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At least 379 records · Page 21

Tissue-Engineered Skeletal Muscle Organoids for Reversible Gene Therapy

Genetically modified murine skeletal myoblasts were tissue engineered in vitro into organ-like structures (organoids) containing only postmitotic myoribers secreting pharmacological levels of recombinant human growth hormone (rhGH). Subcutaneous organoid implantation under tension led to the rapid and stable appearance of physiological sera levels of rhGH for up to 12 weeks, whereas surgical removal led to its rapid disappearance. Reversible delivery of bioactive compounds from postmitotic cells in tissue engineered organs has several advantages over other forms of muscle gene therapy.

Vandenburgh, Herman↗

In Vitro Interleukin-1 and 2 Production and Interleukin 2 Receptor Expression in the Rhesus Monkey

Anti-human monoclonal antibodies were used to detect and quantify interleukins-1 and 2 and interleukin-2 receptor expression in peripheral blood mononuclear cells from a rhesus monkey. Interleukin-1 production could be induced by phorbol esters (PMA) and was potentiated by phytohemagglutinin (PHA). Interleukin-2 secretion could also be induced by the combination of PHA and PMA, but only weakly with PHA alone. Interleukin-2 receptor expression was present in a subpopulation of unstimulated lymphocytes and could be enhanced by PHA or PMA. These data show once again that the rhesus monkey immune system is cross-reactive with the human one and that rhesus macaque could be a good model to study interleukin therapy.

Schmitt, Didier A.↗

The Role of GH/IGF-I Axis in Muscle Homeostasis During Weightlessness

Exposure to reduced gravity during space travel profoundly alters the loads placed on bone and muscle. Astronauts suffer significant losses of muscle and bone strength during weightlessness. Exercise as a countermeasure is only partially effective in remedying severe muscle atrophy and bone demineralization. Similar wasting of muscles and bones affects people on Earth during prolonged bed rest or immobilization due to injury. In the absence of weight bearing activity, atrophy occurs primarily in the muscles that act in low power, routine movements and in maintaining posture. Hormonal disfunction could contribute in part to the loss of muscle and bone during spaceflight. Reduced levels of human Growth Hormone (hGH) were found in astronauts during space flight, as well as reduced GH secretory activity was observed from the anterior pituitary in 7-day space flight rats. Growth hormone has been shown to be required for maintenance of muscle mass and bone mineralization, in part by mediating the biosynthesis IGF-I, a small polypeptide growth factor. IGF biosynthesis and secretion plays an important role in potentiating muscle cell differentiation and has been shown to drive the expression of myogenin, a myogenic specific basic helix-loop-helix factor. IGF-I has also been shown to have an important role in potentiating muscle regeneration, repair and adult muscle hypertrophy.

Schwartz, Robert J.↗

Effects of Microgravity on Quail Eye Development

During embryonic development, the most exposed tissue of the eye, the cornea, becomes differentially bulged outward because of constant intraocular pressure (IOP). The component cells of the cornea secrete a unique, paracrystalline extracellular matrix (the stroma) composed of orthogonal plies of collagen fibrils and proteoglycans. The cornea remains avascular, becomes transparent, and becomes more densely innervated than any other region on the surface of the body. Corneas from chicken embryos that flew on STS-47 contain many more cellular processes in the outermost region of the stroma (Bowman's Layer) than any corresponding region of control corneas. These processes appear to be cross-sections of cytoplasmic extensions of cells and are found in that region of Bowman's Layer immediately beneath the basal lamina of the corneal epithelium. Here, we propose to compare corneas of quail that flew in space on Mir-1 with those of ground controls to determine if the same unusual cellular processes are seen as in the space-flown chicken corneas. In the central regions of such space-flown corneas, the processes appear to be either portions of basal epithelial cells whose pseudopodial extensions have migrated down through their own basal lamina into the stroma, or corneal nerves that have innervated the corneal stroma in an unusual manner. Eyeballs of embryos fixed on Mir-1, control embryos fixed at KSC and clinostated embryos fixed at KSU, will provide corneas for this study. Electron microscopy will be used to assess the distribution of the cellular processes in Bowman's Layer in the central region of each cornea. Attempts also will be made to determine the relative glycosaminoglycan distributions in the corneal stromas by indirect immunofluorscence and to record whole-mount staining patterns of the corneal nerves.

Conrad, Gary W.↗

Tissue-Engineered Skeletal Muscle Organoids for Reversible Gene Therapy

Genetically modified murine skeletal myoblasts were tissue engineered in vitro into organ-like structures (organoids) containing only postmitotic myofibers secreting pharmacological levels of recombinant human growth hormone (rhGH). Subcutaneous organoid Implantation under tension led to the rapid and stable appearance of physiological sera levels of rhGH for up to 12 weeks, whereas surgical removal led to its rapid disappearance. Reversible delivery of bioactive compounds from postimtotic cells in tissue engineered organs has several advantages over other forms of muscle gene therapy.

Vandenburgh, Herman↗

Distinct Secondary Structures of the Leucine-Rich Repeat Proteoglycans Decorin and Biglycan: Glycosylation-Dependent Conformational Stability

Biglycan and decorin, closely related small leucine-rich repeat proteoglycans, have been overexpressed in eukaryotic cers and two major glycoforms isolated under native conditions: a proteoglycan substituted with glycosaminoglycan chains; and a core protein form secreted devoid of glycosaminoglycans. A comparative biophysical study of these glycoforms has revealed that the overall secondary structures of biglycan and decorin are different. Far-UV Circular Dichroism (CD) spectroscopy of decorin and biglycan proteoglycans indicates that, although they are predominantly Beta-sheet, biglycan has a significantly higher content of alpha-helical structure. Decorin proteoglycan and core protein are very similar, whereas the biglycan core protein exhibits closer similarity to the decorin glycoforms than to. the biglycan proteoglycan form. However, enzymatic removal of the chondroitin sulfate chains from biglycan proteoglycan does not induce a shift to the core protein structure, suggesting that the fmal form is influenced by polysaccharide addition only during biosynthesis. Fluorescence emission spectroscopy demonstrated that the single tryptophan residue, which is at a conserved position at the C-terminal domain of both biglycan and decorin, is found in similar microenvironments. This indicates that at least in this specific domain, the different glycoforms do exhibit apparent conservation of structure. Exposure of decorin and biglycan to 10 M urea resulted in an increase in fluorescent intensity, which indicates that the emission from tryptophan in the native state is quenched. Comparison of urea-induced protein unfolding curves provided further evidence that decorin and biglycan assume different structures in solution. Decorin proteoglycan and core protein unfold in a manner similar to a classic two-state model, in which there is a steep transition to an unfolded state between 1-2 M urea. The biglycan core protein also shows a similar steep transition. However, biglycan proteoglycan shows a broad unfolding transition between 1-6 M urea, probably indicating the presence of stable unfolding intermediates.

Krishnan, Priya↗

The Study of Leukocyte Functions in a Rotating Wall Vessel

The objective of this study was to investigate the behavior of leukocytes under free-fall conditions in a rotating wall vessel. In such a vessel, the tendency of a cell to fall in response to gravity is opposed by the rotation of the vessel and the culture medium within, keeping the cells in suspension without fluid shear. Previous reports indicated that such functions as lymphocyte migration through collagen matrix or monocyte cytokine secretion are altered under these conditions, and these changes correlate with similar functional defects of cultured cells seen during spaceflight.

Trial, JoAnn↗

The Effect of Spaceflight on Bone Cell Cultures

Understanding the response of bone to mechanical loading (unloading) is extremely important in defining the means of adaptation of the body to a variety of environmental conditions such as during heightened physical activity or in extended explorations of space or the sea floor. The mechanisms of the adaptive response of bone are not well defined, but undoubtedly they involve changes occurring at the cellular level of bone structure. This proposal has intended to examine the hypothesis that the loading (unloading) response of bone is mediated by specific cells through modifications of their activity cytoskeletal elements, and/or elaboration of their extracellular matrices. For this purpose, this laboratory has utilized the results of a number of previous studies defining molecular biological, biochemical, morphological, and ultrastructural events of the reproducible mineralization of a primary bone cell (osteoblast) culture system under normal loading (1G gravity level). These data and the culture system then were examined following the use of the cultures in two NASA shuttle flights, STS-59 and STS-63. The cells collected from each of the flights were compared to respective synchronous ground (1G) control cells examined as the flight samples were simultaneously analyzed and to other control cells maintained at 1G until the time of shuttle launch, at which point they were terminated and studied (defined as basal cells). Each of the cell cultures was assayed in terms of metabolic markers- gene expression; synthesis and secretion of collagen and non-collagenous proteins, including certain cytoskeletal components; assembly of collagen into macrostructural arrays- formation of mineral; and interaction of collagen and mineral crystals during calcification of the cultures. The work has utilized a combination of biochemical techniques (radiolabeling, electrophoresis, fluorography, Western and Northern Blotting, and light microscopic immunofluorescence) and structural methods (conventional and high voltage electron microscopy, inununocytochemistry, stereomicroscopy, and 3D image reconstruction). The studies have provided new knowledge of aspects of bone cell development and structural regulation, extracellular matrix assembly, and mineralization during spaceflight and under normal gravity. The information has contributed to insights into the means in general by which cells respond and adapt to different conditions of gravity (loading). The data may as well have suggested an underlying basis for the observed loss of bone by vertebrates, including man, in microgravity; and these scientific results may have implications for understanding bone loss following fracture healing and extended periods of inactivity such as during long-term bedrest.

Landis, William J.↗

Effect of Flow on Gene Regulation in Smooth Muscle Cells and Macromolecular Transport Across Endothelial Cell Monolayers

Endothelial cells line all of the vessels of the circulatory system, providing a non-thrombogenic conduit for blood flow; they regulate many complex functions in the vasculature, such as coagulation, fibrinolysis, platelet aggregation, vessel tone and growth, and leukocyte traffic; and they form the principal barrier to transport of substances between the blood and the surrounding tissue space. The permeability of endothelial cell changes with environmental stimuli; shear stress, in particular, applied either in vivo, or in vitro, induces changes in protein expression and secretion of vasoactive factors by endothelial cells. The ability to study the effects of shear on the macromolecular permeability of the cerebral vasculature is particularly important, since in no other place is the barrier function of the endothelium more important than in the brain. The endothelial cells of this organ have developed special barrier properties that keep the cerebral system from experiencing any drastic change in composition; together with glial cells, they form the blood brain barrier (BBB). We have studied the effect of flow on bovine BBB using flow chambers and tissue culture systems.

McIntire, Larry V.↗

Role of Thyroxine in Space-Developed Jellyfish

The Aurelia Metamorphosis Test System was previously used to determine the effects of the space environment on the development and behavior of tiny (1-2 mm) jellyfish ephyrae during the SLS-1 and IML-2 missions. Results from the SLS-1 experiment included the discovery that statolith numbers were significantly reduced in Earth-formed ephyrae flown for nine days in space as compared with ground-based controls. In addition, upon return to Earth, six times more ephyrae which had developed in space than those developed on Earth had pulsing abnormalities, indicating that either these animals did not form their neuromuscular structures normally while in space or they were unable to adapt to the Ig environment upon return to Earth. The metamorphosis process, which enables the formation of ephyrae from polyps is influenced by a hormone, Jf-T4 Oellyfish thyroxine) which is synthesized following iodine administration. Two groups of polyps in space, however, formed ephyrae without iodine administration indicating that Jf-T4 synthesis, utilization, or excretion was different in. the ephyrae. Increased synthesis or build-up in the media of the hormone may also be linked to the increased demineralization of statoliths found in space-exposed ephyrae. In previous experiments, we found that externally administered thyroxine causes increased demineralization of statoliths on Earth. Abnormal pulsina in ephyrae following return to Earth during the SLS-1 mission may also be traced to increased Jf-T4 levels. Thyroxine is known to be important to the normal development and function of the nervous system, heart, and skeletal muscles in higher animals. For this third Jellyfish-in-Space experiment, we proposed to quantitate the levels of Jf- T4 and of T4 receptors in space-developed ephyrae and media and to compare these levels with those of animals developing and at Ig in space and on Earth. We expected to be able to determine whether Jf-T4 synthesis and/or secretion is different in space-flownjellyfish than in controls and to determine which cells (nerve, muscle, lithocytes, etc.)may have enhanced Jf-T4 levels. However, NASA deselected this experiment in August, 1997.

Spangenberg, Dorothy B.↗

Characterization of Human Bone Alkaline Phosphatase in Pichia Pastoris

A soluble form of human bone alkaline phosphatase has been expressed in a recombinant strain of the methylotrophic yeast Pichia pastoris. We constructed a plasmid containing cDNA encoding for human bone alkaline phosphatase, with the hydrophobic carboxyl terminal portion deleted. Alkaline phosphatase was secreted into the medium to a level of 32mg/L when cultured in shake flasks, and enzyme activity was 12U/mg, as measured by a spectrophotometric assay. By conversion to a fermentation system, a yield of 880mg/L has been achieved with an enzyme activity of 968U/mg. By gel electrophoresis analysis, it appears that greater than 50% of the total protein in the fermentation media is alkaline phosphatase. Although purification procedures are not yet completely optimized, they are expected to include filtration, ion exchange and affinity chromatography. Our presentation will focus on the purification and crystallization results up to the time of the conference. Structural data should provide additional information on the role of alkaline phosphatase in normal bone mineralization and in certain bone mineralization anomalies.

Malone, Christine C.↗

Bone Proteoglycan Changes During Skeletal Unloading

Skeletal adaptability to mechanical loads is well known since the last century. Disuse osteopenia due to the microgravity environment is one of the major concerns for space travelers. Several studies have indicated that a retardation of the mineralization process and a delay in matrix maturation occur during the space flight. Mineralizing fibrillar type I collagen possesses distinct cross-linking chemistries and their dynamic changes during mineralization correlate well with its function as a mineral organizer. Our previous studies suggested that a certain group of matrix proteoglycans in bone play an inhibitory role in the mineralization process through their interaction with collagen. Based on these studies, we hypothesized that the altered mineralization during spaceflight is due in part to changes in matrix components secreted by cells in response to microgravity. In this study, we employed hindlimb elevation (tail suspension) rat model to study the effects of skeletal unloading on matrix proteoglycans in bone.

Yamauchi, M.↗

Characterization of Treefoil Peptide Genes in Iron-Ion or X-Irradiated Human Cells

The gastrointestinal (GI) tract is especially sensitive to ionizing radiation, probably because of its high rate of cell turn over. Most of the data in the literature concerns the histological/anatomical description of damage rather than functional studies. In fact, previous reports in humans have shown that, at doses of 2 Gy or more, functional abnormalities appear indicating that in radiation sensitive tissues the effects of radiation are not limited to cell death. GI functions are controlled in particular by GI peptides. One hypothesis is that ionizing radiation may modulate the synthesis and release of these peptides and consequently may contribute largely to abnormalities in GI function. However, no previous studies have been concerned with GI-specific gene expression in irradiated GI tissues. The family of human trefoil peptides comprises three members thus far, all of which are expressed in specific regions of the GI tract. In addition, two trefoil peptides, pS2 (TFFI) and HITF (TFF2) are expressed in breast tissue. Their exact function in GI and breast tissues is unclear but mucosal integrity, repair, mucin secretion and responsiveness to hormones have been shown. We recently isolated and characterized pS2 as a novel p53- and estrogen receptor-independent gene whose MRNA expression in several cells lines was found to be delayed 4 to 7 days after irradiation with X-rays, fission neutrons or 1 GeV/n Fe-ions. The aim of the present study was to determine whether pS2 and HITF have a similar induction kinetics in irradiated gastric and breast cell lines, and whether they have the phorbol ester (TPA) responsive element (TRE).

Balcer-Kubiczek, E. K.↗

GH/IGF-I Transgene Expression on Muscle Homeostasis

We propose to test the hypothesis that the growth hormone/ insulin like growth factor-I axis through autocrine/paracrine mechanisms may provide long term muscle homeostasis under conditions of prolonged weightlessness. As a key alternative to hormone replacement therapy, ectopic production of hGH, growth hormone releasing hormone (GHRH), and IGF-I will be studied for its potential on muscle mass impact in transgenic mice under simulated microgravity. Expression of either hGH or IGF-I would provide a chronic source of a growth-promoting protein whose biosynthesis or secretion is shut down in space. Muscle expression of the IGF-I transgene has demonstrated about a 20% increase in hind limb muscle mass over control nontransgenic litter mates. These recent experiments, also establish the utility of hind-limb suspension in mice as a workable model to study atrophy in weight bearing muscles. Thus, transgenic mice will be used in hind-limb suspension models to determine the role of GH/IGF-I on maintenance of muscle mass and whether concentric exercises might act in synergy with hormone treatment. As a means to engineer and ensure long-term protein production that would be workable in humans, gene therapy technology will be used by to monitor muscle mass preservation during hind-limb suspension, after direct intramuscular injection of a genetically engineered muscle-specific vector expressing GHRH. Effects of this gene-based therapy will be assessed in both fast twitch (medial gastrocnemius) and slow twitch muscle (soleus). End-points include muscle size, ultrastructure, fiber type, and contractile function, in normal animals, hind limb suspension, and reambutation.

Schwartz, Robert J.↗

Sustained Partial Sleep Deprivation: Effects on Immune Modulation and Growth Factors

The vulnerability to medical emergencies is greatest in space where there are real limits to the availability or effectiveness of ground based assistance. Moreover, astronaut safety and health maintenance will be of increasing importance as we venture out into space for extended periods of time. It is therefore critical to understand the mechanisms of the regulatory physiology of homeostatic systems (sleep, circadian, neuroendocrine, fluid and nutritional balance) and the key roles played in adaptation. This synergy project has combined aims of the "Human Performance Factors, Sleep and Chronobiology Team"; the "Immunology, Infection and Hematology Team"; and the "Muscle Alterations and Atrophy Team", to broadly address the effects of long term sleep reduction, as is frequently encountered in space exploration, on neuroendocrine, neuroimmune and circulating growth factors. Astronaut sleep is frequently curtailed to averages of between 4- 6.5 hours per night. There is evidence that this amount of sleep is inadequate for maintaining optimal daytime functioning. However, there is a lack of information concerning the effects of chronic sleep restriction, or reduction, on regulatory physiology in general, and there have been no controlled studies of the cumulative effects of chronic sleep reduction on neuroendocrine and neuroimmune parameters. This synergy project represents a pilot study designed to characterize the effects of chronic partial sleep deprivation (PSD) on neuroendocrine, neuroimmune and growth factors. This project draws its subjects from two (of 18) conditions of the larger NSBRI project, "Countermeasures to Neurobehavioral Deficits from Cumulative Partial Sleep Deprivation During Space Flight", one of the projects on the "Human Performance Factors, Sleep and Chronobiology Team ". For the purposes of this study, to investigate the effects of chronic sleep loss on neuroendocrine and neuroimmune function, we have focused on the two extreme sleep conditions from this larger study: a 4.2 hour per night condition, and a 8.2 hour per night condition. During space flight, muscle mass and bone density are reduced, apparently due to loss of GH and IGF-I, associated with microgravity. Since >70% of growth hormone (GH) is secreted at night in normal adults, we hypothesized that the chronic sleep restriction to 4 hours per night would reduce GH levels as measured in the periphery. In this synergy project, in collaboration with the "Muscle Alterations and Atrophy Team ", we are measuring insulin-like growth factor-I (IGF-I) in peripheral circulation to test the prediction that it will be reduced by chronic sleep restriction. In addition to stress modulation of immune function, recent research suggests that sleep is also involved. While we all have the common experience of being sleepy when suffering from infection, and being susceptible to infection when not getting enough sleep, the mechanisms involved in this process are not understood and until recently have gone largely overlooked. We believe that the immune function changes seen in spaceflight may also be related to the cumulative effects of sleep loss. Moreover, in space flight, the possibility of compromised immune function or of the reactivation of latent viruses are serious potential hazards for the success of long term missions. Confined living conditions, reduced sleep, altered diet and stress are all factors that may compromise immune function, thereby increasing the risks of developing and transmitting disease. Medical complications, which would not pose serious problems on earth, may be disastrous if they emerged in space.

Mullington, Janet M.↗

A Robust Biomarker

Polymers of bacterial origin, either through cell secretion or the degraded product of cell lysis, form isolated mucoidal strands as well as well-developed biofilms on interfaces. Biofilms are structurally and compositionally complex and are readily distinguishable from abiogenic films. These structures range in size from micrometers to decimeters, the latter occurring as the well-known, mineralised biofilms called stromatolites. Compositionally bacterial polymers are greater than 90 % water, with while the majority of the macromolecules forming the framework of the polymers consisting of polysaccharides (with and some nucteic acids and proteins). These macromolecules contain a vaste amount of functional groups, such as carboxyls, hydroxyls, and phosphoryls which are implicated in cation-binding. It is the elevated metal- binding capacity which provides the bacterial polymer with structural support and also helps to preserves it for up to 3.5 b.y. in the terrestrial rock record. The macromolecules, thus, can become rapidly mineralised and trapped in a mineral matrix. Through early and late diagenesis (bacterial degradation, burial, heat, pressure and time) they break down, losing the functional groups and, gradually, their hydrogen atoms. The degraded product is known as "kerogen". With further diagenesis and metamorphism, all the hydrogen atoms are lost and the carbonaceous matter becomes graphite. until the remnant carbonaceous material become graphitised. This last sentence reads a bit as if ALL these macromolecules break down and end up as graphite., but since we find 441 this is not true for all of the macromolecules. We have traced fossilised polymer and biofilms in rocks from throughout Earth's history, to rocks as old as the oldest being 3.5 b.y.-old. Furthermore, Time of Flight Secondary Ion Mass Spectrometry has been able to identify individual macromolecules of bacterial origin, the identities of which are still being investigated, in all the samples containing fossil biofilm, including the 3.5 b.y..-old carbonaceous cherts from South Africa and Australia. As a result of the unique compositional, structural and "mineralisable" properties of bacterial polymer and biofilms, we conclude that bacterial polymers and biofilms constitute a robust and reliable biomarker for life on Earth and could be a potential biomarker for extraterrestrial life.

Westall, F.↗

The ''Rock'': The Role of the Press in Bringing about Change in Aircraft Accident Policy

From 1926 to 1938, the Aeronautics Branch, forerunner of the Federal Aviation Administration (FAA), had been charged with aircraft accident investigation. While the Branch had been investigating accidents since its inception, it had, early in its tenure, put into place a policy making its findings secret. Media and political pressure began to mount in late 1928 over its policy of nondisclosure and the debate brought pressure to bear on the young Aeronautics Branch to reverse its policy and make its findings public. The focusing event for the Branch's policy reversal was the death of Knute Rockne, the famous Notre Dame football coach, in a Transcontinental and Western Airways (TWA) airliner on March 31, 193 1. This paper will examine the role of print media in bringing about a significant, and lasting, change in aircraft accident public-disclosure policy.

Johnson, Randy↗

The Radioimmunoassay of Fluid and Electrolyte Hormones

The subject of the paper will be the assay of fluid/electrolyte hormones. ADH (antidiuretic hormone also referred to as vasopressin) reduces fluid loss by increasing water reabsorption by the kidney. The stimuli for its release from the pituitary are loss of blood, dehydration, or increased salt intake. Angiotensin II is the next hormone of interest. It is "generated" from a blood protein by the release of renin from the kidney. One of its functions is to stimulate the secretion of aldosterone from the adrenal gland. Release of renin is also stimulated by volume and sodium loss.

Keil, Lanny C.↗