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Biomedical subjects

B S Spooner

Publications and source records attributed to B S Spooner.

At least 19 recordsLinked to original sources
PubMed1994

Gravity in mammalian organ development: differentiation of cultured lung and pancreas rudiments during spaceflight.

Organ culture of embryonic mouse lung and pancreas rudiments has been used to investigate development and differentiation, and to assess the effects of microgravity on culture differentiation, during orbital spaceflight of the shuttle Endeavour (mission STS-54). Lung rudiments continue to grow and branch during spaceflight, an initial result that should allow future detailed study of lung morphogenesis in microgravity. Cultured embryonic pancreas undergoes characteristic exocrine acinar tissue and endocrine islet tissue differentiation during spaceflight, and in ground controls. The rudiments developing in the microgravity environment of spaceflight appear to grow larger than their ground counterparts, and they may have differentiated more rapidly than controls, as judged by exocrine zymogen granule presence.

Animals↗
PubMed1994

Pre-metatarsal skeletal development in tissue culture at unit- and microgravity.

Explant organ culture was used to demonstrate that isolated embryonic mouse pre-metatarsal mesenchyme is capable of undergoing a series of differentiative and morphogenetic developmental events. Mesenchyme differentiation into chondrocytes, and concurrent morphogenetic patterning of the cartilage tissue, and terminal chondrocyte differentiation with subsequent matrix mineralization show that cultured tissue closely parallels in vivo development. Whole mount alizarin red staining of the cultured tissue demonstrates that the extracellular matrix around the hypertrophied chondrocytes is competent to support mineralization. Intensely stained mineralized bands are similar to those formed in pre-metatarsals developing in vivo. We have adapted the culture strategy for experimentation in a reduced gravity environment on the Space Shuttle. Spaceflight culture of pre-metatarsals, which have already initiated chondrogenesis and morphogenetic patterning, results in an increase in cartilage rod size and maintenance of rod shape, compared to controls. Older pre-metatarsal tissue, already terminally differentiated to hypertrophied cartilage, maintained rod structure and cartilage phenotype during spaceflight culture.

Animals↗
PubMed1993

Embryonic mouse pre-metatarsal development in organ culture.

Embryonic mouse pre-metatarsals were removed from embryos at 13 days of gestation and cultured in a defined, serum-free medium for up to 15 days. By histological analysis, we observe that the cultured pre-metatarsal tissue undergoes a similar developmental profile as pre-metatarsals growing normally in vivo. The initial mesenchyme condensation regions undergo differentiation and morphogenesis to form distinct rods made up of cartilage tissue. A marker of this differentiation step is the synthesis of type II collagen. Metabolic labelling, pepsin digestion, SDS-PAGE, and autoradiography were used to demonstrate this protein when cartilage tissue is present in the cultures. After additional culture time, terminal chondrocyte differentiation and morphogenesis take place in specific regions of the cartilage rods to form bands of hypertrophied chondrocytes. One marker of this differentiation step is the synthesis of the enzyme alkaline phosphatase. We have measured the activity of this enzyme throughout the culture period and see a substantial increase at the time of terminal chondrocyte differentiation. Another feature of hypertrophied chondrocytes is that the matrix around the cells becomes calcified. Calcified matrix in our cultured pre-metatarsals was visualized by staining with alizarin red. By supplementing the defined culture medium with ITS, we observed that terminal chondrocyte differentiation took place in a shorter culture time. Supplementation of the medium with serum results in a similar acceleration of terminal differentiation, and, with additional culture time, an osteoid-like matrix forms around the central region of the rods.

Alkaline Phosphatase↗
PubMed1993

Utilization of microgravity bioreactors for differentiation of mammalian skeletal tissue.

Bioreactor cell and tissue culture vessels can be used to study bone development in a simulated microgravity environment. These vessels will also provide an advantageous, low maintenance culture system on space station Freedom. Although many types of cells and tissues can potentially utilize this system, our particular interest is in developing bone tissue. We have characterized an organ culture system utilizing embryonic mouse pre-metatarsal mesenchyme, documenting morphogenesis and differentiation as cartilage rods are formed, with subsequent terminal chondrocyte differentiation to hypertrophied cells. Further development to form bone tissue is achieved by supplementation of the culture medium. Research using pre-metatarsal tissue, combined with the bioreactor culture hardware, could give insight into the advantages and/or disadvantages of conditions experienced in microgravity. Studies such as these have the potential to enhance understanding of bone development and adult bone physiology, and may help define the processes of bone demineralization experienced in space and in pathological conditions here on earth.

Animals↗
PubMed1992

Localization of extracellular matrix components in developing mouse salivary glands by confocal microscopy.

The importance of the extracellular matrix (ECM) in epithelial-mesenchymal interactions in developing organisms is well established. Proteoglycans and interstitial collagens are required for the growth, morphogenesis, and differentiation of epithelial organs and the distribution of these molecules has been described. However, much less is known about other ECM macromolecules in developing epithelial organs. We used confocal microscopy to examine the distribution of laminin, heparan sulfate (BM-1) proteoglycan, fibronectin, and collagen types I, IV, and V, in mouse embryonic salivary glands. Organ rudiments were isolated from gestational day 13 mouse embryos and cultured for 24, 48, or 72 hours. Whole mounts were stained by indirect immunofluorescence and then examined using a Zeiss Laser Scan Microscope. We found that each ECM component examined had a distinct distribution and that the distribution of some molecules varied with culture time. Laminin was mainly restricted to the basement membrane. BM-1 proteoglycan was concentrated in the basement membrane and also formed a fine network throughout the mesenchyme. Type IV collagen was mainly located in the basement membrane of the epithelium, but it was also present throughout the mesenchyme. Type V collagen was distributed throughout the mesenchyme at 24 hours, but at 48 hours was principally located in the basement membrane. Type I collagen was distributed throughout the mesenchyme at all culture times, and accumulated in the clefts and particularly at the epithelial-mesenchymal interface as time in culture increased. Fibronectin was observed throughout the mesenchyme at all times.

Animals↗
PubMed1992

Cytokine secretion by immune cells in space.

Cultured, bone marrow-derived macrophages, murine spleen and lymph node cells, and human lymphocytes were tested for their ability to secrete cytokines in space. Lipopolysaccharide-activated bone marrow macrophages were found to secrete significantly more interleukin-1 and tumor necrosis factor when stimulated in space than when stimulated on earth. Murine spleen cells stimulated with poly I:C in space released significantly more interferon-alpha at 1 and 14 hours after stimulation than cells stimulated on earth. Similarly, murine lymph node T cells and human peripheral blood lymphocytes, stimulated with concanavalin A in space, secreted significantly more interferon-gamma than ground controls. These data suggest that space flight has a significant enhancing effect on immune cell release of cytokines in vitro.

Animals↗
PubMed1992

Alterations in biosynthetic accumulation of collagen types I and III during growth and morphogenesis of embryonic mouse salivary glands.

We examined the biosynthetic patterns of interstitial collagens in mouse embryonic submandibular and sublingual glands cultured in vitro. Rudiments explanted on day 13 of gestation and cultured for 24, 48, and 72 h all synthesized collagen types I, III, and V. However, while the total incorporation of label into collagenous proteins did not change over the three-day culture period, the rate of accumulation of newly synthesized types I and III did change. At 24 h, the ratio of newly synthesized collagen types I:III was approximately 2, whereas at 72 h, the ratio was approximately 5. These data suggest that collagen types I and III may be important in initiation of branching in this organ, but that type I may become dominant in the later stages of development and in maintenance of the adult organ.

Animals↗
PubMed1992

Experiments with suspended cells on the Space Shuttle.

Spaceflight experiments since 1981 have demonstrated that certain cell functions are altered by micro-g. Biophysical models suggest that cell membranes and organelles should not be affected directly by gravity, however, the chemical microenvironment surrounding the cell and molecular transport could be altered by reduced gravity. Most experiments have used suspended live cells in small chambers without stirring or medium exchange. Flight results include increased attachment of anchorage-dependent human cells to collagen coated microcarriers, reduced secretion of growth hormone from pituitary cells, decreased mitogenic response of lymphocytes, increased Interferon-alpha by lymphocytes, increased Interleukin-1 and Tumor Necrosis Factor secretion by macrophages. Related experiments on cells immediately postflight and on procaryotic cells have shown significant changes in secretory capacity, cell proliferation, differentiation and development. Postulated mechanism include altered cell-cell interactions, altered calcium ion transport, effects on cell cytoskeleton, transport of transmitters and interactions with receptors. The discussion includes use of new molecular methods, considerations for cell environmental control and a preview of several experiments planned for the Shuttle and Spacelab flights to study the basic effects of microgravity on cellular physiology and potential interactions of spaceflight with radiation damage and cellular repair mechanisms.

Animals↗
PubMed1990

Binding of alpha-fetoprotein by immobilized monoclonal antibodies during episodes of zero-gravity obtained by parabolic flight.

Alpha-fetoprotein (AFP), a single chain polypeptide which is synthesized by the liver and yolk sac of the human fetus, provided a model ligand for assessing the effects of microgravity on ligand binding to surface-immobilized model receptor molecules. Monoclonal antibodies, used as receptors for AFP, were immobilized by covalent attachment to latex microparticles. Zero gravity environment was obtained by parabolic flight aboard NASA 930, a modified KC-135 aircraft. During the onset of an episode of zero gravity, ligand and receptor were mixed. Timed incubation (20 s) was terminated by centrifugation, the supernatant removed, and microparticles were assessed for bound AFP by immunochemical methods. The extent of binding was not influenced by microgravity, when compared with 1-G controls, which suggests that aberrant cellular activities observed in microgravity are not the simple expression of altered macromolecular interactions.

Antibodies, Monoclonal↗
PubMed1990

EMG analysis of human postural responses during parabolic flight microgravity episodes.

Anticipatory postural activity in the trunk and legs precedes rapid shoulder flexion in unit gravity. We tested the hypothesis that anticipatory activity is a component of a single neural command for arm movement by monitoring the surface electromyographic activity of the biceps femoris, paraspinals and deltoid muscles of three subjects during the microgravity phase of parabolic flight. If part of a single command, anticipatory postural activity would be expected to remain intact despite the absence of the body's center of gravity in a reduced gravity environment. However, in at least 75% of the microgravity trials anticipatory biceps femoris activity was absent, indicating a separation of postural and agonist muscle activity. Such a finding suggests that anticipatory postural biceps femoris activity may be initiated independently of agonist (deltoid) activity.

Adult↗
PubMed1989

Effects of microgravity on liposome-reconstituted cardiac gap junction channeling activity.

Effects of microgravity on cardiac gap junction channeling activity were investigated aboard NASA zero-gravity aircraft. Liposome-reconstituted gap junctions were assayed for channel function during free-fall, and the data were compared with channeling at 1 g. Control experiments tested for 0 g effects on the structural stability of liposomes, and on the enzyme-substrate signalling system of the assay. The results demonstrate that short periods of microgravity do not perturb reconstituted cardiac gap junction channeling activity.

Animals↗
PubMed1989

beta-Xyloside effects on basal lamina structure and anionic site distribution the embryonic mouse submandibular salivary gland.

beta-D-Xyloside is a proteoglycan biosynthesis inhibitor. Previous studies on embryonic salivary glands have demonstrated that 0.5 mM beta-xyloside (1) inhibits proteoglycan synthesis by 50%; (2) severely depresses sulphated glycosaminoglycan deposition at the basal epithelial surface, and (3) dramatically inhibits epithelial branching morphogenesis. Electron microscopy revealed a conventional three-layered basal lamina that is altered in the presence of beta-xyloside by a 35% reduction in the number of tannic acid-resolved particles in the lamina densa. Basal lamina anionic sites, resolved with ruthenium red (RR) and polyethyleneimine (PEI) cationic probes, were also reduced in the presence of beta-xyloside. PEI particles were reduced by 28%, and RR particles by 24%, per two-dimensional unit of basal lamina. These beta-xyloside effects on anionic sites are consistent with an hypothesis that sulphated glycosaminoglycans account for 50% of the basal lamina anionic sites and a predicted 25% decrease in anionic sites in the presence of beta-xyloside.

Animals↗
PubMed1989

Embryonic salivary gland epithelial branching activity is experimentally independent of epithelial expansion activity.

Embryonic mouse submandibular salivary gland rudiments undergo morphogenesis in organ culture, characterized by extensive epithelial growth and expansion and repetitive branching activity. Tunicamycin, at a concentration of 25 ng/ml culture medium, decreases the degree of net protein accumulation by 83% and the degree of epithelial expansion by 70% compared to controls, over a 48-hr culture tenure. These decreases correlate with reduced incorporation of [3H]thymidine into DNA. Nevertheless, epithelial branching activity is uncompromised, undergoing an approximately 10-fold increase in lobe numbers, in both controls and tunicamycin-treated rudiments, during the same 48-hr period. The effect is most striking during the 24- to 48-hr culture interval, when controls and tunicamycin-treated rudiments each triple their lobe numbers and controls approximately double epithelial area, while tunicamycin virtually stops all epithelial expansion.

Animals↗
PubMed1988

Reconstitution of cardiac gap junction channeling activity into liposomes: a functional assay for gap junctions.

Cardiac gap junctions were reconstituted into liposomes. To determine if reconstitution resulted in membrane channel formation, we developed an assay for channel function that used a liposome-entrapped peroxidase to detect entry of a substrate into the liposome. The data demonstrate, for the first time, that reconstituted gap junctions from heart are capable of channel-forming activity in artificial membranes.

Animals↗
PubMed1987

An autoradiographic analysis of N-linked glycoconjugates in embryonic salivary gland morphogenesis.

The synthesis, deposition, and loss of mannose-bearing glycoconjugates during branching morphogenesis of embryonic mouse salivary glands has been evaluated. Day 13 embryonic mouse salivary glands were cultured for 44 hr, pulse labeled 4 hr with [3H]mannose, then fixed after 0, 2, 4, 8, or 24 hr of chase in nonradioactive medium, and processed for autoradiography. Light microscopic autoradiograms of sectioned rudiments reveal extensive label within the epithelium, little label over the mesenchyme, and a concentration of radioactivity at the basal surface of the epithelium. Autoradiograms of "chased" rudiments reveal a) no detectable loss of label from the epithelium or the basal epithelial surface over the first 8 hr, and b) significant label loss by 24 hr of chase at the basal epithelial surface, while moderate amounts of radioactivity remain throughout the rest of the epithelium. The [3H]bound material is insensitive to chondroitinase ABC, a glycosaminoglycan degradative enzyme, but is sensitive to tunicamycin presence in the culture medium. Earlier studies showed that embryonic mouse salivary glands cultured in medium containing tunicamycin (25 ng/ml) continued normal epithelial branching while epithelial growth was inhibited. The present autoradiographic studies of [3H]mannose-labeled rudiments demonstrate that tunicamycin causes a significant decrease in radioactivity, relative to controls. Thus, our results suggest that epithelial branching activity is independent of control levels of mannose-containing/tunicamycin-sensitive, glycoconjugate deposition.

Animals↗