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At least 55 records · Page 3
All sourcesNASA NTRSDOE OSTI
NASA NTRS1999

Detection of endogenous alkaline phosphatase activity in intact cells by flow cytometry using the fluorogenic ELF-97 phosphatase substrate

BACKGROUND: The alkaline phosphatase (AP) substrate 2-(5'-chloro-2'-phosphoryloxyphenyl)-6-chloro-4-(3H)-quinazolinone (ELF((R))-97 for enzyme-labeled fluorescence) has been found useful for the histochemical detection of endogenous AP activity and AP-tagged proteins and oligonucleotide probes. In this study, we evaluated its effectiveness at detecting endogenous AP activity by flow cytometry. METHODS: The ELF-97 phosphatase substrate was used to detect endogenous AP activity in UMR-106 rat osteosarcoma cells and primary cultures of chick chondrocytes. Cells were labeled with the ELF-97 reagent and analyzed by flow cytometry using an argon ultraviolet (UV) laser. For comparison purposes, cells were also assayed for AP using a Fast Red Violet LB azo dye assay previously described for use in detecting AP activity by flow cytometry. RESULTS: The ELF-97 phosphatase substrate effectively detected endogenous AP activity in UMR-106 cells, with over 95% of the resulting fluorescent signal resulting from AP-specific activity (as determined by levamisole inhibition of AP activity). In contrast, less than 70% of the fluorescent signal from the Fast Red Violet LB (FRV) assay was AP-dependent, reflecting the high intrinsic fluorescence of the unreacted components. The ELF-97 phosphatase assay was also able to detect very low AP activity in chick chondrocytes that was undetectable by the azo dye method. CONCLUSIONS: The ELF-97 phosphatase assay was able to detect endogenous AP activity in fixed mammalian and avian cells by flow cytometry with superior sensitivity to previously described assays. This work also shows the applicability of ELF-97 to flow cytometry, supplementing its previously demonstrated histochemical applications. Copyright 1999 Wiley-Liss, Inc.

Non-NASA Center↗
NASA NTRS1999

Myocardialization of the cardiac outflow tract

During development, the single-circuited cardiac tube transforms into a double-circuited four-chambered heart by a complex process of remodeling, differential growth, and septation. In this process the endocardial cushion tissues of the atrioventricular junction and outflow tract (OFT) play a crucial role as they contribute to the mesenchymal components of the developing septa and valves in the developing heart. After fusion, the endocardial ridges in the proximal portion of the OFT initially form a mesenchymal outlet septum. In the adult heart, however, this outlet septum is basically a muscular structure. Hence, the mesenchyme of the proximal outlet septum has to be replaced by cardiomyocytes. We have dubbed this process "myocardialization." Our immunohistochemical analysis of staged chicken hearts demonstrates that myocardialization takes place by ingrowth of existing myocardium into the mesenchymal outlet septum. Compared to other events in cardiac septation, it is a relatively late process, being initialized around stage H/H28 and being basically completed around stage H/H38. To unravel the molecular mechanisms that are responsible for the induction and regulation of myocardialization, an in vitro culture system in which myocardialization could be mimicked and manipulated was developed. Using this in vitro myocardialization assay it was observed that under the standard culture conditions (i) whole OFT explants from stage H/H20 and younger did not spontaneously myocardialize the collagen matrix, (ii) explants from stage H/H21 and older spontaneously formed extensive myocardial networks, (iii) the myocardium of the OFT could be induced to myocardialize and was therefore "myocardialization-competent" at all stages tested (H/H16-30), (iv) myocardialization was induced by factors produced by, most likely, the nonmyocardial component of the outflow tract, (v) at none of the embryonic stages analyzed was ventricular myocardium myocardialization-competent, and finally, (vi) ventricular myocardium did not produce factors capable of supporting myocardialization. Copyright 1999 Academic Press.

NASA Discipline Cardiopulmonary↗
NASA NTRS1998

Bioreactor perfusion system for the long-term maintenance of tissue-engineered skeletal muscle organoids

Three-dimensional skeletal muscle organ-like structures (organoids) formed in tissue culture by fusion of proliferating myoblasts into parallel networks of long, unbranched myofibers provide an in vivo-like model for examining the effects of growth factors, tension, and space flight on muscle cell growth and metabolism. To determine the feasibility of maintaining either avian or mammalian muscle organoids in a commercial perfusion bioreactor system, we measured metabolism, protein turnover. and autocrine/paracrine growth factor release rates. Medium glucose was metabolized at a constant rate in both low-serum- and serum-free media for up to 30 d. Total organoid noncollagenous protein and DNA content decreased approximately 22-28% (P < 0.05) over a 13-d period. Total protein synthesis rates could be determined accurately in the bioreactors for up to 30 h and total protein degradation rates could be measured for up to 3 wk. Special fixation and storage conditions necessary for space flight studies were validated as part of the studies. For example, the anabolic autocrine/paracrine skeletal muscle growth factors prostaglandin F2alpha (PGF2alpha) and insulin-like growth factor-1 (IGF-1) could be measured accurately in collected media fractions, even after storage at 37 degrees C for up to 10 d. In contrast, creatine kinase activity (a marker of cell damage) in collected media fractions was unreliable. These results provide initial benchmarks for long-term ex vivo studies of tissue-engineered skeletal muscle.

NASA Discipline Musculoskeletal↗
NASA NTRS1997

Effect of atrophy and contractions on myogenin mRNA concentration in chick and rat myoblast omega muscle cells

The skeletal rat myoblast omega (RMo) cell line forms myotubes that exhibit spontaneous contractions under appropriate conditions in culture. We examined if the RMo cells would provide a model for studying atrophy and muscle contraction. To better understand how to obtain contractile cultures, we examined levels of contraction under different growing conditions. The proliferation medium and density of plating affected the subsequent proportion of spontaneously contracting myotubes. Using a ribonuclease protection assay, we found that exponentially growing RMo myoblasts contained no detectable myogenin or herculin mRNA, while differentiating myoblasts contained high levels of myogenin mRNA but no herculin mRNA. There was no increase in myogenin mRNA concentration in either primary chick or RMo myotubes whose contractions were inhibited by depolarizing concentrations of potassium (K+). Thus, altered myogenin mRNA concentrations are not involved in atrophy of chick myotubes. Depolarizing concentrations of potassium inhibited spontaneous contractions in both RMo cultures and primary chick myotube cultures. However, we found that the myosin concentration of 6-d-old contracting RMo cells fed medium plus AraC was 11 +/- 3 micrograms myosin/microgram DNA, not significantly different from 12 +/- 4 micrograms myosin/microgram DNA (n = 3), the myosin concentration of noncontracting RMo cells (treated with 12 mM K+ for 6 d). Resolving how RMo cells maintained their myosin content when contraction is inhibited may be important for understanding atrophy.

Muscular Atrophy↗
NASA NTRS1996

Structural relations between collagen and mineral in bone as determined by high voltage electron microscopic tomography

Aspects of the ultrastructural interaction between collagen and mineral crystals in embryonic chick bone have been examined by the novel technique of high voltage electron microscopic tomography to obtain three-dimensional information concerning extracellular calcification in this tissue. Newly mineralizing osteoid along periosteal surfaces of mid-diaphyseal regions from normal chick tibiae was embedded, cut into 0.25 microns thick sections, and documented at 1.0 MV in the Albany AEI-EM7 high voltage electron microscope. The areas of the tissue studied contained electron dense mineral crystals associated with collagen fibrils, some marked by crystals disposed along their cylindrically shaped lengths. Tomographic reconstructions of one site with two mineralizing fibrils were computed from a 5 degrees tilt series of micrographs over a +/- 60 degrees range. Reconstructions showed that the mineral crystals were platelets of irregular shape. Their sizes were variable, measured here up to 80 x 30 x 8 nm in length, width, and thickness, respectively. The longest crystal dimension, corresponding to the c-axis crystallographically, was generally parallel to the collagen fibril long axis. Individual crystals were oriented parallel to one another in each fibril examined. They were also parallel in the neighboring but apparently spatially separate fibrils. Crystals were periodically (approximately 67 nm repeat distance) arranged along the fibrils and their location appeared to correspond to collagen hole and overlap zones defined by geometrical imaging techniques. The crystals appeared to be continuously distributed along a fibril, their size and number increasing in a tapered fashion from a relatively narrow tip containing smaller and infrequent crystals to wider regions having more densely packed and larger crystals. Defined for the first time by direct visual 3D imaging, these data describe the size, shape, location, orientation, and development of early crystals in normal bone collagen. The results suggest that platelet-shaped crystals are arranged in channels or grooves which are formed by collagen hole zones in register and that crystal sizes may exceed the dimensions of hole zones. Such data agree with those from mineral-matrix interaction in normally calcifying avian tendon obtained by similar high voltage tomographic means, but in addition they indicate a possible gradual and continuous deposition of crystals in collagen of bone unlike tendon and imply that individual collagen fibrils in local regions of osteoid are organized such that they all may be aligned in a coherent manner.

NASA Discipline Number 40-40↗
NASA NTRS1995

Fibronectin gene expression, synthesis and accumulation during in vitro differentiation of chicken osteoblasts

A well-defined chicken osteoblast culture system(18) has been used to examine fibronectin (FN) mRNA levels, synthesis, and accumulation during in vitro differentiation and matrix mineralization. Immunofluorescent staining of cells after 6 or 18 days in culture revealed that FN was initially associated with the cell surface and in partial coalignment with cytoskeletal elements while at the latter time most FN was associated with the extracellular matrix as a ubiquitous fibrillar network. Western blot analysis of total cell-associated proteins also detected FN at all culture times. However, when results were normalized to cellular DNA, FN levels increased until 12-16 and remained relatively constant thereafter. Similarly, FN synthesis as measured by [35S]-methionine labeling, and immunoprecipitation was greatest in early cultures (culture day 3) and then declined such that synthesis decreased 60% at day 18 and 94% after 24-31 days. FN mRNA levels as measured by Northern blot analysis were well correlated with FN synthesis. These results clearly show that FN is made by primary osteoblasts during their in vitro maturation. In contrast to other osteoblast markers such as alkaline phosphatase, osteocalcin, and osteopontin, whose expression increases as cells differentiate, FN accumulates in the matrix during periods of early cell growth and attachment and then remains proportional to cell number. Results with FN differ from those obtained with collagen which continues to accumulate in the extracellular matrix during osteoblast maturation. These results are consistent with FN being important for the initial attachment of early osteoblasts or osteoblast precursors to the pericellular matrix.

NASA Discipline Musculoskeletal↗
NASA NTRS1995

Differential localization of cytoplasmic myosin II isoforms A and B in avian interphase and dividing embryonic and immortalized cardiomyocytes and other cell types in vitro

Two principal isoforms of cytoplasmic myosin II, A and B (CMIIA and CMIIB), are present in different proportions in different tissues. Isoform-specific monoclonal and polyclonal antibodies to avian CMIIA and CMIIB reveal the cellular distributions of these isoforms in interphase and dividing embryonic avian cardiac, intestinal epithelial, spleen, and dorsal root ganglia cells in primary cell culture. Embryonic cardiomyocytes react with antibodies to CMIIB but not to CMIIA, localize CMIIB in stress-fiber-like-structures during interphase, and markedly concentrate CMIIB in networks in the cleavage furrow during cytokinesis. In contrast, cardiac fibroblasts localize both CMIIA and CMIIB in stress fibers and networks during interphase, and demonstrate slight and independently regulated concentration of CMIIA and CMIIB in networks in their cleavage furrows. V-myc-immortalized cardiomyocytes, an established cell line, have regained the ability to express CMIIA, as well as CMIIB, and localize both CMIIA and CMIIB in stress fibers and networks in interphase cells and in cleavage furrows in dividing cells. Conversely, some intestinal epithelial, spleen, and dorsal root ganglia interphase cells express only CMIIA, organized primarily in networks. Of these, intestinal epithelial cells express both CMIIA and CMIIB when they divide, whereas some dividing cells from both spleen and dorsal root ganglia express only CMIIA and concentrate it in their cleavage furrows. These results suggest that within a given tissue, different cell types express different isoforms of CMII, and that cells expressing either CMIIA or CMIIB alone, or simultaneously, can form a cleavage furrow and divide.

NASA Discipline Developmental Biology↗
NASA NTRS1994

Mechanical stimulation of skeletal muscle cells mitigates glucocorticoid-induced decreases in prostaglandin production and prostaglandin synthase activity

The glucocorticoid dexamethasone (Dex) induces a decline in protein synthesis and protein content in tissue cultured, avian skeletal muscle cells, and this atrophy is attenuated by repetitive mechanical stretch. Since the prostaglandin synthesis inhibitor indomethacin mitigated this stretch attenuation of muscle atrophy, the effects of Dex and mechanical stretch on prostaglandin production and prostaglandin H synthase (PGHS) activity were examined. In static cultures, 10(-8) M Dex reduced PGF2 alpha production 55-65% and PGE2 production 84-90% after 24-72 h of incubation. Repetitive 10% stretch-relaxations of non-Dex-treated cultures increased PGF2 alpha efflux 41% at 24 h and 276% at 72 h, and increased PGE2 production 51% at 24 h and 236% at 72 h. Mechanical stimulation of Dex-treated cultures increased PGF2 alpha production 162% after 24 h, returning PGF2 alpha efflux to the level of non-Dex-treated cultures. At 72 h, stretch increased PGF2 alpha efflux 65% in Dex-treated cultures. Mechanical stimulation of Dex-treated cultures also increased PGE2 production at 24 h, but not at 72 h. Dex reduced PGHS activity in the muscle cultures by 70% after 8-24 h of incubation, and mechanical stimulation of the Dex-treated cultures increased PGHS activity by 98% after 24 h. Repetitive mechanical stimulation attenuates the catabolic effects of Dex on cultured skeletal muscle cells in part by mitigating the Dex-induced declines in PGHS activity and prostaglandin production.

NASA Discipline Musculoskeletal↗
NASA NTRS1992

Colour thresholding and objective quantification in bioimaging

Computer imaging is rapidly becoming an indispensable tool for the quantification of variables in research and medicine. Whilst its use in medicine has largely been limited to qualitative observations, imaging in applied basic sciences, medical research and biotechnology demands objective quantification of the variables in question. In black and white densitometry (0-256 levels of intensity) the separation of subtle differences between closely related hues from stains is sometimes very difficult. True-colour and real-time video microscopy analysis offer choices not previously available with monochrome systems. In this paper we demonstrate the usefulness of colour thresholding, which has so far proven indispensable for proper objective quantification of the products of histochemical reactions and/or subtle differences in tissue and cells. In addition, we provide interested, but untrained readers with basic information that may assist decisions regarding the most suitable set-up for a project under consideration. Data from projects in progress at Tulane are shown to illustrate the advantage of colour thresholding over monochrome densitometry and for objective quantification of subtle colour differences between experimental and control samples.

NASA Discipline Neuroscience↗
NASA NTRS1992

Production and characterization of monoclonal antibodies to budgerigar fledgling disease virus major capsid protein VP

Eleven hybridoma cell lines producing monoclonal antibodies (MAbs) against intact budgerigar fledgling disease (BFD) virions were produced and characterized. These antibodies were selected for their ability to react with BFD virions in an enzyme-linked immunosorbent assay. Each of these antibodies was reactive in the immunofluorescent detection of BFD virus-infected cells. These antibodies immunoprecipitated intact virions and specifically recognized the major capsid protein, VP1, of the dissociated virion. The MAbs were found to preferentially recognize native BFD virus capsid protein when compared with denatured virus protein. These MAbs were capable of detecting BFD virus protein in chicken embryonated cell-culture lysates by dot-blot analysis.

Non-NASA Center↗
NASA NTRS1992

Glucocorticoid-induced skeletal muscle atrophy in vitro is attenuated by mechanical stimulation

Glucocorticoids induce rapid atrophy of fast skeletal myofibers in vivo, and either weight lifting or endurance exercise reduces this atrophy by unknown mechanisms. We examined the effects of the synthetic glucocorticoid dexamethasone (Dex) on protein turnover in tissue-cultured avian fast skeletal myofibers and determined whether repetitive mechanical stretch altered the myofiber response to Dex. In static cultures after 3-5 days, 10(-8) M Dex decreased total protein content 42-74%, total protein synthesis rates 38-56%, mean myofiber diameter 35%, myosin heavy chain (MHC) content 86%, MHC synthesis rate 44%, and fibronectin synthesis rate 29%. Repetitive 10% stretch-relaxations of the cultured myofibers for 60 s every 5 min for 3-4 days prevented 52% of the Dex-induced decrease in protein content, 42% of the decrease in total protein synthesis rate, 77% of the decrease in MHC content, 42% of the decrease in MHC synthesis rate, and 67% of the decrease in fibronectin synthesis rate. This in vitro model system will complement in vivo studies in understanding the mechanism by which mechanical activity and glucocorticoids interact to regulate skeletal muscle growth.

Non-NASA Center↗
NASA NTRS1990

Reproduction in the space environment: Part I. Animal reproductive studies

Mankind's exploration and colonization of the frontier of space will ultimately depend on men's and women's ability to live, work, and reproduce in the space environment. This paper reviews animal studies, from microorganisms to mammals, done in space or under space-simulated conditions, which identify some of the key areas which might interfere with human reproductive physiology and/or embryonic development. Those space environmental factors which impacted almost all species included: microgravity, artificial gravity, radiation, and closed life support systems. These factors may act independently and in combination to produce their effects. To date, there have been no studies which have looked at the entire process of reproduction in any animal species. This type of investigation will be critical in understanding and preventing the problems which will affect human reproduction. Part II will discuss these problems directly as they relate to human physiology.

Review, Tutorial↗
NASA NTRS2004

Experiments with osteoblasts cultured under hypergravity conditions

To understand further the role of gravity in osteoblast attachment, osteoblasts were subjected to hypergravity conditions in vitro. Scanning electron microscopy of all confluent coverslips from FPA units show that the number of attached osteoblasts was similar among gravitational levels and growth durations (~90 cells/microscopic field). Specifically, confluent 1.0 G control cultures contained an average of 91 +/- 8 cells/field, 3.3 G samples had 88 +/- 8 cells/field, and 4.0 G cultures averaged 90 +/- 7 cells/field. The sparsely plated cultures assessed by immunohistochemistry also had similar numbers of cells at each time point (l.0 G was similar to 3.3 and 4.0 G), but cell number changed from one time point to the next as those cells proliferated. Immunohistochemistry of centrifuged samples showed an increase in number (up to 160% increase) and thickness (up to 49% increase) of actin fibers, a decrease in intensity of fibronectin fluorescence (18-23% decrease) and an increase in number of vinculin bulbs (202-374% increase in number of vinculin bulbs/area). While hypergravity exposure did not alter the number of attached osteoblasts, it did result in altered actin, fibronectin, and vinculin elements, changing some aspects of osteoblast- substrate adhesion.

NASA Program Fundamental Space Biology↗
NASA NTRS1995

In vitro chick pre-cardiac explant tissue differentiation during spaceflight on SpaceHab-02

Chick precardiac tissue explants were cultured on the 8-day mission of STS-60, space shuttle Discovery. Development of in vitro cultures of precardiac chick tissue from embryo stages 5 though 8 (H-H) were initiated during orbit and were terminated after approximately fifteen hours of 37 degree C culture. Transmission electron microscopy and tritiated thymidine studies were performed postflight. No significant differences in cell proliferation were observed between flight and ground controls. Electron-microscopic studies revealed stage 8 explants were capable of differentiation during flight in a pattern which matched ground control tissues. As anticipated, stage 7 explant tissues had differentiated to a lesser extent compared to stage 8 tissues. Interestingly, stage 7 precardiac explant flight tissue differentiation was less than ground control tissue. This difference in differentiation between flight and ground cultures was enhanced in stage 6 tissues, as high levels of myofibril organization were only seen in ground controls. Other cellular components such as Golgi apparatus, junctional complexes, and mitochondria were present and appeared normal and healthy.

NASA Program NSCORT↗
NASA NTRS1991

Weightlessness and the ontogeny of vestibular function - Evidence for persistent vestibular threshold shifts in chicks incubated in space

The effect of microgravity on the embryonic development of the vestibular function was investigated by comparing the vestibular function parameters measured in 21-old chicks incubated as embryos aboard the Space Shuttle Discovery with those of age-matched synchronous controls incubated on earth. Measurements of the electrophysiological responses in both groups of chicks indicated that the exposure of embryos to weightlessness resulted in altered sensitivity of embryonic vestibular sensors. Moreover, the effect of space flight was persistent: it was still present one month after the spacecraft landed on earth.

Jones, Timothy A.↗
NASA NTRS1985

Development of otoconia in the embryonic chick (Gallus domesticus)

In the chick (Gallus domesticus) embryo, otoconium formation started first over the macula sacculi around the 4th day of incubation, and a day later over the macula utriculi. It was determined that each otoconium formed as a result of the segmentation of the immature otolithic membrane, and that the calcium responsible for otoconium calcification was incorporated into the organic matrix of each otoconium in the form of small electron-dense granules (20-150 nm in. diameter). The presence of calcium in these granules was confirmed by histochemical staining with osmic-potassium pyroantimonate, by EDTA chelation, and by X-ray micronanalysis under the electron microscope.

Fermin, C. D.↗
NASA NTRS1998

Chicken (Gallus domesticus) inner ear afferents

Neurons from the vestibular (VG) and the statoacoustic (SAG) ganglion of the chick (Gallus domesticus) were evaluated histologically and morphometrically. Embryos at stages 34 (E8 days), 39 (E13 days) and 44 (E18 days) were sacrificed and temporal bones microdissected. Specimens were embedded in JB-4 methacrylate plastic, and stained with a mixture of 0.2% toluidine blue (TB) and 0.1% basic Fuschin in 25% ethanol or with a mixture of 2% TB and 1% paraphenylenediamine (PDA) for axon and myelin measurement study. Images of the VIIIth nerve were produced by a V150 (R) color imaging system and the contour of 200-300 neuronal bodies (perikarya) was traced directly on a video screen with a mouse in real time. The cross-sectional area of VG perikarya was 67.29 micrometers2 at stage 34 (E8), 128.46 micrometers2 at stage 39 (E13) and 275.85 micrometers2 at stage 44 (E18). The cross-sectional area of SAG perikarya was 62.44 micrometers2 at stage 34 (E8), 102.05 micrometers2 at stage 39 (E13) and 165.02 micrometers2 at stage 44 (E18). A significant cross-sectional area increase of the VG perikarya between stage 39 (E13) and stage 44 (E18) was determined. We randomly measured the cross-sectional area of myelin and axoplasm of hatchling afferent nerves, and found a correspondence between axoplasmic and myelin cross-sectional area in the utricular, saccular and semicircular canal nerve branches of the nerve. The results suggest that the period between stage 34 (E8) and 39 (E13) is a critical period for afferent neuronal development. Physiological and behavioral vestibular properties of developing and maturing hatchlings may change accordingly. The results compliment previous work by other investigators and provide valuable anatomical measures useful to correlate physiological data obtained from stimulation of the whole nerve or its parts.

NASA Discipline Neuroscience↗
NASA NTRS1992

Studies of mineralization in tissue culture: optimal conditions for cartilage calcification

The optimal conditions for obtaining a calcified cartilage matrix approximating that which exists in situ were established in a differentiating chick limb bud mesenchymal cell culture system. Using cells from stage 21-24 embryos in a micro-mass culture, at an optimal density of 0.5 million cells/20 microliters spot, the deposition of small crystals of hydroxyapatite on a collagenous matrix and matrix vesicles was detected by day 21 using X-ray diffraction, FT-IR microscopy, and electron microscopy. Optimal media, containing 1.1 mM Ca, 4 mM P, 25 micrograms/ml vitamin C, 0.3 mg/ml glutamine, no Hepes buffer, and 10% fetal bovine serum, produced matrix resembling the calcifying cartilage matrix of fetal chick long bones. Interestingly, higher concentrations of fetal bovine serum had an inhibitory effect on calcification. The cartilage phenotype was confirmed based on the cellular expression of cartilage collagen and proteoglycan mRNAs, the presence of type II and type X collagen, and cartilage type proteoglycan at the light microscopic level, and the presence of chondrocytes and matrix vesicles at the EM level. The system is proposed as a model for evaluating the events in cell mediated cartilage calcification.

Non-NASA Center↗