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NASA NTRS1999

Osteoblast fibronectin mRNA, protein synthesis, and matrix are unchanged after exposure to microgravity

The well-defined osteoblast line, MC3T3-E1 was used to examine fibronectin (FN) mRNA levels, protein synthesis, and extracellular FN matrix accumulation after growth activation in spaceflight. These osteoblasts produce FN extracellular matrix (ECM) known to regulate adhesion, differentiation, and function in adherent cells. Changes in bone ECM and osteoblast cell shape occur in spaceflight. To determine whether altered FN matrix is a factor in causing these changes in spaceflight, quiescent osteoblasts were launched into microgravity and were then sera activated with and without a 1-gravity field. Synthesis of FN mRNA, protein, and matrix were measured after activation in microgravity. FN mRNA synthesis is significantly reduced in microgravity (0-G) when compared to ground (GR) osteoblasts flown in a centrifuge simulating earth's gravity (1-G) field 2.5 h after activation. However, 27.5 h after activation there were no significant differences in mRNA synthesis. A small but significant reduction of FN protein was found in the 0-G samples 2.5 h after activation. Total FN protein 27.5 h after activation showed no significant difference between any of the gravity conditions, however, there was a fourfold increase in absolute amount of protein synthesized during the incubation. Using immunofluorescence, we found no significant differences in the amount or in the orientation of the FN matrix after 27.5 h in microgravity. These results demonstrate that FN is made by sera-activated osteoblasts even during exposure to microgravity. These data also suggest that after a total period of 43 h of spaceflight FN transcription, translation, or altered matrix assembly is not responsible for the altered cell shape or altered matrix formation of osteoblasts.

Flight Experiment↗

NASA NTRS1998

Decorin and biglycan of normal and pathologic human corneas

PURPOSE: Corneas with scars and certain chronic pathologic conditions contain highly sulfated dermatan sulfate, but little is known of the core proteins that carry these atypical glycosaminoglycans. In this study the proteoglycan proteins attached to dermatan sulfate in normal and pathologic human corneas were examined to identify primary genes involved in the pathobiology of corneal scarring. METHODS: Proteoglycans from human corneas with chronic edema, bullous keratopathy, and keratoconus and from normal corneas were analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), quantitative immunoblotting, and immunohistology with peptide antibodies to decorin and biglycan. RESULTS: Proteoglycans from pathologic corneas exhibit increased size heterogeneity and binding of the cationic dye alcian blue compared with those in normal corneas. Decorin and biglycan extracted from normal and diseased corneas exhibited similar molecular size distribution patterns. In approximately half of the pathologic corneas, the level of biglycan was elevated an average of seven times above normal, and decorin was elevated approximately three times above normal. The increases were associated with highly charged molecular forms of decorin and biglycan, indicating modification of the proteins with dermatan sulfate chains of increased sulfation. Immunostaining of corneal sections showed an abnormal stromal localization of biglycan in pathologic corneas. CONCLUSIONS: The increased dermatan sulfate associated with chronic corneal pathologic conditions results from stromal accumulation of decorin and particularly of biglycan in the affected corneas. These proteins bear dermatan sulfate chains with increased sulfation compared with normal stromal proteoglycans.

Non-NASA Center↗

NASA NTRS1996

Immunohistochemical evidence of rapid extracellular matrix remodeling after iron-particle irradiation of mouse mammary gland

High-LET radiation has unique physical and biological properties compared to sparsely ionizing radiation. Recent studies demonstrate that sparsely ionizing radiation rapidly alters the pattern of extracellular matrix expression in several tissues, but little is known about the effect of heavy-ion radiation. This study investigates densely ionizing radiation-induced changes in extracellular matrix localization in the mammary glands of adult female BALB/c mice after whole-body irradiation with 0.8 Gy 600 MeV iron particles. The basement membrane and interstitial extracellular matrix proteins of the mammary gland stroma were mapped with respect to time postirradiation using immunofluorescence. Collagen III was induced in the adipose stroma within 1 day, continued to increase through day 9 and was resolved by day 14. Immunoreactive tenascin was induced in the epithelium by day 1, was evident at the epithelial-stromal interface by day 5-9 and persisted as a condensed layer beneath the basement membrane through day 14. These findings parallel similar changes induced by gamma irradiation but demonstrate different onset and chronicity. In contrast, the integrity of epithelial basement membrane, which was unaffected by sparsely ionizing radiation, was disrupted by iron-particle irradiation. Laminin immunoreactivity was mildly irregular at 1 h postirradiation and showed discontinuities and thickening from days 1 to 9. Continuity was restored by day 14. Thus high-LET radiation, like sparsely ionizing radiation, induces rapid-remodeling of the stromal extracellular matrix but also appears to alter the integrity of the epithelial basement membrane, which is an important regulator of epithelial cell proliferation and differentiation.

NASA Discipline Radiation Health↗

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 NTRS1993

The major nucleoside triphosphatase in pea (Pisum sativum L.) nuclei and in rat liver nuclei share common epitopes also present in nuclear lamins

The major nucleoside triphosphatase (NTPase) activities in mammalian and pea (Pisum sativum L.) nuclei are associated with enzymes that are very similar both biochemically and immunochemically. The major NTPase from rat liver nuclei appears to be a 46-kD enzyme that represents the N-terminal portion of lamins A and C, two lamina proteins that apparently arise from the same gene by alternate splicing. Monoclonal antibody (MAb) G2, raised to human lamin C, both immunoprecipitates the major (47 kD) NTPase in pea nuclei and recognizes it in western blot analyses. A polyclonal antibody preparation raised to the 47-kD pea NTPase (pc480) reacts with the same lamin bands that are recognized by MAb G2 in mammalian nuclei. The pc480 antibodies also bind to the same lamin-like bands in pea nuclear envelope-matrix preparations that are recognized by G2 and three other MAbs known to bind to mammalian lamins. In immunofluorescence assays, pc480 and anti-lamin antibodies stain both cytoplasmic and nuclear antigens in plant cells, with slightly enhanced staining along the periphery of the nuclei. These results indicate that the pea and rat liver NTPases are structurally similar and that, in pea nuclei as in rat liver nuclei, the major NTPase is probably derived from a lamin precursor by proteolysis.

Non-NASA Center↗

NASA NTRS1993

The 'tubulin-like' S1 protein of Spirochaeta is a member of the hsp65 stress protein family

A 65-kDa protein (called S1) from Spirochaeta bajacaliforniensis was identified as 'tubulin-like' because it cross-reacted with at least four different antisera raised against tubulin and was isolated, with a co-polymerizing 45-kDa protein, by warm-cold cycling procedures used to purify tubulin from mammalian brain. Furthermore, at least three genera of non-cultivable symbiotic spirochetes (Pillotina, Diplocalyx, and Hollandina) that contain conspicuous 24-nm cytoplasmic tubules displayed a strong fluorescence in situ when treated with polyclonal antisera raised against tubulin. Here we summarize results that lead to the conclusion that this 65-kDa protein has no homology to tubulin. S1 is an hsp65 stress protein homologue. Hsp65 is a highly immunogenic family of hsp60 proteins which includes the 65-kDa antigens of Mycobacterium tuberculosis (an active component of Freund's complete adjuvant), Borrelia, Treponema, Chlamydia, Legionella, and Salmonella. The hsp60s, also known as chaperonins, include E. coli GroEL, mitochondrial and chloroplast chaperonins, the pea aphid 'symbionin' and many other proteins involved in protein folding and the stress response.

NASA Discipline Exobiology↗

NASA NTRS1992

Identification of a nuclear localization sequence in the polyomavirus capsid protein VP2

A nuclear localization signal (NLS) has been identified in the C-terminal (Glu307-Glu-Asp-Gly-Pro-Gln-Lys-Lys-Lys-Arg-Arg-Leu318) amino acid sequence of the polyomavirus minor capsid protein VP2. The importance of this amino acid sequence for nuclear transport of newly synthesized VP2 was demonstrated by a genetic "subtractive" study using the constructs pSG5VP2 (expressing full-length VP2) and pSG5 delta 3VP2 (expressing truncated VP2, lacking amino acids Glu307-Leu318). These constructs were transfected into COS-7 cells, and the intracellular localization of the VP2 protein was determined by indirect immunofluorescence. These studies revealed that the full-length VP2 was localized in the nucleus, while the truncated VP2 protein was localized in the cytoplasm and not transported to the nucleus. A biochemical "additive" approach was also used to determine whether this sequence could target nonnuclear proteins to the nucleus. A synthetic peptide identical to VP2 amino acids Glu307-Leu318 was cross-linked to the nonnuclear proteins bovine serum albumin (BSA) or immunoglobulin G (IgG). The conjugates were then labeled with fluorescein isothiocyanate and microinjected into the cytoplasm of NIH 3T6 cells. Both conjugates localized in the nucleus of the microinjected cells, whereas unconjugated BSA and IgG remained in the cytoplasm. Taken together, these genetic subtractive and biochemical additive approaches have identified the C-terminal sequence of polyoma-virus VP2 (containing amino acids Glu307-Leu318) as the NLS of this protein.

Non-NASA Center↗

NASA NTRS1993

Fluorescent-Antibody Measurement Of Cancer-Cell Urokinase

Combination of laboratory techniques provides measurements of amounts of urokinase in and between normal and cancer cells. Includes use of fluorescent antibodies specific against different forms of urokinase-type plasminogen activator, (uPA), fluorescence microscopy, quantitative analysis of images of sections of tumor tissue, and flow cytometry of different uPA's and deoxyribonucleic acid (DNA) found in suspended-tumor-cell preparations. Measurements provide statistical method for indicating or predicting metastatic potentials of some invasive tumors. Assessments of metastatic potentials based on such measurements used in determining appropriate follow-up procedures after surgical removal of tumors.

Morrison, Dennis R.↗

NASA NTRS2007

Bench-Top Antigen Detection Technique that Utilizes Nanofiltration and Fluorescent Dyes which Emit and Absorb Light in the Near Infrared

This article discusses the development of a bench-top technique to detect antigens in fluids. The technique involves the use of near infrared NIR fluorescent dyes conjugated to antibodies, centrifugation, nanofilters, and spectrometry. The system used to detect the antigens utilizes a spectrometer, fiber optic cables, NIR laser, and laptop computer thus making it portable and ideally suited for desk top analysis. Using IgM as an antigen and the secondary antibody, anti-IgM conjugated to the near infrared dye, IRDye (trademark) 800, for detection, we show that nanofiltration can efficiently and specifically separate antibody-antigen complexes in solution and that the complexes can be detected by a spectrometer and software using NIR laser excitation at 778 nm and NIR dye offset emission at 804 nm. The peak power detected at 778 nm for the excitation emission and at 804 nm for the offset emission is 879 pW (-60.06 dBm) and 35.7 pW (-74.5 dBm), respectively.

Varaljay-Spence, Vanessa A.↗

NASA NTRS1980

Immunomicrospheres - Reagents for cell labeling and separation

Immunomicrospheres are specially designed microscopic particles that have antibodies or similar molecules chemically bound to their surfaces. The antibody-coated microspheres react in a highly specific way with target cells, viruses, or other antigenic agents. Immunomicrospheres may be synthesized so that they incorporate compounds that are highly radioactive, intensely fluorescent, magnetic, electron opaque, highly colored, or pharmacologically active. These various types of microspheres may be coated with pure, highly specific monoclonal antibodies obtained by the new hybridoma cell cloning techniques or with conventional antibody preparations. Some of the many present and potential applications for these new reagents are (1) new types of radioimmune or immunofluorescent assays, (2) improved fluorescence microscopy, (3) separation of cells on the basis of the fluorescent, electrophoretic, or magnetic properties of bound immunomicrospheres, (4) markers for use in several types of electron or standard light microscopy, and (5) delivery of lethal compouds to specific undesirable living cells. The combination of the various new types of synthetic microspheres and the newly available homogeneous antibodies offers new opportunities in research, diagnosis, and therapy.

Rembaum, A.↗

NASA NTRS1978

Fluorescent microspheres

Latex particles with attached antibodies have potential biochemical and environmental applications. Human red blood cells and lymphocytes have been labeled with fluorescent microspheres by either direct or indirect immunological technique. Immunolatex spheres can also be used for detecting and localizing specific cell surface receptors. Hormones and toxins may also be bondable.

Rembaum, A.↗

NASA NTRS2007

Benchtop Detection of Proteins

A process, and a benchtop-scale apparatus for implementing the process, have been developed to detect proteins associated with specific microbes in water. The process and apparatus may also be useful for detection of proteins in other, more complex liquids. There may be numerous potential applications, including monitoring lakes and streams for contamination, testing of blood and other bodily fluids in medical laboratories, and testing for microbial contamination of liquids in restaurants and industrial food-processing facilities. A sample can be prepared and analyzed by use of this process and apparatus within minutes, whereas an equivalent analysis performed by use of other processes and equipment can often take hours to days. The process begins with the conjugation of near-infrared-fluorescent dyes to antibodies that are specific to a particular protein. Initially, the research has focused on using near-infrared dyes to detect antigens or associated proteins in solution, which has proven successful vs. microbial cells, and streamlining the technique in use for surface protein detection on microbes would theoretically render similar results. However, it is noted that additional work is needed to transition protein-based techniques to microbial cell detection. Consequently, multiple such dye/antibody pairs could be prepared to enable detection of multiple selected microbial species, using a different dye for each species. When excited by near-infrared light of a suitable wavelength, each dye fluoresces at a unique longer wavelength that differs from those of the other dyes, enabling discrimination among the various species. In initial tests, the dye/antibody pairs are mixed into a solution suspected of containing the selected proteins, causing the binding of the dye/antibody pairs to such suspect proteins that may be present. The solution is then run through a microcentrifuge that includes a membrane that acts as a filter in that it retains the dye/antibody/protein complexes while allowing any remaining unbound dye/antibody pairs to flow away. The retained dye/antibody/protein complexes are transferred to a cuvette, wherein they are irradiated with light from a miniature near-infrared laser delivered via a fiber-optic cable. The resulting fluorescence from the dye(s) is measured by use of a miniature spectrometer, the output of which is digitized, then analyzed by laptop computer. The software running in the computer identifies the protein species by the wavelengths of their spectral peaks and determines the amounts of the proteins, and thus, one day, microbes of the various species from the intensities of the peaks. The abovementioned removal of the unbound dye/antibody pairs during centrifugation prevents false positive readings. The process proves successful in detecting proteins in solution and thus can now be employed for use in microbe detection.

Scardelletti, Maximilian C.↗

NASA NTRS2009

Benchtop Antigen Detection Technique using Nanofiltration and Fluorescent Dyes

The designed benchtop technique is primed to detect bacteria and viruses from antigenic surface marker proteins in solutions, initially water. This inclusive bio-immunoassay uniquely combines nanofiltration and near infrared (NIR) dyes conjugated to antibodies to isolate and distinguish microbial antigens, using laser excitation and spectrometric analysis. The project goals include detecting microorganisms aboard the International Space Station, space shuttle, Crew Exploration Vehicle (CEV), and human habitats on future Moon and Mars missions, ensuring astronaut safety. The technique is intended to improve and advance water contamination testing both commercially and environmentally as well. Lastly, this streamlined technique poses to greatly simplify and expedite testing of pathogens in complex matrices, such as blood, in hospital and laboratory clinics.

Scardelletti, Maximilian C.↗

NASA NTRS2013

Medicine Delivery Device with Integrated Sterilization and Detection

Sterile delivery devices can be created by integrating a medicine delivery instrument with surfaces that are coated with germicidal and anti-fouling material. This requires that a large-surface-area template be developed within a constrained volume to ensure good contact between the delivered medicine and the germicidal material. Both of these can be integrated using JPL-developed silicon nanotip or cryo-etch black silicon technologies with atomic layer deposition (ALD) coating of specific germicidal layers. The application of semiconductor processing techniques and technologies to the problems of fluid manipulation and delivery has enabled the integration of chemical, electrical, and mechanical manipulation of samples all within a single microfluidic device. This approach has been successfully applied at JPL to the automated processing, detection, and analysis of minute quantities (parts per trillion level) of biomaterials to develop instruments for in situ exploration or extraterrestrial bodies. The same nanofabrication techniques that are used to produce a microfluidics device are also capable of synthesizing extremely high-surface-area templates in precise locations, and coating those surfaces with conformal films to manipulate their surface properties. This methodology has been successfully applied at JPL to produce patterned and coated silicon nanotips (also known as black silicon) to manipulate the hydrophilicity of surfaces to direct the spreading of fluids in microdevices. JPL's ALD technique is an ideal method to produce the highly conformal coatings required for this type of application. Certain materials, such as TiO2, have germicidal and anti-fouling properties when they are illuminated with UV light. The proposed delivery device contacts medicine with this high-surface-area black silicon surface coated with a thin-film germicidal deposited conformally with ALD. The coating can also be illuminated with ultraviolet light for the purpose of sterilization or identification of the medicine itself. This constrained volume that is located immediately prior to delivery into a patient, ensures that the medicine delivery device is inherently sterile. An additional benefit to integrating a high-surface-area template within the fluid channel of a medicine delivery device is that one can envision a number of different functional coatings that could facilitate the capture and analysis of either microbial contaminants or the medicine itself. For example, one could attach antibodies or some other binding agent with a specific affinity to the silicon nanotip template. Once a target molecule or microbe is bound to the high-surface- area template, one could use an optical analytical technique such as fluorescence or adsorption to determine the identity and potentially the concentration of the species of interest. By illuminating the bound species from the back, it may also be possible to probe only the molecules with an evanescent wave, making detection of the species from the front side of the device much simpler.

Shearn, Michael J.↗

NASA NTRS1982

Rapid diagnostic methods for influenza virus in clinical specimens - A comparative study

A comparison of five rapid viral diagnostic techniques for identifying influenza virus in nasopharyngeal aspirates has been made on patients with influenza-like illnesses. Initial results with immune electron microscopy were positive in only one of 11 specimens from which virus was isolated and further work abandoned. Four other rapid tests were carried out on 39 specimens from which influenza virus had been isolated in tissue culture in 28. Of these 28 specimens yielding virus, 24 (85.7 percent) were positive by an indirect fluorescent antibody test (IFAT) on nasopharyngeal cells, 18 (64.3 percent) by enzyme-linked immunosorbent assay (ELISA), 19 (67.8 percent) by enzyme-linked fluorescent assay (ELFA), and 26 (92.8 percent) by a rapid tissue culture amplification method (TCA) in a continuous Rhesus monkey kidney line (LLC-MK2) with identification of virus by fluorescent antibody. In terms of sensitivity, simplicity, and rapidity, a combination of the IFAT and TCA methods seems to be very useful.

Evans, A. S.↗

NASA NTRS2002

Demonstration of four immunoassay formats using the array biosensor

The ability of a fluorescence-based array biosensor to measure and quantify the binding of an antigen to an immobilized antibody has been demonstrated using the four different immunoassay formats: direct, competitive, displacement, and sandwich. A patterned array of antibodies specific for 2,4,6-trinitrotoluene (TNT) immobilized onto the surface of a planar waveguide and used to measure signals from different antigen concentrations simultaneously. For direct, competitive, and displacement assays, which are one-step assays, measurements were obtained in real time. Dose-response curves were calculated for all four assay formats, demonstrating the array biosensor's ability to quantify the amount of antigen present in solution.

NASA Discipline Life Sciences Technologies↗

NASA NTRS2001

Biotechnology Approaches to Life Detection

The direct detection of organic biomarkers for living or fossil microbes on Mars by an in situ instrument is a worthy goal for future lander missions. Several new and innovative biotechnology approaches are being explored. Firstly we have proposed an instrument based on immunological reactions to specific antibodies to cause activation of fluorescent stains. Antibodies are raised or acquired to a variety of general and specific substances that might be in Mars soil. These antibodies are then combined with various fluorescent stains and applied to micron sized numbered spots on a small (2-3 cm) test plate where they become firmly attached after freeze drying. Using technology that has been developed for gene mining in DNA technology up to 10,000 tests per square inch can now be applied to a test plate. On Mars or the planet/moon of interest, a sample of soil from a trench or drill core is extracted with water and/or an organic solvent and ultrasonication and then applied to the test plate. Any substance, which has an antibody on the test plate, will react with its antibody and activate its fluorescent stain. At the moment a small UV light source will illuminate the test plate, which is observed with a small CCD camera, although other detection systems will be applied. The numbered spots that fluoresce indicate the presence of the tested-for substance, and the intensity indicates relative amounts. Furthermore with up to a thousand test plates available false positives and several variations of antibody can also be screened for. The entire instrument can be quite small and light, on the order of 10 cm in each dimension. A possible choice for light source may be small UV lasers at several wavelengths. Some of the wells or spots can contain simply standard fluorescent stains used to detect live cells, dead cells, DNA, etc. The stains in these spots may be directly activated, with no antibodies being necessary. The proposed system will look for three classes of biomarkers: those from extant life, such as DNA, those from extinct life such as hopanes, and those from organic compounds not necessarily associated with life such as PAHs, rocket exhaust contamination and other a/pre-biotic chemicals. Both monoclonal and polyclonal antibodies can be used. Monoclonal antibodies react with a very specific compound, but polyclonal antibodies may react to any of a whole family of compounds. Furthermore the technique of phage display to raise antibodies against classically non-antigenic molecules is also being considered. Additional information is contained in the original extended abstract.

Steele, Andrew↗

NASA NTRS2003

Physical association and functional interaction between beta1 integrin and CD98 on human T lymphocytes

CD98 is a cell surface protein previously characterized as a cell activation marker, an amino acid transporter, and has recently been implicated in integrin-related functions. Integrins are cell surface proteins, important for homotypic cell aggregation, cell adhesion, and coactivation of T lymphocytes. We have previously shown that the anti-CD98 mAb 80A10, when coimmobilized with anti-CD3 mAb OKT3, is able to mediate human T cell coactivation that is inhibited by anti-beta1 integrin specific mAb 18D3. These results indicated a functional association of CD98 and beta1 integrin signaling but left open the question of a physical association. We now show the induction of homotypic aggregation through CD98 among human T cells and this aggregation was inhibited by anti-beta1 integrin mAb. Therefore, CD98-dependent lymphocyte proliferation and adhesion may involve integrins. Competitive binding assays and fluorescence colocalization analysis suggested that CD98 and beta1 integrin were physically associated. Differential extraction techniques and immunoprecipitations provided the first evidence that the alpha4beta1 integrin and CD98 are specifically associated on human T lymphocytes.

NASA Discipline Cell Biology↗