Glucocorticoid-induced differentiation of fetal rat calvarial osteoblasts is mediated by bone morphogenetic protein-6.

Glucocorticoids (GCs) at physiological concentrations promote osteoblast differentiation from fetal calvarial cells, calvarial organ cultures, and bone marrow stromal cells; however, the cellular pathways involved are not known. Bone morphogenetic proteins (BMPs) are recognized as important mediators of osteoblast differentiation. Specific roles for individual BMPs during postembryonic membranous bone formation have yet to be determined. We recently reported that GC potentiated the osteoblast differentiation effects of BMP-2 and BMP-4, but not of BMP-6, which, by itself, was the most potent of the three. In the present study, we used fetal rat secondary calvarial cultures to study the role of BMP-6 during early osteoblast differentiation. Treatment with the GC triamcinolone (10(-9) M) resulted in a 5- to 8-fold increase in BMP-6 steady-state messenger RNA levels, peaking at 12 h. In contrast, BMPs -2, -4, -5, -7, and transforming growth factor (TGF)-beta1 messenger RNA levels increased by less than 2-fold, after GC treatment, compared with untreated control cultures at 24 h. BMP-6 protein secretion increased 6- to 7-fold by 12 h and 12-fold (from 7.5 to 90 ng/ml) by 24 h, as measured by quantitative Western analysis. Treatment of cells with oligodeoxynucleotides antisense to BMP-6 diminished secretion of BMP-6 protein and significantly inhibited the GC-induced differentiation, as determined by a 10-fold decrease in the number of mineralized bone nodules, compared with controls that were treated with sense oligonucleotides or no oligonucleotides (ANOVA, P < 0.05). The antisense oligonucleotide inhibition of differentiation was rescued by treatment with exogenous recombinant human BMP-6. We conclude that GC-induced differentiation of osteoblasts from the pluripotent precursors is mediated, in part, by BMP-6. These results suggest that BMP-6 has an important and unique role during early osteoblast differentiation.

Differential effects and glucocorticoid potentiation of bone morphogenetic protein action during rat osteoblast differentiation in vitro.

Bone morphogenetic proteins (BMPs) induce cartilage and bone differentiation in vivo and promote osteoblast differentiation from calvarial and marrow stromal cell preparations. Functional differences between BMP-2, -4, and -6 are not well understood. Recent investigations find that these three closely related osteoinductive proteins may exert different effects in primary rat calvarial cell cultures, suggesting the possibility of unique functions in vivo. In this study, we use a fetal rat secondary calvarial cell culture system to examine the differential effects of BMP-2, -4, and -6 on early osteoblast differentiation. These cells do not spontaneously differentiate into osteoblasts, as do cells in primary calvarial cultures, but rather require exposure to a differentiation initiator such as glucocorticoid or BMP. We determined that BMP-6 is a 2- to 2.5-fold more potent inducer of osteoblast differentiation than BMP-2 or -4. BMP-6 induced the formation of more and larger bone nodules as well as increased osteocalcin secretion. The effects of all three of these BMPs were potentiated up to 10-fold by cotreatment or pretreatment with the glucocorticoid triamcinolone (Trm). The Trm effects were synergistic with those of BMP-2 or -4, suggesting that this glucocorticoid may increase the cell responsiveness to these BMPs. Finally, BMP-6 did not require either cotreatment or pretreatment with Trm to achieve greater amounts of osteoblast differentiation than seen with BMP-2 or BMP-4 treatment, suggesting that BMP-6 may act at an earlier stage of cell differentiation.

Expression of the Bgp gene and characterization of mouse colon biliary glycoprotein isoforms.

The biliary glycoprotein (BGP)-encoding gene is a member of the human carcinoembryonic antigen (CEA) gene family. We have now cloned several mouse Bgp cDNAs from an outbred CDR-1 mouse colon cDNA library, as well as by reverse transcription-PCR amplification of colon RNA. The distinguishing features of the deduced Bgp protein isoforms are found in the two divergent N-terminal domains, the highly conserved internal C2-set immunoglobulin domains, and an intracytoplasmic domain of either 10 or 73 amino acids (aa). The cDNA structures suggest that these mRNAs are produced through alternative splicing of a Bgp gene and the usage of multiple transcriptional terminators. The Bgp deduced aa sequences are highly homologous to several well characterized rat hepatocyte proteins such as the cell CAM105/ecto-ATPase/pp120/HA4 proteins. Oligodeoxyribonucleotide probes representing the various cDNA isoform domains revealed predominant transcripts of 1.8, 3.1 and 4.0 kb on Northern analyses of mouse colon RNA; some of these bands are actually composed of several co-migrating transcripts. The transcripts encoding the long intracytoplasmic-tailed Bgp proteins are expressed at one-tenth the relative abundance of the shorter-tailed species. We have previously demonstrated that several mouse Bgp cDNAs, when transfected into eukaryotic cells, express BGP proteins at the cell surface and function in vitro as cell adhesion molecules, much like their human and rat counterparts. The expression of the many Bgp isoforms at the surface of epithelial cells, such as colon, suggests that these proteins play a determinant role, through self- or heterologous contact, in renewal and/or differentiation of their epithelia.

Surgical problems in space: an overview.

Terrestrial experience indicates that surgical emergencies may cause significant morbidity in space, and may present unique problems. Microgravity physiologic changes that may increase susceptibility to, and complications from, injury include fluid redistribution and diuresis, bone demineralization, and immune system alterations. Conventional diagnostic tests may be unavailable or unreliable--e.g. the radiologic finding of "air under the diaphragm", which conventionally indicates perforation of an air-containing hollow viscus, such as the stomach, with a gravity-dependent rising of gas in the abdominal cavity to collect under the diaphragm. Microgravity surgical experience is limited, but studies conducted in the U.S.S.R. and on KC-135 aircraft indicate that, although surgical procedures are possible, weight and volume constraints, lack of gravity, altered fluid mechanics, and risk of environmental contamination mandate considerable modifications of conventional equipment and technique.

Simulation of blood flow in microgravity.

Knowledge of venous, capillary, and arterial blood flow in microgravity is required to modify hemostatic techniques for control of bleeding in traumatic injuries or surgical procedures in space. To simulate human arterial, venous, and capillary bleeding, fresh whole bovine blood was injected by two operators at calculated flow rates (3.5, 7, and 14 mL for venous and 14 and 28 mL for arterial) in 10 seconds with empirical controls in a lucent glove box during zero gravity parabolic flight on NASA's KC-135 aircraft. A pig's foot was used to mimic capillary bleeding. Hemostasis with sponges and laerdal suction was evaluated by video and still photography. Evaluations of the arterial and venous bleeding were conducted at 3 rates x 3 parabolas, and capillary bleeding was evaluated with 5 parabolas x 2 methods (pig's foot and sponge). Influenced by surface tension, the slow venous bleeding coated syringe surfaces and formed a dome over the skin laceration bleeding site. Arterial and venous bleeders broke into uniform spheres with low-velocity spheres bouncing off an absorbent pad and suction tip. Conventional dabbing with gauze fragmented blood into small spheres. Capillary oozing was better controlled by "wicking" up blood with gauze. Repeated arterial bleeding opacified the glove box wall. This stimulation demonstrated unique characteristics of extracorporeal blood flow and inadequacies of common methods of hemostasis in microgravity.

A simple and sensitive high-throughput assay for steroid agonists and antagonists.

We have developed a simple and highly sensitive tissue culture-based assay for the biological activity of steroids and synthetic steroidal compounds. A DNA cassette, containing a synthetic steroid-inducible promoter controlling the expression of a bacterial chloramphenicol acetyltransferase gene (GRE5-CAT), was inserted into an Epstein-Barr virus (EBV) episomal vector which replicates autonomously in primate and human cells. We then used this promoter/reporter system to generate two stably transfected human cell lines. In the cervical carcinoma cell line HeLa, which expresses high levels of glucocorticoid receptor, the GRE5 promoter is inducible over 100-fold by the synthetic glucocorticoid dexamethasone. In the breast carcinoma cell line T47D, which expresses progesterone and androgen receptors, the GRE5 promoter is inducible over 100-fold by either progesterone or dihydrotestosterone. In both cell lines basal expression of CAT activity is strictly dependent on the presence of steroid, so that very low levels of induction can be detected. Thus, the cell lines can be used to test for low levels of agonist activity in steroid antagonists. These cell lines can be used to screen compounds for steroid agonist or antagonist activity by testing extracts of cells grown in microtiter wells directly using a colorimetric CAT assay. This system should provide a sensitive and efficient method for screening and analysis of the activity of large numbers of natural or synthetic steroid agonists or antagonists.

Aseptic technique in microgravity.

Within the next decade, the United States will launch a space station into low Earth orbit as a preliminary step toward a manned mission to Mars. Provision of asepsis in the unique microgravity environment, essential in operative and invasive procedures, is addressed. An assessment of conventional terrestrial aseptic methods and possible modifications for a microgravity environment was done during the microgravity portion of parabolic flight on NASA KC-135 aircraft. During 110 parabolas on three flight days, a "surgical team" (surgeon, scrub nurse and circulating nurse) using a life size mannequin fastened to a prototype surgical "work station" (operating table), evaluated open and closed gloving (ten parabolas), skin preparation (six parabolas), surgical scrub methods (24 parabolas), gowning (22 parabolas) and draping (48 parabolas). Evaluated were povidone iodine solution, 1 percent povidone iodine detergent, Chloroxylenol with detergent, wet prep soap sponge, a water insoluble iodophor polymer (DuraPrep, 3M), disposable towels, disposable and reusable gowns, large and small disposable drapes with and without adhesive edges, disposable latex surgeon's gloves with and without packaging modifications and restraint mechanisms (tether, swiss seat, waist and foot restraint devices, fairfield and wire clamps and clips). Ease of use, provision of restraint for supplies and personnel and waste disposal were assessed. The literature was reviewed and its relevance to the space environment discussed, including risk factors, environmental contamination, immune status and microbiology. The microgravity environment, limited water supply and restricted operating area mandated that modifications of fabrication and packaging of supplies and technique be made to create and preserve asepsis. Material must meet stringent flammability and off-gassing standards. Either a chlorhexidine or povidone iodine detergent prepackaged brush and sponge would provide an adequate scrub plus preliminary cleansing of a dirty wound. Choice may depend on ease of removal from the water supply as well as sensitivity to each compound of individual crew members. Rinsing was achieved with sterile water soaked gauze. Drying would be more efficient with two small hand towels, which would be easier to manipulate in microgravity and require less stowage volume. Skin preparation highlighted unexpected packaging problems, as centrifugal force was required to "shake" the solution out of the container on to the mannequin. To minimize contamination, a gown should be folded in an accordion manner and fastened to the base of its sterile wrapper, so that an assistant can compensate for the lack of gravity by applying constant tension.(ABSTRACT TRUNCATED AT 400 WORDS)

Management of trauma and emergency surgery in space.

Trauma may cause morbidity or mortality in expeditionary spaceflight settings. Physiologic and mechanical changes related to microgravity may increase susceptibility to and complicate the management of injuries in spaceflight. Limited surgical experience in microgravity suggests that special apparatuses and techniques will be needed to maintain the stability of patients, surgeons, and equipment, and to control fluids. A prototype microgravity surgical workstation and suction unit and modifications of standard procedures were devised to address these needs. Using these devices and methods and selected surgical supplies during repeated 25-second intervals of microgravity generated by parabolic arc flight, the "ABCs" of trauma management, limb traction and immobilization, and minor surgical procedures were performed in flight and problems were identified. Convincing "qualification" of spaceflight surgical equipment and protocols will require evaluations in continuous microgravity. As on Earth, the major determinant of emergency surgical care in spaceflight may be the presence or absence of a well-trained surgeon.

mmCGM1a: a mouse carcinoembryonic antigen gene family member, generated by alternative splicing, functions as an adhesion molecule.

Carcinoembryonic antigen is a human tumor marker and the prototype of a large family of immunoglobulin-like proteins. We have been developing a mouse model for this large protein family and have cloned a complementary DNA (cDNA) for a mouse carcinoembryonic antigen gene family member (mmCGM1a). Two transcripts expressed in several different adult mouse tissues hybridize to this cDNA, a 1.8-kilobase and a 4.6-kilobase mRNA. Sequences of many related cDNA clones indicate that they are most likely encoded by a single gene which undergoes alternative splicing. The protein encoded by the mmCGM1a cDNA shares 69% of the amino acid residues in the NH2-terminal domain with a rat liver ecto-ATPase and with the human biliary glycoprotein. Mouse fibroblast transfectant cells expressing the mmCGM1a protein on their cell surface exhibit calcium- and temperature-independent adhesion in vitro which can be specifically inhibited by an antibody raised against a carcinoembryonic antigen-related 120 kilodalton protein.

Molecular cloning of the gene encoding the mouse parathyroid hormone/parathyroid hormone-related peptide receptor.

The parathyroid hormone/parathyroid hormone-related peptide receptor (PTHR) is a G-protein-coupled receptor containing seven predicted transmembrane domains. We have isolated and characterized recombinant bacteriophage lambda EMBL3 genomic clones containing the mouse PTHR gene, including 10 kilobases of the promoter region. The gene spans > 32 kilobases and is divided into 15 exons, 8 of which contain the transmembrane domains. The PTHR exons containing the predicted membrane-spanning domains are heterogeneous in length and three of the exon-intron boundaries fall within putative transmembrane sequences, suggesting that the exons did not arise from duplication events. This arrangement is closely related to that of the growth hormone releasing factor receptor gene, particularly in the transmembrane region, providing strong evidence that the two genes evolved from a common precursor. Transcription is initiated principally at a series of sites over a 15-base-pair region. The proximal promoter region is highly (G+C)-rich and lacks an apparent TATA box or initiator element homologies but does contain CCGCCC motifs. The presumptive amino acid sequence of the encoded receptor is 99%, 91%, and 76% identical to those of the rat, human, and opossum receptors, respectively. There is no consensus polyadenylation signal in the 3' untranslated region. The poly(A) tail of the PTHR transcript begins 32 bases downstream of a 35-base-long A-rich sequence, suggesting that this region directs polyadenylylation.

Highly potent transcriptional activation by 16-ene derivatives of 1,25-dihydroxyvitamin D3. Lack of modulation by 9-cis-retinoic acid of response to 1,25-dihydroxyvitamin D3 or its derivatives.

Although several studies have been performed on the biological activities of analogs of 1,25-dihydroxyvitamin D3 (1,25-(OH)2 D3) at the whole animal and cellular levels, little work has been done to analyze their transcriptional activation properties. A highly inducible 1,25-(OH)2 D3-responsive promoter composed of three copies of the mouse osteopontin vitamin D3 response element (VDRE3) inserted upstream of a herpes simplex virus thymidine kinase promoter has been constructed, and its transcriptional properties have been analyzed by transient transfection into the monkey kidney cell line COS-7 and the rat osteoblast-like osteosarcoma line ROS 17/2.8. We have studied systematically transcriptional activation by a number of 1,25-(OH)2 D3 analogs, particularly those substituted at positions 16, 23, 26, and 27, sites that are targets for metabolism. Strikingly, except for derivatives that bind the 1,25-(OH)2 D3 receptor (VDR) very weakly, we find no parallel between the potency of action of a derivative as a transcriptional inducer and its affinity for the VDR. Derivatives substituted by multiple bonds at positions 16 and/or 23, although having varying affinities for the VDR, all stimulate transcription more potently than D3, in some cases at 100-fold lower concentrations. The peak transcriptional activity observed varies by only approximately 20% among different active analogs, indicating little difference in the activity of the VDR once bound to ligand. Gel retardation assays with ROS 17/2.8 nuclear extracts suggest that the VDR binds to the mouse osteopontin VDRE predominantly as a heterodimer with retinoid X receptor(s) (RXR(s)). We find that 9-cis-retinoic acid, the cognate ligand for RXRs, does not have a significant effect on the response of the VDRE3 promoter to 1,25-(OH)2 D3 or a number of its derivatives in ROS 17/2.8 or in COS-7 cells, under conditions in which promoters containing retinoid X response elements are activated. This suggests that 9-cis-retinoic acid may not act on the response to 1,25-(OH)2 D3 or its derivatives by directly influencing the transcriptional activity of VDR/RXR heterodimers. This promoter/reporter system should be useful for analyzing the tissue-specific transcriptional activity of 1,25-(OH)2 D3 and its derivatives in any cell type amenable to transient transfection.

Parathyroid hormone/parathyroid hormone related peptide receptor gene transcripts are expressed from tissue-specific and ubiquitous promoters.

Parathyroid hormone (PTH) and PTH related peptide (PTHrP) stimulate diverse physiological responses in a number of tissues by binding to the same receptor. We have previously cloned the gene encoding the mouse PTH/PTHrP receptor (PTHR), and have identified a promoter region. The first exon transcribed from this promoter contains untranslated sequence and is followed by an exon encoding signal sequence and the first amino acids of the mature polypeptide. We have now identified and characterized a second promoter region, located > 3 kb upstream of the original. Four partial cDNA clones, amplified from mouse kidney RNA by reverse transcription followed by the polymerase chain reaction, contain sequence corresponding to two previously unidentified exons composed of untranslated sequence. The second (3') of the two exons is spliced to the previously identified signal sequence exon. These cDNAs are highly homologous to the 5' end of a cDNA isolated from human kidney, strongly suggesting that the promoter region is conserved between mouse and humans. RNase protection and primer extension experiments have identified several transcriptional start sites extending over a region of approximately 100 bp. Unlike the previously identified promoter, this promoter is not (G+C)-rich. It lacks a consensus TATA element, but does contain a consensus CCAAT box. We have determined the expression patterns of both promoters by RNase protection with total and poly A+ RNA from several mouse tissues. The newly identified promoter is highly tissue specific, being strongly active in kidney and weakly active in liver, but not expressed in the other tissues studied. The previously identified (G+C)-rich promoter is expressed in all tissues studied. This indicates that the PTHR gene expression is controlled by regulatory signals specific to kidney and liver, as well as signals functioning in a wide variety of cell types. These results may provide insight into certain defects in PTH signalling found in humans.

Variability in measurements of pressure-volume curves in normal subjects.

Changes in lung elasticity as measured by the pressure-volume curve are used in clinical investigative studies to diagnose abnormalities in lung function and to evaluate changes in a patient either over time or with an acute intervention. To assess the intrinsic variability of parameters derived from this technique, 4 static deflation curves per day on 5 separate days during a 2-month period were constructed for 10 healthy adults. The pressure-volume data were fitted to the exponential equation: V = A-Be-KP. The coefficients of variation for maximal elastic recoil pressure, transpulmonary pressures at 90, 80, 70, and 60% total lung capacity, static expiratory compliance, and the constants A, B, and k were determined. No significant correlation was found between the variability of daily curves and that of curves performed on separate occasions. The natural log of the exponential constant showed the lowest coefficient of variation, indicating that this parameter is the most reproducible.

Several members of the mouse carcinoembryonic antigen-related glycoprotein family are functional receptors for the coronavirus mouse hepatitis virus-A59.

Mouse hepatitis virus-A59 (MHV-A59), a murine coronavirus, can utilize as a cellular receptor MHVR, a murine glycoprotein in the biliary glycoprotein (BGP) subfamily of the carcinoembryonic antigen (CEA) family in the immunoglobulin superfamily (G.S. Dveksler, M. N. Pensiero, C. B. Cardellichio, R. K. Williams, G.-S. Jiang, K. V. Holmes, and C. W. Dieffenbach, J. Virol. 65:6881-6891, 1991). Several different BGP isoforms are expressed in tissues of different mouse strains, and we have explored which of these glycoproteins can serve as functional receptors for MHV-A59. cDNA cloning, RNA-mediated polymerase chain reaction analysis, and Western immunoblotting with a monoclonal antibody, CC1, specific for the N-terminal domain of MHVR showed that the inbred mouse strains BALB/c, C3H, and C57BL/6 expressed transcripts and proteins of the MHVR isoform and/or its splice variants but not the mmCGM2 isoform. In contrast, adult SJL/J mice, which are resistant to infection with MHV-A59, express transcripts and proteins only of the mmCGM2-related isoforms, not MHVR. These data are compatible with the hypothesis that the MHVR and mmCGM2 glycoproteins may be encoded by different alleles of the same gene. We studied binding of anti-MHVR antibodies or MHV-A59 virions to proteins encoded by transcripts of MHVR and mmCGM2 and two splice variants of MHVR, one containing two immunoglobulin-like domains [MHVR(2d)] and the other with four domains as in MHVR but with a longer cytoplasmic domain [MHVR(4d)L]. We found that the three isoforms tested could serve as functional receptors for MHV-A59, although only isoforms that include the N-terminal domain of MHVR were recognized by monoclonal antibody CC1 in immunoblots or by MHV-A59 virions in virus overlay protein blot assays. Thus, in addition to MHVR, both the two-domain isoforms, mmCGM2 and MHVR(2d), and the MHVR(4d)L isoform served as functional virus receptors for MHV-A59. This is the first report of multiple related glycoprotein isoforms that can serve as functional receptors for a single enveloped virus.