Continuous in vitro evolution of catalytic function.

A population of RNA molecules that catalyze the template-directed ligation of RNA substrates was made to evolve in a continuous manner in the test tube. A simple serial transfer procedure was used to achieve approximately 300 successive rounds of catalysis and selective amplification in 52 hours. During this time, the population size was maintained against an overall dilution of 3 x 10(298). Both the catalytic rate and amplification rate of the RNAs improved substantially as a consequence of mutations that accumulated during the evolution process. Continuous in vitro evolution makes it possible to maintain laboratory "cultures" of catalytic molecules that can be perpetuated indefinitely.

A simple procedure for constructing 5'-amino-terminated oligodeoxynucleotides in aqueous solution.

A rapid method for the synthesis of oligodeoxynucleotides (ODNs) terminated by 5'-amino-5'-deoxythymidine is described. A 3'-phosphorylated ODN (the donor) is incubated in aqueous solution with 5'-amino- 5'-deoxythymidine in the presence of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC), extending the donor by one residue via a phosphoramidate bond. Template- directed ligation of the extended donor and an acceptor ODN, followed by acid hydrolysis, yields the acceptor ODN extended by a single 5'-amino-5'-deoxythymidine residue at its 5'terminus.

The effect of space flight on monoclonal antibody synthesis in a hybridoma mouse cell line.

The hybridoma cell line, 3G10G5, producing a monoclonal antibody to the major capsid protein VP1 from the avian polyomavirus budgerigar fledgling disease virus, was produced from a Balb/C mouse. This cell line was used to test the effects of microgravity on cellular processes, specifically protein synthesis. A time course study utilizing incorporation of [35S]methionine into newly synthesized monoclonal antibody was performed on STS-77. After 5.5 days, it was observed that cell counts for the samples exposed to microgravity were lower than those of ground-based samples. However, radiolabel incorporation of the synthesized monoclonal antibody was similar in both orbiter and ground control samples. Overall, microgravity does not seem to have an effect on this cell line's ability to synthesize IgG protein.

Composition of the carbohydrate granules of the cyanobacterium, Cyanothece sp. strain ATCC 51142.

Cyanothece sp. strain ATCC 51142 is an aerobic, unicellular, diazotrophic cyanobacterium that temporally separates O2-sensitive N2 fixation from oxygenic photosynthesis. The energy and reducing power needed for N2 fixation appears to be generated by an active respiratory apparatus that utilizes the contents of large interthylakoidal carbohydrate granules. We report here on the carbohydrate and protein composition of the granules of Cyanothece sp. strain ATCC 51142. The carbohydrate component is a glucose homopolymer with branches every nine residues and is chemically identical to glycogen. Granule-associated protein fractions showed temporal changes in the number of proteins and their abundance during the metabolic oscillations observed under diazotrophic conditions. There also were temporal changes in the protein pattern of the granule-depleted supernatant fractions from diazotrophic cultures. None of the granule-associated proteins crossreacted with antisera directed against several glycogen-metabolizing enzymes or nitrogenase, although these proteins were tentatively identified in supernatant fractions. It is suggested that the granule-associated proteins are structural proteins required to maintain a complex granule architecture.

Diminished forearm vasomotor response to central hypervolemic loading in aerobically fit individuals.

The aim of this study was to test the hypothesis that cardiopulmonary baroreflex control of forearm vascular resistance (FVR) during central hypervolemic loading was less sensitive in exercise trained high fit individuals (HF) compared to untrained average fit individuals (AF). Eight AF (age: 24 +/- 1 yr and weight: 78.9 +/- 1.7 kg) and eight HF (22 +/- 1 yr 79.5 +/- 2.4 kg) voluntarily participated in the investigation. Maximal aerobic power (determined on a treadmill), plasma volume and blood volume (Evans blue dilution method) were significantly greater in the HF than AF (60.8 +/- 0.7 vs. 41.2 +/- 1.9 ml.kg-1.min-1, 3.96 +/- 0.17 vs 3.36 +/- 0.08 1, and 6.33 +/- 0.23 vs 5.28 +/- 0.13 1). Baseline heart rate (HR), central venous pressure (CVP), mean arterial pressure (MAP, measured by an intraradial catheter or a Finapres finger cuff), forearm blood flow (FBF, plethysmography), and FVR, calculated from the ratio (MAP-CVP)/FBF, were not different between the HF and the AF. Lower body negative pressure (LBNP, -5, -10, -15, and -20 torr) and passive leg elevation (LE, 50 cm) combined with lower body positive pressure (LBPP, +5, +10, and +20 torr) were utilized to elicit central hypovolemia and hypervolemia, respectively. Range of CVP (from LBNP to LE+LBPP) was similar in the AF (from -3.9 to +1.9 mm Hg) and HF (from -4.0 to +2.2 mm Hg). However, FVR/CVP was significantly less in the HF (-1.8 +/- 0.1 unit.mm Hg-1) than AF (-34 +/- 0.1 unit.mm Hg-1). The FVR decrease in response to increase in CVP was significantly diminished in the HF (-1.46 +/- 0.45 unit.mm Hg-1) compared to the AF (-4.40 +/- 0.97 unit.mm Hg-1), and during LBNP induced unloading the FVR/CVP of the HF (-2.01 +/- 0.49 unit.mm Hg-1) was less (P < 0.08) than the AF (-3.28 +/- 0.69 unit.mm Hg-1). We concluded that the cardiopulmonary baroreceptor mediated FVR reflex response was significantly less sensitive to changes in CVP in individuals who practice exercise training.

Orthostatic intolerance after spaceflight.

Orthostatic intolerance occurs commonly after spaceflight, and important aspects of the underlying mechanisms remain unclear. We studied 14 individuals supine and standing before and after three space shuttle missions of 9-14 days. After spaceflight, 9 of the 14 (64%) crew members could not complete a 10-min stand test that all completed preflight. Pre- and postflight supine hemodynamics were similar in both groups except for slightly higher systolic and mean arterial pressures preflight in the finishers [15 +/- 3.7 and 8 +/- 1.2 (SE) mmHg, respectively; P < 0.05]. Postflight, finishers and nonfinishers had equally large postural reductions in stroke volume (-47 +/- 3.7 and -48 +/- 3.3 ml, respectively) and increases in heart rate (35 +/- 6.6 and 51 +/- 5.2 beats/min, respectively). Cardiac output during standing was also similar (3.6 +/- 0.4 and 4.1 +/- 0.3 l/min, respectively). However, the finishers had a greater postflight vasoconstrictor response with higher total peripheral resistance during standing (22.3 +/- 1.2 units preflight and 29.4 +/- 2.3 units postflight) than did the nonfinishers (20.1 +/- 1.1 units preflight and 19.9 +/- 1.4 units postflight). We conclude that 1) the primary systemic hemodynamic event, i.e., the postural decrease in stroke volume, was similar in finishers and nonfinishers and 2) the heart rate response and cardiac output during standing were not significantly different, but 3) the postural vasoconstrictor response was significantly greater among the finishers (P < 0.01).

Identifying yield-optimizing environments for two cowpea breeding lines by manipulating photoperiod and harvest scenario.

Photoperiod and harvest scenario of cowpea (Vigna unguiculata L. Walp) canopies were manipulated to optimize productivity for use in future controlled ecological life-support systems. Productivity was measured by edible yield rate (EYR:g m-2 day-1), shoot harvest index (SHI: g edible biomass [g total shoot dry weight]), and yield-efficiency rate (YER:g edible biomass m-2 day-1 per[g nonedible shoot dry weight]). Breeding lines 'IT84S-2246' (S-2246) and "IT82D-889' (D-889) were grown in a greenhouse under 8-, 12-, or 24-h photoperiods. S-2246 was short-day and D-889 was day-neutral for flowering. Under each photoperiod, cowpeas were harvested either for leaves only, seeds only, or leaves plus seeds (mixed harvest). Photoperiod did not affect EYR of either breeding line for any harvest scenario tested. Averaged over both breeding lines, seed harvest gave the highest EYR at 6.7 g m-2 day-1. The highest SHI (65%) and YER (94 mg m-2 day-1 g-1) were achieved for leaf-only harvest of D-889 under an 8-h photoperiod. For leaf-only harvest of S-2246, both SHI and YER increased with increasing photoperiod, but declined for seed-only and mixed harvests. However, photoperiod had no effect on SHI or YER for D-889 for any harvest scenario. A second experiment utilized the short-day cowpea breeding line 'IT89KD-288' (D-288) and the day-neutral breeding line 'IT87D-941-1' (D-941) to compare yield parameters using photoperiod extension under differing lamp types. This experiment confirmed the photoperiod responses of D-889 and S-2246 to a mixed-harvest scenario and indicated that daylength extension with higher irradiance from high pressure sodium lamps further suppressed EYR, SHI, and YER of the short-day breeding line D-288.

A mutation in protein phosphatase 2A regulatory subunit A affects auxin transport in Arabidopsis.

The phytohormone auxin controls processes such as cell elongation, root hair development and root branching. Tropisms, growth curvatures triggered by gravity, light and touch, are also auxin-mediated responses. Auxin is synthesized in the shoot apex and transported through the stem, but the molecular mechanism of auxin transport is not well understood. Naphthylphthalamic acid (NPA) and other inhibitors of auxin transport block tropic curvature responses and inhibit root and shoot elongation. We have isolated a novel Arabidopsis thaliana mutant designated roots curl in NPA (rcn1). Mutant seedlings exhibit altered responses to NPA in root curling and hypocotyl elongation. Auxin efflux in mutant seedlings displays increased sensitivity to NPA. The rcn1 mutation was transferred-DNA (T-DNA) tagged and sequences flanking the T-DNA insert were cloned. Analysis of the RCN1 cDNA reveals that the T-DNA insertion disrupts a gene for the regulatory A subunit of protein phosphatase 2A (PP2A-A). The RCN1 gene rescues the rcn1 mutant phenotype and also complements the temperature-sensitive phenotype of the Saccharomyces cerevisiae PP2A-A mutation, tpd3-1. These data implicate protein phosphatase 2A in the regulation of auxin transport in Arabidopsis.

Synthesis of long prebiotic oligomers on mineral surfaces.

Most theories of the origin of biological organization assume that polymers with lengths in the range of 30-60 monomers are needed to make a genetic system viable. But it has not proved possible to synthesize plausibly prebiotic polymers this long by condensation in aqueous solution, because hydrolysis competes with polymerization. The potential of mineral surfaces to facilitate prebiotic polymerization was pointed out long ago. Here we describe a system that models prebiotic polymerization by the oligomerization of activated monomers--both nucleotides and amino acids. We find that whereas the reactions in solution produce only short oligomers (the longest typically being a 10-mer), the presence of mineral surfaces (montmorillonite for nucleotides, illite and hydroxylapatite for amino acids) induces the formation of oligomers up to 55 monomers long. These are formed by successive 'feedings' with the monomers; polymerization takes place on the mineral surfaces in a manner akin to solid-phase synthesis of biopolymers.

Varying plant density and harvest time to optimize cowpea leaf yield and nutrient content.

Plant density and harvest time were manipulated to optimize vegetative (foliar) productivity of cowpea [Vigna unguiculata (L.) Walp.] canopies for future dietary use in controlled ecological life-support systems as vegetables or salad greens. Productivity was measured as total shoot and edible dry weights (DW), edible yield rate [(EYR) grams DW per square meter per day], shoot harvest index [(SHI) grams DW per edible gram DW total shoot], and yield-efficiency rate [(YER) grams DW edible per square meter per day per grams DW nonedible]. Cowpeas were grown in a greenhouse for leaf-only harvest at 14, 28, 42, 56, 84, or 99 plants/m2 and were harvested 20, 30, 40, or 50 days after planting (DAP). Shoot and edible dry weights increased as plant density and time to harvest increased. A maximum of 1189 g shoot DW/m2 and 594 g edible DW/m2 were achieved at an estimated plant density of 85 plants/m2 and harvest 50 DAP. EYR also increased as plant density and time to harvest increased. An EYR of 11 g m-2 day-1 was predicted to occur at 86 plants/m2 and harvest 50 DAP. SHI and YER were not affected by plant density. However, the highest values of SHI (64%) and YER (1.3 g m-2 day-1 g-1) were attained when cowpeas were harvested 20 DAP. The average fat and ash contents [dry-weight basis (dwb)] of harvested leaves remained constant regardless of harvest time. Average protein content increased from 25% DW at 30 DAP to 45% DW at 50 DAP. Carbohydrate content declined from 50% DW at 30 DAP to 45% DW at 50 DAP. Total dietary fiber content (dwb) of the leaves increased from 19% to 26% as time to harvest increased from 20 to 50 days.

Extraterrestrial helium trapped in fullerenes in the Sudbury impact structure.

Fullerenes (C60 and C70) in the Sudbury impact structure contain trapped helium with a 3He/4He ratio of 5.5 x 10(-4) to 5.9 x 10(-4). The 3He/4He ratio exceeds the accepted solar wind value by 20 to 30 percent and is higher by an order of magnitude than the maximum reported mantle value. Terrestrial nuclear reactions or cosmic-ray bombardment are not sufficient to generate such a high ratio. The 3He/4He ratios in the Sudbury fullerenes are similar to those found in meteorites and in some interplanetary dust particles. The implication is that the helium within the C60 molecules at Sudbury is of extraterrestrial origin.

Clusters of DNA damage induced by ionizing radiation: formation of short DNA fragments. II. Experimental detection.

The basic 30-nm chromatin fiber in the mammalian cell consists of an unknown (possibly helical) arrangement of nucleosomes, with about 1.2 kb of DNA per 10-nm length of fiber. Track-structure considerations suggest that interactions of single delta rays or high-LET particles with the chromatin fiber might result in the formation of multiple lesions spread over a few kilobases of DNA (see the accompanying paper: W.R. Holley and A. Chatterjee, Radiat. Res. 145, 188-199, 1996). In particular, multiple DNA double-strand breaks and single-strand breaks may form. To test this experimentally, primary human fibroblasts were labeled with [3H]thymidine and exposed at 0 degrees C to X rays or accelerated nitrogen or iron ions in the LET range of 97-440 keV/microns. DNA was isolated inside agarose plugs and subjected to agarose gel electrophoresis under conditions that allowed good separation of 0.1-2 kb size DNA. The bulk of DNA remained in the well or migrated only a small distance into the gel. It was found that DNA fragments in the expected size range were formed linearly with dose with an efficiency that increased with LET. A comparison of the yield of such fragments with the yield of total DNA double-strand breaks suggests that for the high-LET ions a substantial proportion (20-90%) of DNA double-strand breaks are accompanied within 0.1-2 kb by at least one additional DNA double-strand break. It is shown that these results are in good agreement with theoretical calculations based on treating the 30-nm chromatin fiber as the target for ionizing particles. Theoretical considerations also predict that the clusters will contain numerous single-strand breaks and base damages. It is proposed that such clusters be designated "regionally multiply damaged sites." Postirradiation incubation at 37 degrees C resulted in a decline in the number of short DNA fragments, suggesting a repair activity. The biological significance of regionally multiply damaged sites is presently unknown.

Potential contribution of the diazotrophic cyanobacterium, Cyanothece sp. strain 51142, to a bioregenerative life support system.

Long-duration manned space missions will likely require the development of bioregenerative means of life support. Such a Controlled Ecological Life Support System (CELSS) would use higher plants to provide food and a breathable atmosphere for the crew and employ a waste processing system to recover elements for recycling. The current study identifies ways in which a cyanobacterial component may enhance the sustainability of a space-deployed CELSS, including balancing CO2/O2 gas exchange, production of bioavailable N, dietary supplementation, and contingency against catastrophic failure of the higher plant crops. Relevant quantitative data have been collected about the cyanobacterium, Cyanothece sp. strain ATCC 51142, a large, aerobic, unicellular diazotroph. This organism grew rapidly (466 g dry wt. m-3 d-1) and under diverse environmental conditions, was amenable to large-scale culture, could be grown with relative energy efficiency (3.8% conversion), could actively fix atmospheric N2 (35.0 g m-3 d-1), could survive extreme environmental insults, and exhibited gas exchange properties (assimilatory quotient of 0.49) that may be useful for correcting the gas exchange ratio imbalances observed between humans and higher plants. It is suggested that a diazotrophic cyanobacterium, like Cyanothece sp. strain ATCC 51142, may be a safe, effective, and renewable complement or alternative to physicochemical backup systems in a CELSS.

Liposome formation in microgravity.

Liposomes are artificial vesicles with a phospholipid bilayer membrane. The formation of liposomes is a self-assembly process that is driven by the amphipathic nature of phospholipid molecules and can be observed during the removal of detergent from phospholipids dissolved in detergent micelles. As detergent concentration in the mixed micelles decreases, the non-polar tail regions of phospholipids produce a hydrophobic effect that drives the micelles to fuse and form planar bilayers in which phospholipids orient with tail regions to the center of the bilayer and polar head regions to the external surface. Remaining detergent molecules shield exposed edges of the bilayer sheet from the aqueous environment. Further removal of detergent leads to intramembrane folding and membrane folding and membrane vesiculation, forming liposomes. We have observed that the formation of liposomes is altered in microgravity. Liposomes that were formed at 1-g did not exceed 150 nm in diameter, whereas liposomes that were formed during spaceflight exhibited diameters up to 2000 nm. Using detergent-stabilized planar bilayers, we determined that the stage of liposome formation most influenced by gravity is membrane vesiculation. In addition, we found that small, equipment-induced fluid disturbances increased vesiculation and negated the size-enhancing effects of microgravity. However, these small disturbances had no effect on liposome size at 1-g, likely due to the presence of gravity-induced buoyancy-driven fluid flows (e.g., convection currents). Our results indicate that fluid disturbances, induced by gravity, influence the vesiculation of membranes and limit the diameter of forming liposomes.

Theories and models on the biology of cells in space.

A wide variety of observations on cells in space, admittedly made under constraining and unnatural conditions in many cases, have led to experimental results that were surprising or unexpected. Reproducibility, freedom from artifacts, and plausibility must be considered in all cases, even when results are not surprising. The papers in the symposium on "Theories and Models on the Biology of Cells in Space" are dedicated to the subject of the plausibility of cellular responses to gravity--inertial accelerations between 0 and 9.8 m/s2 and higher. The mechanical phenomena inside the cell, the gravitactic locomotion of single eukaryotic and prokaryotic cells, and the effects of inertial unloading on cellular physiology are addressed in theoretical and experimental studies.

Evaluation of Cyanothece sp. ATCC 51142 as a candidate for inclusion in a CELSS.

Controlled ecological life support systems (CELSS) have been proposed to make long-duration manned space flights more cost-effective. Higher plants will presumably provide food and a breathable atmosphere for the crew. It has been suggested that imbalances between the CO2/O2 gas exchange ratios of the heterotrophic and autotrophic components of the system will inevitably lead to an unstable system, and the loss of O2 from the atmosphere. Ratio imbalances may be corrected by including a second autotroph with an appropriate CO2/O2 gas exchange ratio. Cyanothece sp. ATCC 51142 is a large unicellular N2-fixing cyanobacterium, exhibiting high growth rates under diverse physiological conditions. A rat-feeding study showed the biomass to be edible. Furthermore, it may have a CO2/O2 gas exchange ratio that theoretically can compensate for ratio imbalances. It is suggested that Cyanothece spp. could fulfill several roles in a CELSS: supplementing atmosphere recycling, generating fixed N from the air, providing a balanced protein supplement, and protecting a CELSS in case of catastrophic crop failure.