EXPRESS Rack Facilities. Sean W. Thompson and Robert E. Lake. NASA Marshall Space Flight Center, Huntsville, AL, USA. Eight "Expedite the Processing of Experiments to Space Station" (EXPRESS) Rack facilities are located within the International Space Station (ISS) laboratories to provide standard resources and interfaces for the simultaneous and independent operation of multiple experiments within each rack. Each EXPRESS Rack provides eight Middeck Locker Equivalent locations and two drawer locations for powered experiment equipment, also referred to as sub-rack payloads. Payload developers may provide their own structure to occupy the equivalent volume of one, two, or four lockers as a single unit. Resources provided for each location include power (28 Vdc, 0-500 W), command and data handling (Ethernet, RS-422, 5 Vdc discrete, +/- 5 Vdc analog), video (NTSC/RS 170A), and air cooling (0-200 W). Each rack also provides water cooling (500 W) for two locations, one vacuum exhaust interface, and one gaseous nitrogen interface. Standard interfacing cables and hoses are provided on-orbit. One laptop computer is provided with each rack to control the rack and to accommodate payload application software. Four of the racks are equipped with the Active Rack Isolation System to reduce vibration between the ISS and the rack. EXPRESS Racks are operated by the Payload Operations Integration Center at Marshall Space Flight Center and the sub-rack experiments are operated remotely by the investigating organization. Payload Integration Managers serve as a focal to assist organizations developing payloads for an EXPRESS Rack. NASA provides EXPRESS Rack simulator software for payload developers to checkout payload command and data handling at the development site before integrating the payload with the EXPRESS Functional Checkout Unit for an end-to-end test before flight. EXPRESS Racks began supporting investigations onboard ISS on April 24, 2001 and will continue through the life of the ISS.

Thompson, Sean W.↗

NASA NTRS2014

Conducting Research on the International Space Station Using the EXPRESS Rack Facilities

EXPRESS Racks provide capability for payload access to ISS resources. The successful on-orbit operations and versatility of the EXPRESS Rack has facilitated the operations of many scientific areas, with the promise of continued payload support for years to come. EXPRESS Racks are currently deployed in the US Lab, Columbus and JEM. Process improvements and enhancements continue to improve the accommodations and make the integration and operations process more efficient. Payload Integration Managers serve as the primary interface between the ISS Program and EXPRESS Payload Developers. EXPRESS Project coordinates across multiple functional areas and organizations to ensure integrated EXPRESS Rack and subrack products and hardware are complete, accurate, on time, safe, and certified for flight. NASA is planning to expand the EXPRESS payload capacity by developing new Basic Express Racks expected to be on ISS in 2018.

Thompson, Sean W.↗

DOE OSTI2024

Streamlining heterologous expression of top carbonic anhydrases in Escherichia coli : bioinformatic and experimental approaches

Carbonic anhydrase (CA) enzymes facilitate the reversible hydration of CO 2 to bicarbonate ions and protons. Identifying efficient and robust CAs and expressing them in model host cells, such as Escherichia coli, enables more efficient engineering of these enzymes for industrial CO 2 capture. However, expression of CAs in E. coli is challenging due to the possible formation of insoluble protein aggregates, or inclusion bodies. This makes the production of soluble and active CA protein a prerequisite for downstream applications. In this study, we streamlined the process of CA expression by selecting seven top CA candidates and used two bioinformatic tools to predict their solubility for expression in E. coli. The prediction results place these enzymes in two categories: low and high solubility. Our expression of high solubility score CAs (namely CA5-SspCA, CA6-SazCAtrunc, CA7-PabCA and CA8-PhoCA) led to significantly higher protein yields (5 to 75 mg purified protein per liter) in flask cultures, indicating a strong correlation between the solubility prediction score and protein expression yields. Furthermore, phylogenetic tree analysis demonstrated CA class-specific clustering patterns for protein solubility and production yields. Unexpectedly, we also found that the unique N-terminal, 11-amino acid segment found after the signal sequence (not present in its homologs), was essential for CA6-SazCA activity. Overall, this work demonstrated that protein solubility prediction, phylogenetic tree analysis, and experimental validation are potent tools for identifying top CA candidates and then producing soluble, active forms of these enzymes in E. coli. The comprehensive approaches we report here should be extendable to the expression of other heterogeneous proteins in E. coli.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

DOE OSTI2025

Creb5 controls its own expression and directly induces the joint interzone regulatory program

Prior studies have indicated that the transcription factor Creb5 is expressed in the joint interzone, which contains the progenitors for all synovial joint tissues in both mouse and human embryos. In the absence of Creb5 function, most synovial joint interzones fail to form and the cartilage templates in the long bones remain fused. This earlier work did not clarify whether Creb5 initiates a cascade of signaling molecules, such as growth and differentiation factor 5 (Gdf5) and Wnt-family members, that in turn induce the formation of the joint interzone, or instead directly activates the expression of joint interzone markers. In the present study, an integrative analysis of the transcriptome, chromatin accessibility, and Creb5-occupancy in joint progenitors revealed that Creb5 directly binds to both its own two promoters and to the regulatory regions of Gdf5 and Sfrp2, each of whose expression in the joint interzone is Creb5-dependent. Functional enhancer analysis indicated that Creb5 binding sites in either the two Creb5 promoters, or in Gdf5 and Sfrp2 regulatory elements are necessary for these sequences to drive transgene expression in the developing synovial joints. While Creb5 directly drives Gdf5 and Sfrp2 expression in the inner joint interzone, Creb5 activates Barx1 expression specifically in the outer joint interzone. Our findings indicate that Creb5 initiates a regulatory network that both promotes the formation of synovial joints, and subsequently activates distinct transcriptional targets in the inner versus the outer regions of the joint interzone, thus regionalizing gene expression in the developing joint.

Zhang, Cheng-Hai↗

DOE OSTI2025

Amino acid substrate specificities and tissue expression profiles of the nine CYP79A encoding genes in Sorghum bicolor

Cytochrome P450s of the CYP79 family catalyze two N-hydroxylation reactions, converting a selected number of amino acids into the corresponding oximes. The sorghum genome (Sorghum bicolor) harbours nine CYP79A encoding genes, and here sequence comparisons of the CYP79As along with their substrate recognition sites (SRSs) are provided. The substrate specificity of previously uncharacterized CYP79As was investigated by transient expression in Nicotiana benthamiana and subsequent transformation of the oximes formed into the corresponding stable oxime glucosides catalyzed by endogenous UDPG-glucosyltransferases (UGTs). CYP79A61 uses phenylalanine as a substrate, whereas CYP79A91, CYP79A93, and CYP79A95 use valine and isoleucine as substrates, with CYP79A93 showing the ability also to use phenylalanine. CYP79A94 uses isoleucine as a substrate. Analysis of 249 sorghum transcriptomes from two different sorghum cultivars showed the expression levels and tissue-specific expression of the CYP79As. CYP79A1 is the committed gene in dhurrin formation and was the highest expressed gene in most tissues/organs. CYP79A61 was primarily expressed in fully developed leaf blades and leaf sheaths. CYP79A91 and CYP79A92 were expressed mainly in roots >200 cm below ground, while CYP79A93 and CYP79A94 were most highly expressed in the leaf collar and leaf sheath, respectively. Here, the possible signalling effects of the oximes and their metabolites produced in different sorghum tissues are discussed.

59 BASIC BIOLOGICAL SCIENCES↗

NASA NTRS1996

Regulated Expression of a Calmodulin Isoform Alters Growth and Development in Potato

A transgene approach was taken to study the consequences of altered expression of a calmodutin iso-form on plant growth and development. Eight genomic clones of potato calmodulin (PCM 1 to 8) have been isolated and characterized. Among the potato calmodulin isoforms studied, PCM 1 differs from the other isoforms because of its unique amino acid substitutions. Transgenic potato plants were produced carrying sense construct of PCM 1 fused to the CAMV 35S promoter. Transgenic plants showing a moderate increase in PCM 1 MRNA exhibited strong apical dominance, produced elongated tubers, and were taller than the controls. Interestingly, the plants expressing the highest level of PCM 1 MRNA did not form underground tubers. Instead, these transgenic plants produced aerial tubers when allowed to grow for longer periods. The expression of different calmodulin isoforms (PCM 1, 5, 6, and 8) was studied in transgenic plants. Among the four potato calmodulin isoforms, only the expression of PCM 1 MRNA was altered in transgenic plants, while the expression of other isoforms was not significantly altered. Western analysis revealed increased PCM 1 protein in transgenic plants, indicating that the expression of both MRNA and protein are altered in transgenic plants. These results suggest that increasing the expression of PCM 1 alters growth and development in potato plants.

Poovaiah, B. W.↗

NASA NTRS2000

The ISS EXPRESS Rack: An Innovative Approach of Rapid Integration

The EXpedite the PRocessing of Experiments to Space Station or EXPRESS Rack System, was developed to provide Space Station accommodations for small, subrack payloads. The EXPRESS Rack accepts Space Shuttle middeck locker type payloads and International Subrack Interface Standard (ISIS) Drawer payloads, allowing previously flown payloads an opportunity to transition to the International Space Station. The EXPRESS Rack provides power, data, command and control, video, water cooling, air cooling, vacuum exhaust, and Nitrogen supply to payloads. The EXPRESS Rack system also includes transportation racks to transport payloads to and from the Space Station, Suitcase Simulators to allow a payload developer to verify power and data interfaces at the development site, Functional Checkout Units to allow Payload checkout at KSC prior to launch, and trainer racks for the astronauts to learn how to operate the EXPRESS Racks prior to flight. Standard hardware and software interfaces provided by the EXPRESS Rack simplify the analytical and physical integration processes, and facilitates simpler ISS payload development. The EXPRESS Rack has also formed the basis for the U.S. Life Sciences payload racks and the Window Observational Research Facility on Space Station.

Sledd, Annette M.↗

NASA NTRS1999

EXPRESS Rack Overview

Sledd, Annette M.↗

NASA NTRS2001

Expression of the Acyl-Coenzyme A: Cholesterol Acyltransferase GFP Fusion Protein in Sf21 Insect Cells

The enzyme acyl-coenzyme A:cholesterol acyltransferase (ACAT) is an important contributor to the pathological expression of plaque leading to artherosclerosis n a major health problem. Adequate knowledge of the structure of this protein will enable pharmaceutical companies to design drugs specific to the enzyme. ACAT is a membrane protein located in the endoplasmic reticulum.t The protein has never been purified to homogeneity.T.Y. Chang's laboratory at Dartmouth College provided a 4-kb cDNA clone (K1) coding for a structural gene of the protein. We have modified the gene sequence and inserted the cDNA into the BioGreen His Baculovirus transfer vector. This was successfully expressed in Sf2l insect cells as a GFP-labeled ACAT protein. The advantage to this ACAT-GFP fusion protein (abbreviated GCAT) is that one can easily monitor its expression as a function of GFP excitation at 395 nm and emission at 509 nm. Moreover, the fusion protein GCAT can be detected on Western blots with the use of commercially available GFP antibodies. Antibodies against ACAT are not readily available. The presence of the 6xHis tag in the transfer vector facilitates purification of the recombinant protein since 6xHis fusion proteins bind with high affinity to Ni-NTA agarose. Obtaining highly pure protein in large quantities is essential for subsequent crystallization. The purified GCAT fusion protein can readily be cleaved into distinct GFP and ACAT proteins in the presence of thrombin. Thrombin digests the 6xHis tag linking the two protein sequences. Preliminary experiments have indicated that both GCAT and ACAT are expressed as functional proteins. The ultimate aim is to obtain large quantities of the ACAT protein in pure and functional form appropriate for protein crystal growth. Determining protein structure is the key to the design and development of effective drugs. X-ray analysis requires large homogeneous crystals that are difficult to obtain in the gravity environment of earth. Protein crystals grown in microgravity are often larger and have fewer defects than those grown on earth. The analysis of higher quality space-grown crystals will assist in structure-based drug design. We have successfully grown GCAT-infected Sf21 cells in both adhesion and suspension cultures. Expression levels of GCAT in cell lines such as Sf9 and High Five appear to be reduced. We intend to replicate GCAT expression in all three cell lines using the NASA rotating wall bioreactor which effectively duplicates a microgravity environment. The bioreactor itself could be launched to study the expression of the GFP and GCAT proteins in the actual microgravity environment achieved in orbit.

Mahtani, H. K.↗

NASA NTRS2003

Id expression in amphioxus and lamprey highlights the role of gene cooption during neural crest evolution

Neural crest cells are unique to vertebrates and generate many of the adult structures that differentiate them from their closest invertebrate relatives, the cephalochordates. Id genes are robust markers of neural crest cells at all stages of development. We compared Id gene expression in amphioxus and lamprey to ask if cephalochordates deploy Id genes at the neural plate border and dorsal neural tube in a manner similar to vertebrates. Furthermore, we examined whether Id expression in these cells is a basal vertebrate trait or a derived feature of gnathostomes. We found that while expression of Id genes in the mesoderm and endoderm is conserved between amphioxus and vertebrates, expression in the lateral neural plate border and dorsal neural tube is a vertebrate novelty. Furthermore, expression of lamprey Id implies that recruitment of Id genes to these cells occurred very early in the vertebrate lineage. Based on expression in amphioxus we postulate that Id cooption conferred sensory cell progenitor-like properties upon the lateral neurectoderm, and pharyngeal mesoderm-like properties upon cranial neural crest. Amphioxus Id expression is also consistent with homology between the anterior neurectoderm of amphioxus and the presumptive placodal ectoderm of vertebrates. These observations support the idea that neural crest evolution was driven in large part by cooption of multipurpose transcriptional regulators from other tissues and cell types.

NASA Program Fundamental Space Biology↗

NASA NTRS2001

Differential global gene expression in red and white skeletal muscle

The differences in gene expression among the fiber types of skeletal muscle have long fascinated scientists, but for the most part, previous experiments have only reported differences of one or two genes at a time. The evolving technology of global mRNA expression analysis was employed to determine the potential differential expression of approximately 3,000 mRNAs between the white quad (white muscle) and the red soleus muscle (mixed red muscle) of female ICR mice (30-35 g). Microarray analysis identified 49 mRNA sequences that were differentially expressed between white and mixed red skeletal muscle, including newly identified differential expressions between muscle types. For example, the current findings increase the number of known, differentially expressed mRNAs for transcription factors/coregulators by nine and signaling proteins by three. The expanding knowledge of the diversity of mRNA expression between white and mixed red muscle suggests that there could be quite a complex regulation of phenotype between muscles of different fiber types.

Non-NASA Center↗

NASA NTRS2001

Expression of extracellular calcium-sensing receptor in human osteoblastic MG-63 cell line

We have previously shown the expression of the extracellular calcium (Ca2+o)-sensing receptor (CaR) in osteoblast-like cell lines, and others have documented its expression in sections of murine, bovine, and rat bone. The existence of the CaR in osteoblasts remains controversial, however, since some studies have failed to document its expression in the same osteoblast-like cell lines. The goals of the present study were twofold. 1) We sought to determine whether the CaR is expressed in the human osteoblast-like cell line, MG-63, which has recently been reported by others not to express this receptor. 2) We investigated whether the CaR, if present in MG-63 cells, is functionally active, since most previous studies have not proven the role of the CaR in mediating known actions of Ca2+o on osteoblast-like cells. We used immunocytochemistry and Western blotting with the specific, affinity-purified anti-CaR antiserum 4637 as well as Northern blot analysis and RT-PCR using a riboprobe and PCR primers specific for the human CaR, respectively, to show readily detectable CaR protein and mRNA expression in MG-63 cells. Finally, we employed the patch-clamp technique to show that an elevation in Ca2+o as well as the specific, allosteric CaR activator NPS R-467 (0.5 microM), but not its less active stereoisomer NPS S-467 (0.5 microM), activate an outward K+ channel in MG-63 cells, strongly suggesting that the CaR in MG-63 cells is not only expressed but is functionally active.

NASA Discipline Regulatory Physiology↗