A sensitive and specific isotopic assay for the estimation of tyrosine transaminase.
Tyrosine transaminase activity estimation by radioactive isotopic assay method, discussing application to rat liver and other organs
Larin, F.↗
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Z-DNA binding protein from chicken blood nuclei
A protein (Z alpha) that appears to be highly specific for the left-handed Z-DNA conformer has been identified in chicken blood nuclear extracts. Z alpha activity is measured in a band-shift assay by using a radioactive probe consisting of a (dC-dG)35 oligomer that has 50% of the deoxycytosines replaced with 5-bromodeoxycytosine. In the presence of 10 mM Mg2+, the probe converts to the Z-DNA conformation and is bound by Z alpha. The binding of Z alpha to the radioactive probe is specifically blocked by competition with linear poly(dC-dG) stabilized in the Z-DNA form by chemical bromination but not by B-form poly(dC-dG) or boiled salmon-sperm DNA. In addition, the binding activity of Z alpha is competitively blocked by supercoiled plasmids containing a Z-DNA insert but not by either the linearized plasmid or by an equivalent amount of the parental supercoiled plasmid without the Z-DNA-forming insert. Z alpha can be crosslinked to the 32P-labeled brominated probe with UV light, allowing us to estimate that the minimal molecular mass of Z alpha is 39 kDa.
NASA Discipline Exobiology↗
Evidence for zinc binding by two structural proteins of Plodia interpunctella granulosis virus
Workers in our laboratory previously reported the possibility of cation involvement in the in vitro dissociation of the Plodia interpunctella granulosis virus nucleocapsids (K. A. Tweeten, L. A. Bulla, Jr., and R. A. Consigli, J. Virol. 33:866-876, 1980; M. E. Wilson and R. A. Consigli, Virology 143:516-525, 1985). The current study found zinc associated with both granulosis virus nucleocapsids and granulin by atomic absorption analysis. A blotting assay with 65Zn2+ specifically identified the radioactive cation as binding to two viral structural proteins, granulin and VP12. These findings indicate that zinc may have a critical role in maintaining virus stability.
NASA Discipline Cell Biology↗
A quarantine protocol for analysis of returned extraterrestrial samples
A protocol is presented for the analysis at an earth-orbiting quarantine facility of return samples of extraterrestrial material that might contain (nonterrestrial) life forms. The protocol consists of a series of tests designed to determine whether the sample, conceptualized as a 1-kg sample of Martian soil, is free from nonterrestrial biologically active agents and so may safely be sent to a terrestrial containment facility, or it exhibits biological activity requiring further (second-order) testing outside the biosphere. The first-order testing procedure seeks to detect the presence of any replicating organisms or toxic substances through a series of experiments including gas sampling, analysis of radioactivity, stereomicroscopic inspection, chemical analysis, microscopic examination, the search for metabolic products under growth conditions, microbiologicl assays, and the challenge of cultured cells with any agents found or with the extraterrestrial material as is. Detailed plans for the second-order testing would be developed in response to the actual data received from primary testing.
Bagby, J. R.↗
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.↗
Measurement of transpiration in Pinus taeda L. and Liquidambar styraciflua L. in an environmental chamber using tritiated water
Transpiration rates of loblolly pine (Pinus taeda L.) and sweetgum (Liquidambar styraciflua L.) were measured at two different atmospheric water vapor pressure deficits (V.P.D.) in a controlled environment growth chamber using tritiated water as a tracer. The trees were maintained in a sealed plant bed containing a hydroponic nutrient solution into which labeled water (spike) was introduced. Samples of leaves, chamber air, spiked nutrient solution and control water were assayed for ratio-activity using liquid scintillation techniques to determine transpiration rates. The transpiration rate of sweetgum in ml./hr./gm. (4.95) was found to be 5 times greater than that of loblolly pine (1.03) at 1.84 V.P.D. and 8 times greater at 6.74 V.P.D. (15.99 for sweetgum vs. 2.19 for pine). Transpiration (based on measurements of leaf radioactivity) in both species rose with increasing deficit; however sweetgum increased its output by 3 times while pine only doubled its rate. Cyclical changes in transpiration rates were noted in both species; the sweetgum cycle required a 6 hour interval whereas the pine cycle required a 9 hour interval.
Levy, G. F.↗
In vivo synthesis of phosphatidylcholine in rat brain via the phospholipid methylation pathway
The in vivo synthesis of brain phosphatidylcholine (PC) by the methylation of phosphatidylethanolamine (PE) was examined. (H-3)methyl)methionine was infused i.c.v., by indwelling cannula, and brain samples were taken 0.5-18 h thereafter and assayed for (H-3)PC, as well as for its biosynthetic intermediates (H-3)phosphatidyl monomethylethanolamine ((H-3)PMME) and (H-3)phosphatidyl dimethylethanolamine ((H-3)PDME), and for (H-3)lysophosphatidylcholine ((H-3)LPC) and S-(H-3)adenosylmethionine ((H-3)SAM). Most of the (H-3)PC (79-94 percent) was present ipsilateral to the infusion site; indicating that the radioactivity in the (H-3)PC was primarily of intracerebral origin, and not taken up from the blood. Moreover, only very low levels of (H-3)PC were attained in brains of animals receiving (H-3)methionine i.p. and these levels were symmetrically distributed. (H-3)PMME and (H-3)PDME turned over with apparent half-lives of 2.2 h and 2.4 h. In contrast, the accumulation of brain (H-3)PC was biphasic, suggesting the existence of two pools, the more labile of which turned over rapidly (t(sub 1/2) = 5 h) and was formed for as long as (H-3)PMME and (H-3)PDME are present in the brain, and another, which was distinguishable only at 18 h after the (H-3)methionine infusion. (The latter pool may have been synthesized from (H-3)choline that was released via the hydrolysis of some of the brain (H-3)PC previously formed by the methylation of PE.) Subcellular fractionation of brain tissue obtained after in vivo labelling with (H-3)methionine revealed that mitochondrial PC had the highest specific radioactivity (dpm per micromol total lipid phosphorus), and myelin the least. These observations affirm that rat brain does synthesize PC in vivo by methylating PE, and the technique provides an experimental system which may be useful for examining the physiological regulation of this process.
Lakher, Michael↗
Automated life-detection experiments for the Viking mission to Mars
As part of the Viking mission to Mars in 1975, an automated set of instruments is being built to test for the presence of metabolizing organisms on that planet. Three separate modules are combined in this instrument so that samples of the Martian surface can be subjected to a broad array of experimental conditions so as to measure biological activity. The first, the Pyrolytic Release Module, will expose surface samples to a mixture of C-14O and C-14O2 in the presence of Martian atmosphere and a light source that simulates the Martian visible spectrum. The assay system is designed to determine the extent of assimilation of CO or CO2 into organic compounds. The Gas Exchange Module will incubate surface samples in a humidified CO2 atmosphere. At specified times, portions of the incubation atmosphere will be analyzed by gas chromatography to detect the release or uptake of CO2 and several additional gases. The Label Release Module will incubate surface samples with a dilute aqueous solution of simple radioactive organic substrates in Martian atmosphere, and the gas phase will be monitored continuously for the release of labeled CO2.
Klein, H. P.↗
Techniques for determining total body water using deuterium oxide
The measurement of total body water (TBW) is fundamental to the study of body fluid changes consequent to microgravity exposure or treatment with microgravity countermeasures. Often, the use of radioactive isotopes is prohibited for safety or other reasons. It was selected and implemented for use by some Johnson Space Center (JCS) laboratories, which permitted serial measurements over a 14 day period which was accurate enough to serve as a criterion method for validating new techniques. These requirements resulted in the selection of deuterium oxide dilution as the method of choice for TBW measurement. The development of this technique at JSC is reviewed. The recommended dosage, body fluid sampling techniques, and deuterium assay options are described.
Bishop, Phillip A.↗
Measuring Surface Bulk Elemental Composition on Venus
The extreme surface environment (462 C, 93 bars pressure) of Venus makes subsurface measurements of its bulk elemental composition extremely challenging. Instruments landed on the surface of Venus must be enclosed in a pressure vessel. The high surface temperatures also require a thermal control system to keep the instrumentation temperatures within their operational range for as long as possible. Since Venus surface probes can currently operate for only a few hours, it is crucial that the lander instrumentation be able to make statistically significant measurements in a short time. An instrument is described that can achieve such a measurement over a volume of thousands of cubic centimeters of material by using high energy penetrating neutron and gamma radiation. The instrument consists of a Pulsed Neutron Generator (PNG) and a Gamma-Ray Spectrometer (GRS). The PNG emits isotropic pulses of 14.1 MeV neutrons that penetrate the pressure vessel walls, the dense atmosphere and the surface rock. The neutrons induce nuclear reactions in the rock to produce gamma rays with energies specific to the element and nuclear process involved. Thus the energies of the detected gamma rays identify the elements present and their intensities provide the abundance of each element. The GRS spectra are analyzed to determine the Venus elemental composition from the spectral signature of individual major, minor, and trace radioactive elements. As a test of such an instrument, a Schlumberger Litho Scanner oil well logging tool was used in a series of experiments at NASA's Goddard Space Flight Center. The Litho Scanner tool was mounted above large (1.8 m x 1.8 m x.9 m) granite and basalt monuments and made a series of one-hour elemental composition measurements in a planar geometry more similar to a planetary lander measurement. Initial analysis of the results shows good agreement with target elemental assays
Venus↗
A miniaturized fibrinolytic assay for plasminogen activators
This report describes a micro-clot lysis assay (MCLA) for evaluating fibrinolytic activity of plasminogen activators (PA). Fibrin clots were formed in wells of microtiter plates. Lysis of the clots by PA, indicated by change in turbidity (optical density, OD), was monitored with a microplate reader at five minutes intervals. Log-log plots of PA dilution versus endpoint, the time at which the OD value was halfway between the maximum and minimum value for each well, were linear over a broad range of PA concentrations (2-200 International units/ml). The MCLA is a modification and miniaturization of well established fibrinolytic methods. The significant practical advantages of the MCLA are that it is a simple, relatively sensitive, non-radioactive, quantitative, kinetic, fibrinolytic micro-technique which can be automated.
NASA Discipline Cell Biology↗
Gibberellins and gravitropism in maize shoots: endogenous gibberellin-like substances and movement and metabolism of [3H]Gibberellin A20
[3H]Gibberellin A20 (GA20) of high specific radioactivity (49.9 gigabecquerel per millimole) was applied equilaterally in a ring of microdrops to the internodal pulvinus of shoots of 3-week-old gravistimulated and vertical normal maize (Zea mays L.), and to a pleiogravitropic (prostrate) maize mutant, lazy (la). All plants converted the [3H]GA20 to [3H]GA1- and [3H]GA29-like metabolites as well as to several metabolites with the partitioning and chromatographic behavior of glucosyl conjugates of [3H]GA1, [3H]GA29, and [3H]GA8. The tentative identification of these putative [3H]GA glucosyl conjugates was further supported by the release of the free [3H]GA moiety after cleavage with cellulase. Within 12 hours of the [3H]GA20 feed, there was a significantly higher proportion of total radioactivity in lower than in upper halves of internode and leaf sheath pulvini in gravistimulated normal maize. Further, there was a significantly higher proportion of putative free GA metabolites of [3H]GA20, especially [3H]GA1, in the lower halves of normal maize relative to upper halves. The differential localization of the metabolites between upper and lower halves was not apparent in the pleiogravitropic mutant, la. Endogenous GA-like substances were also examined in gravistimulated maize shoots. Forty-eight hours after gravistimulation of 3-week-old maize seedlings, endogenous free GA-like substances in upper and lower leaf sheath and internode pulvini halves were extracted, chromatographed, and bioassayed using the "Tanginbozu" dwarf rice microdrop assay. Lower halves contained consistently higher total levels of GA-like activity. The qualitative elution profile of GA-like substances differed consistently, upper halves containing principally a GA20-like substance and lower halves containing principally a GA20-like substance and lower halves containing mainly GA1-like and GA19-like substances. Gibberellins A1 (10 nanograms per gram) and A20 (5 nanograms per gram) were identified from these lower leaf sheath pulvini by capillary gas chromatography-selected ion monitoring. Results from all of these experiments are consistent with a role for GAs in the differential shoot growth that follows gravitropism, although the results do not eliminate the possibility that the redistribution of GAs results from the gravitropic response.
NASA Discipline Plant Biology↗
Turnover of Biogenic Amines in the Hypothalamus of Rats during Pyrogen Fever
Many pharmacological studies have implicated the biogenic amines in the hypothalamus as playing a role in the production of fever, but few investigations of endogenous neurochemicals have been made during fever. Turnover rates of transmitters utilizing radioactive precursors may be one of the most accurate measurements of activity in brain regions. The present study was designed to measure the turnover of 5-hydroxytryptamine (5-HT) norepinephrine (NE) and dopamine (DA) in the hypothalamus of rats during pyrogen fever. Salmonella typhosa (Wyeth, 8 units) was previously found in our laboratory to produce a significant hyperthermia in most rats by 2.5 hours. This pyrogen (N = l2) or saline control (N = 8) was injected intraperitoneally and the rats killed 2.75 hours later. Rectal temperatures (Tr) were monitored continuously with thermocouples taped to the tail and recorded automatically every 3 minutes. Half of each group received an injection of radioactive precursors, (3)H-tryptophan (0.5 mCi) and (3)H-tryptophan (1.0 mCi), via an indwelling jugular catheter 60 minutes before killing, and the other half at 90 minutes. The rats were killed by near freezing in liquid nitrogen and the brains dissected in the cold. Turnover was measured by the method of Lane (Life Sci 21, 1101, 1977). At the time of killing most of the pyrogen group showed a significant (p < .02) increase (mean +/- s.e.m.) in Tr above pre injection levels (0.75 +/- 0.13 C, N = 10). The saline group showed no change (-0.025 +/- 0.16, N = 8), and the difference between groups was also significant. No significant differences were found in the levels of the amines between the pyrogen and saline groups. A significant difference was found in the specific activity of NE between the 60 minute pyrogen and saline groups (4.41 +/- 0.41 vs 2.6 +/- 0.51 dpm/pmole) but no change in turnover. This suggests an increased accumulation of (3)H-NE in the pyrogen group, but no change in utilization. An increased turnover of DA for the pyrogen group (44.5 vs 19.2 pmole/mg protein/hr) was found. However, DA is mainly a precursor in the hypothalamus and measurement was near the limit of sensitivity for the assay; these limitations Must be considered in interpreting this data. The most significant finding was an increase in the turnover of 5-HT in the pyrogen group (41.3 vs 7.3 pmole/mg protein/hr), indicating a resynthetization rate of 77% of the total pool per hour. These results suggest that at the time point measured, an increase in the utilization of 5-HT in the hypothalamus is-correlated with pyrogen fever.
Penn, P. E.↗