ALBERT

Description: This paper describes the affects of microgravity upon three key cellular levels (functional, protein, and mRNA) that are linked to one another. It is clear that at each of these levels, microgravity produces rapid and substantial alterations. One of the key challenges facing the life science community is the development of effective countermeasures that prevent the loss of muscle function as described in this paper. The development of optimal countermeasures, however, awaits a clearer understanding of events occurring at the levels of transcription, translation, and degradation.

Description: Transition from a normal gravitational environment to that of microgravity eventually results in decreased plasma and blood volumes, increasing with duration of exposure to microgravity. This loss of vascular fluid is presumably due to negative fluid and electrolyte balance and most likely contributes to the orthostatic intolerance associated with the return to gravity. The decrease in plasma volume is presumed to be a reflection of a concurrent decrease in extracellular fluid volume with maintenance of normal plasma-interstitial fluid balance. In addition, the specific alterations in renal function contributing to these changes in fluid and electrolyte homeostasis are potentially responding to neuro-humoral signals that are not consistent with systemic fluid volume status. We have previously demonstrated an early increase in both glomerular filtration rate and extracellular fluid volume and that this decreases towards control values by 7 days of simulated microgravity. However, longer duration studies relating these changes to plasma volume alterations and the response to return to orthostasis have not been fully addressed. Male Wistar rats were chronically cannulated, submitted to 30 days heat-down tilt (HDT) and followed for 7 days after return to orthostasis from HDT. Measurements of renal function and extracellular and blood volumes were performed in the awake rat.

Description: Electrical stimulation of vestibular-nerve afferents innervating the semicircular canals has been used to identify the extraocular muscles receiving activation or inhibition by individual ampullary nerves. This technique was originally developed by Szentagothai (1950) and led to the description of three neuron reflex arcs that connect each semicircular canal through an interneuron traversing in the region of the medial longitudinal fasciculus to one ipsilateral and one contralateral eye muscle. Selective ampullary nerve stimulation was subsequently used by Cohen and colleagues (Cohen and Suzuki, 1963; Cohen et al., 1964; Suzuki et al., 1964; Cohen et al., 1966) to study movements of the eyes and activation of individual extraocular muscles in response to stimulation of combinations of ampullary nerves. This work led to a description of the now familiar relationships between activation of a semicircular canal ampullary nerves and the anticipated movement in each eye. Disconjugacy of eye movements induced by individual vertical canal stimulation and dependence of the pulling direction of vertical recti and oblique muscles on eye position were also defined in these experiments. Subsequent studies have defined the mechanisms by which externally applied galvanic currents result in a change in vestibular-nerve afferent discharge. The currents appear to act at the spike trigger site. Perilymphatic cathodal currents depolarize the trigger site and lead to excitation whereas anodal currents hyperpolarize and result in inhibition. Afferents innervating all five vestibular endorgans appear to be affected equally by the currents (Goldberg et al., 1984). Irregularly discharging afferents are about 5-10 times more sensitive than regularly discharging ones because of the steeper slope of the former's faster postspike recovery of excitability in encoder sensitivity (Smith and Goldberg, 1986). Response adaptation similar to that noted during acceleration steps is apparent for longer periods of current administration. This adaptation is manifested as a perstimulus return toward resting discharge and poststimulus after-response in the opposite direction (Goldberg et al., 1984; Minor and Goldberg, l991). Cathodal currents (with respect to the perilymphatic space of the vestibule) are excitatory whereas anodal currents are inhibitory. Horizontal eye movements evoked by unilateral galvanic polarizations administered through chronically implanted labyrinthine stimulating electrodes have been studied in alert squirrel monkeys (Minor and Goldberg, 1991). We sought to extend this analysis by recording three-dimensional eye movements during galvanic stimulation. As predicted based upon roughly equal stimulation of ampullary nerves innervating the vertical canals, a substantial torsional component to the nystagmus is noted. The trajectory of torsional slow phases and nystagmus profile after the polarization provide insight into the central mechanisms that influence these responses.

Description: Administration of 2-deoxy-D-glucose (2-DG), an analog of glucose which inhibits glycolysis by competitive antagonism for phosphohexose isomerase, results in acute periods of intracellular glucoprivation and hyperglycemia resulting in hyperphagia. In addition to these changes in the carbohydrate metabolism, injection of 2-DG results in alterations of both the endocrine and neurological systems as suggested by modifications in oxytocin and glucocorticoid levels and norepinephrine production. Moreover, alterations of the immune response, such as a decrease in the in vitro proliferation of splenocytes after mitogen-stimulation, were observed in mice injected with 2-DG. Sex, genotype and environment are among the factors that may modulate effects of catecholamines and hypothalamo-pituitary-adrenal axis on these immune changes. Sexual dimorphism in immune function resulting from the effects of sex hormones on immune effector cells has been shown in both animals and humans. These observations have important implications, especially with regard to higher incidence of many autoimmune diseases in females. Evidence exists that reproductive hormones influence the immune system and increase the risk of immunologically related disorders in both animals and humans. Indeed, immunological responses in stressful situations may also be confounded by fluctuations of sex hormones especially in females. Lymphocyte distribution, cytoldne production, and the ability of lymphocyte to proliferate in vitro were analyzed in male and female mice to determine if sex influenced 2-DG immunomodulation. In addition, the influence of hormones, especially sex hormones, on these changes were evaluated.

Description: NASA Ames Research Center is responsible for the development of the Space Station Biological Research Project (SSBRP) which will support non-human life sciences research on the International Space Station Alpha (ISSA). The SSBRP is designed to support both basic research to understand the effect of altered gravity fields on biological systems and applied research to investigate the effects of space flight on biological systems. The SSBRP will provide the necessary habitats to support avian and reptile eggs, cells and tissues, plants and rodents. In addition a habitat to support aquatic specimens will be provided by our international partners. Habitats will be mounted in ISSA compatible racks at u-g and will also be mounted on a 2.5 m diameter centrifuge except for the egg incubator which has an internal centrifuge. The 2.5 m centrifuge will provide artificial gravity levels over the range of 0.01 G to 2 G. The current schedule is to launch the first rack in 1999, the Life Sciences glovebox and a second rack early in 2001, a 4 habitat 2.5 in centrifuge later the same year in its own module, and to upgrade the centrifuge to 8 habitats in 2004. The rodent habitats will be derived from the Advanced Animal Habitat currently under development for the Shuttle program and will be capable of housing either rats or mice individually or in groups (6 rats/group and at least 12 mice/group). The egg incubator will be an upgraded Avian Development Facility also developed for the Shuttle program through a Small Business and Innovative Research grant. The Space Tissue Loss cell culture apparatus, developed by Walter Reed Army Institute of Research, is being considered for the cell and tissue culture habitat. The Life Sciences Glovebox is crucial to all life sciences experiments for specimen manipulation and performance of science procedures. It will provide two levels of containment between the work volume and the crew through the use of seals and negative pressure. The glovebox will accommodate use by two crew persons simultaneously and the capability for real time video down-link and data acquisition. In house testbeds and Phase B studies of the centrifuge validated the concepts of vibration isolation and autobalancing systems to meet the ISSA microgravity requirements. The vibration isolation system is effective above the centrifuge rotation frequency while the autobalancing system on the rotor removes vibration at and below the rotation rate. Torque of the Station, induced by spin-up/spindown of the centrifuge, can be minimized by controlling spin-up/spin-down rates. The SSBRP and ISSA will provide the opportunity to perform long-term, repeatable and high quality science. The long duration increments available on the Station will permit multigeneration studies of both plants and animals which have not previously been possible. The u-g habitat racks and the eight habitat centrifuge will accommodate sufficient number of specimens to permit statistically significant sampling of specimens to investigate the time course of adaptation to altered gravity environments. The centrifuge will, for the first time, permit investigators to use gravity itself as a tool to investigate fundamental processes, to investigate the intensity and duration of gravity to maintain normal structure and function, to separate the effects of u-g from other environmental factors and to examine artificial gravity as a potential countermeasure for the physical deconditioning observed during space flight.

Description: In the terrestrial environment multiple time cues exist. Zeitgebers have been identified and studied for their ability to convey temporal information to various physiological systems. In the microgravity experiment it is necessary to define time cues within the flight hardware prior to flight. During flight if changes in the Circadian System (e.g., mean, phase angle, period) occur this would indicate that the gravity vector is important relative to biological timing. This presentation is concerned with the environmental parameter: to support rodent experiments in microgravity. The Animal Enclosure Module (AEM) provides solid food bars and water via lixits and ad libitum. Flight animals (Sprague-Dawley rats, 60 - 300g) when compared to ground controls show similar growth (mean growth per day g, plus or minus SD; flight 5.4 plus or minus 2.0, ground 5.9 plus or minus 2.1). Current AEMs use incandescent lighting (approx. 5 Lux). Light emitting diode (LED) arrays are being developed that provide a similar light environment as cool-white fluorescent sources (40 Lux). In ground based tests (12L:12D), these arrays show normal circadian entrainment (Tau = 24.0) with respect to the behavioral responses, measured (drinking, eating, gross locomotor activity). A newly developed ultra high efficiency filter system can entrap all feces, urine and odors from 6 rats for 24 days. Maximum sound level exposure limits (per octave band 22 Hz - 179 kHz) have been established. The AEM will effectively support animal experiments in microgravity.

Description: Ross 3.0 software was developed in the Biocomputation Center for semi-automated reconstruction of objects from serial thin sections. Data are captured directly from a transmission electron microscope via a video camera to a graphics workstation where the sections are mosaicked and contours are traced, registered and displayed by semi-automated methods. For the first time, macular type II cells are described completely for their innervation patterns. The purposes are to learn more about the fundamental circuitry of the macula and to demonstrate whether the terminals are altered morphologically by space flight. Current examples, from the medial part of the macula, are from maculas collected in-flight on the Space Life Sciences-2 mission, 4.5 hrs post-flight, and from a ground control. Results show that the typical type 11 cell receives processes from up to six nearby calyces or afferents. Nearly all the processes are elongated; some have bouton-like swellings and numerous vesicles. Multiple (2 to 4) processes from a single calyx to a type II cell are common, and approx. 1/3 of the processes innervate 2 type II cells of a neighboring cluster of 3 cells. About 2% of type II cells resemble type I cells morphologically and are surrounded by demicalyces. Differences in size or shape of the terminals under flight conditions could not be determined because the sample size is still too small; but it is clear that reconstruction methods provide insights into macular circuitry not obtainable by other techniques. The results demonstrate a morphological basis for interactions between adjacent receptive fields, through feedback-feedforward connections, during preprocessing of linear acceleratory information by the maculas. While the methods are currently being tested using vestibular maculas as the model system, it is clear that the technology is applicable to any tissue that can be physically or optically sectioned. ROSS software has already been implemented for reconstructing objects from tissues studied by confocal and by transmitted light microscopy, and research into magnetic resonance imaging-computational tomography combined visualization are underway.

Description: The vacuolar-like ATPase from Halobacterium saccha vorum is inhibited by N-ethylmaleimide and p-chloromercudphenylsulfonate. The failure of adenine nucleotides to protect against p-chloromercuriphenyisulfonate inhibition, of p-chloromercuriphenylsulfonate to protect against N-ethylmaleimide inhibition, and the difference in the temperature dependence of inactivation infers that the enzyme contains at least two thiols that are essential for enzyme activity. CNBr cleavage of C-14-N-ethylmaleimide labeled subunit results in two radioactive peptides that locates the N-ethylmaleimide-reactive cysteinyl residue as cysteine-262 in the H. salinarium sequence.

Description: A semiautomated method for reconstructing objects from serial thin sections has been developed in the Biocomputation Center. The method is being used to completely, for the first time, type II hair cells and their innervations. The purposes are to learn more about the fundamental circuitry of the macula on Earth and to determine whether changes in connectivities occur under space flight conditions. Data captured directly from a transmission electron microscope via a video camera are sent to a graphics workstation. There, the digitized micrographs are mosaicked into sections and contours are traced, registered and displayed by semiautomated methods. Current reconstructions are of type II cells from the medial part of rat maculas collected in-flight on the Space Life Sciences-2 mission, 4.5 hrs post-flight, and from a ground control. Results show that typical type II cells receive processes from tip to six nearby calyces or afferents. Nearly all processes are elongated and have bouton-like enlargements; some have numerous vesicles. Multiple (2 to 4) processes from a single calyx to a type II cell are common, and approximately 1/3 of the processes innervale 2 or 3 type II cells or a neighboring cluster. From 2% to 6% of the cells resemble type I cells morphologically but have demi-calyces. Thus far, increments in synaptic number in type II cells of flight rats are prominent along processes that supply two hair cells. It is clear that reconstruction methods provide insights into details of macular circuitry not obtainable by other techniques. The results demonstrate a morphological basis for interactions between adjacent receptive fields through feed back-feed forward connections, and for dynamic alterations in receptive field range and activity during preprocessing of linear acceleratory information by the maculas. The reconstruction method we have developed will find further applications in the study of the details of neuronal architecture of more complex systems, to seek out shared organizational properties or neuronal networks and to understand better localization of synaptic changes in altered environments.

Description: When Pinus taeda cell suspension cultures are exposed to 8% sucrose solution, the cells undergo significant intracellular disruption, irregular wall thickening/lignification with concomitant formation of an 'extracellular lignin precipitate. However, addition of potassium iodide (KI), an H202 scavenger, inhibits this lignification response, while the ability to synthesize the monolignols, p-coumaryl and coniferyl alcohols, is retained. Lignin synthesis (i.e. polymerization) is thus temporarily correlated with H202 generation, strongly implying a regulatory role for the latter. Time course analyses of extracellular metabolites leading up to polymer formation reveal that coniferyl alcohol, but not p-coumaryl alcohol, undergoes substantial coupling reactions to give various lignans. Of these, the metabolites, dihydrodehydrodiconiferyl alcohol, shonanin (divanillyl tetrahydrofuran) and its apparent aryl tetralin derivative, cannot be explained simply on the basis of phenolic coupling. It is proposed that these moieties are the precursors of so-called reduced substructures in the lignin macromolecule. This adds a new perspective to the lignin assembly mechanism.

Description: The effect of pH and Ca2+ on net NO3- uptake, influx, and efflux by intact roots of barley (Hordeum vulgare L.) seedlings was studied. Seedlings were induced with NO3- or NO2-. Net NO3- uptake and efflux, respectively, were determined by following its depletion from, and accumulation in, the external solution. Since roots of both uninduced and NO2(-)-induced seedlings contain little internal NO3- initial net uptake rates are equivalent to influx (M. Aslam, R.L. Travis, R.C. Huffaker [1994] Plant Physiol 106: 1293-1301). NO3-, uptake (influx) by these roots was little affected at acidic pH. In contrast, in NO3(-)-induced roots, which accumulate NO3-, net uptake rates decreased in response to acidic pH. Under these conditions, NO3- efflux was stimulated and was a function of root NO3- concentration. Conversely, at basic pH, NO3- uptake by NO3- and NO2(-)-induced and uninduced roots decreased, apparently because of the inhibition of influx. Calcium had little effect on NO3- uptake (influx) by NO2(-)-induced roots at either pH 3 or 6. However, in NO3(-)-induced roots, lack of Ca2+ at pH 3 significantly decreased net NO3- uptake and stimulated efflux. The results indicate that at acidic pH the decrease in net NO3- uptake is due to the stimulation of efflux, whereas at basic pH, it is due to the inhibition of influx.

Description: Microfilaments were visualized in dark-grown protonemata of the moss Ceratodon to assess their possible role in tip growth and gravitropism. The relative effectiveness of rhodamine phalloidin (with or without m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS)) and of immunofluorescence (using the C4 antibody) was evaluated for actin localization in the same cell type. Using immunofluorescence, microfilaments were primarily in an axial orientation within the apical cell. However, a more complex network of microfilaments was observed using rhodamine phalloidin after MBS pretreatment, especially when viewed by confocal laser scanning microscopy. This method revealed a rich three dimensional network of fine microfilaments throughout the apical cell, including the extreme apex. Although there were numerous internal microfilaments, peripheral microfilaments were more abundant. No major redistribution of microfilaments was detected after gravistimulation. The combination of MBS, rhodamine phalloidin, and confocal laser scanning microscopy preserves and reveals microfilaments remarkably well and documents perhaps the most extensive F-actin network visualized to date in any tip-growing cell.

Description: We conducted a series of gravitropic experiments on Avena coleoptiles in the weightlessness environment of Spacelab. The purpose was to test the threshold stimulus, reciprocity rule and autotropic reactions to a range of g-force stimulations of different intensities and durations The tests avoided the potentially complicating effects of earth's gravity and the interference from clinostat ambiguities. Using slow-speed centrifuges, coleoptiles received transversal accelerations in the hypogravity range between 0.l and 1.0 g over periods that ranged from 2 to 130 min. All responses that occurred in weightlessness were compared to clinostat experiments on earth using the same apparatus. Characteristic gravitropistic response patterns of Atuena were not substantially different from those observed in ground-based experiments. Gravitropic presentation times were extrapolated. The threshold at 1.0 g was less than 1 min (shortest stimulation time 2 min), in agreement with values obtained on the ground. The least stimulus tested, 0.1 g for 130 min, produced a significant response. Therefore the absolute threshold for a gravitropic response is less than 0.1 g.

Description: Experiments were undertaken to determine if the reciprocity rule is valid for gravitropic responses of oat coleoptiles in the acceleration region below 1 g. The rule predicts that the gravitropic response should be proportional to the product of the applied acceleration and the stimulation time. Seedlings were cultivated on 1 g centrifuges and transferred to test centrifuges to apply a transverse g-stimulation. Since responses occurred in microgravity, the uncertainties about the validity of clinostat simulation of weightlessness was avoided. Plants at two stages of coleoptile development were tested. Plant responses were obtained using time-lapse video recordings that were analyzed after the flight. Stimulus intensities and durations were varied and ranged from 0.1 to 1.0 g and from 2 to 130 min, respectively. For threshold g-doses the reciprocity rule was obeyed. The threshold dose was of the order of 55 g s and 120 g s, respectively, for two groups of plants investigated. Reciprocity was studied also at bending responses which are from just above the detectable level to about 10 degrees. The validity of the rule could not be confirmed for higher g-doses, chiefly because the data were more variable. It was investigated whether the uniformity of the overall response data increased when the gravitropic dose was defined as (gm x t) with m-values different from unity. This was not the case and the reciprocity concept is, therefore, valid also in the hypogravity region. The concept of gravitropic dose, the product of the transverse acceleration and the stimulation time, is also well-defined in the acceleration region studied. With the same hardware, tests were done on earth where responses occurred on clinostats. The results did not contradict the reciprocity rule but scatter in the data was large.

Description: No straightforward method exists for separating the proportion of ion exchange and respiration due to rhizoplane microbial organisms from that of root ion exchange and respiration. We examined several antibiotics that might be used for the temporary elimination of rhizoplane bacteria from hydroponically grown wheat roots (Triticum aestivum cv. Veery 10). Each antibiotic was tested for herbicidal activity and plate counts were used to enumerate bacteria and evaluate antibiotic kinetics. Only lactam antibiotics (penicillins and cephalosporins) did not reduce wheat growth rates. Aminoglycosides, the pyrimidine trimethoprim, colistin and rifampicin reduced growth rates substantially. Antibiotics acted slowly, with maximum reductions in rhizoplane bacteria occurring after more than 48 h of exposure. Combinations of nonphytotoxic antibiotics reduced platable rhizoplane bacteria by as much as 98%; however, this was generally a reduction from about 10(9) to 10(6) colony forming units per gram of dry root mass, so that many viable bacteria remained on root surfaces. We present evidence which suggests that insufficient bacterial biomass exists on root surfaces of nonstressed plants grown under well-aerated conditions to quantitatively interfere with root nitrogen absorption measurements.

Description: The enzyme nitric oxide synthase is present in the macula densa and may participate in the control of renin secretion by the adjacent juxtagiomerular cells. In the present study, we investigated the effect of inhibiting nitric oxide synthase on the renin secretory response to frusemide, which stimulates renin secretion by blocking Na(+)-K(+)-2Cl(-) co-transport in the macula densa. Injection of frusemide in 12 conscious rabbits elicited a transient increase in mean arterial pressure from 84 +/- 2 to 92 +/-3 mm Hg at 5 min (P less than 0.01) and a sustained increase in heart rate from 246 +/- 6 to 281 +/- 10 beats/min at 45 min (P less than 0.01). Plasma renin activity increased from 8.0 +/- 1.2 to 14.3 +/- 1.8, 12.4 +/- 1.6 and 11.6 +/- 1.5 pmol/2h ml at 15, 30 and 45min respectively (P less than 0.01). There were no changes in plasma sodium and potassium concentrations or osmoiality. Inhibition of nitric oxide synthase with N(sup G)-nitro-L- arginine methyl ester increased mean arterial pressure by 9 mm Hg, decreased heart rate and plasma renin activity, and markedly suppressed the renin response to frusemide (from 4.6 +/- 0.7 to 7.6 +/- 1.7, 4.7 +/- 1.0 and 4.6 +/- 0.7pmol/2h ml at 15, 30 and 45 min respectively). By contrast, infusion of an equipressor dose of phenylephrine did not suppress the renin response to frusemide. Histochemical studies with the NADPH diaphorase technique confirmed the presence of nitric oxide synthase in the macula densa, and suggested that enzyme activity is increased by treatment with frusemide. These results are consistent with a role for the L- arginine-nitric oxide pathway in the modulation of renin secretion by the macula densa.

Description: Eleven anesthetized rhesus monkeys were used to study cardiovascular, renal, and endocrine alterations associated with 120 min of head-out water immersion. Five animals underwent complete intrapericardial denervation using the Randall technique, while the remaining six monkeys served as intact controls. Each animal was chronically instrumented with an electromagnetic flow probe on the ascending aorta, a strain gauge pressure transducer implanted in the apex of the left ventricle (LV), and electrocardiogram leads anchored to the chest wall and LV. During immersion, LV end-diastolic pressure, urine flow, glomerular filtration rate, sodium excretion, and circulating atrial natriuretic peptide (ANP) each increased (P less than 0.05) for intact and denervated monkeys. There were no alterations in free water clearance in either group during immersion, yet fractional excretion of free water increased (P less than 0.05) in the intact monkeys. Plasma renin activity (PRA) decreased (P less than 0.05) during immersion in intact monkeys but not the denervated animals. Plasma vasopressin (PVP) concentration decreased (P less than 0.05) during the first 30 min of immersion in both groups but was not distinguishable from control by 60 min of immersion in denervated monkeys. These data demonstrate that complete cardiac denervation does not block the rise in plasma ANP or prevent the natriuresis associated with head-out water immersion. The suppression of PVP during the first minutes of immersion after complete cardiac denervation suggests that extracardiac sensing mechanisms associated with the induced fluid shifts may be responsible for the findings.

Description: The results of several experiments were disseminated during this semiannual period. This publication and each of these presented papers represent investigations of the continuity in psychological processes between monkeys and humans. Thus, each serves to support the animal model of behavior and performance research.

Description: Sensors 2000! (S2K!) is a NASA Ames Research Center engineering initiative designed to provide biosensor and bio-instrumentation systems technology expertise to NASA's life sciences spaceflight programs. S2K! covers the full spectrum of sensor technology applications, ranging from spaceflight hardware design and fabrication to advanced technology development, transfer and commercialization. S2K! is currently developing sensor systems for space biomedical applications on BION (a Russian biosatellite focused on Rhesus Monkey physiology) and NEUROLAB (a Space Shuttle flight devoted to neuroscience). It's Advanced Technology Development-Biosensors (ATD-B) project focuses efforts in five principle areas: biotelemetry Systems, chemical and biological sensors, physiological sensors, advanced instrumentation architectures, and data and information management. Technologies already developed and tested included, application-specific sensors, preamplifier hybrids, modular programmable signal conditioners, power conditioning and distribution systems, and a fully implantable dual channel biotelemeter. Systems currently under development include a portable receiver system compatible with an off-the-shelf analog biotelemeter, a 4 channel digital biotelemetry system which monitors pH, a multichannel, g-processor based PCM biotelemetry system, and hand-held personal monitoring systems. S2K! technology easily lends itself to telescience and telemedicine applications as a front-end measurement and data acquisition device, suitable for obtaining and configuring physiological information, and processing that information under control from a remote location.

Description: The successful utilization of higher plants in space flight is likely to require the effective transition of plants through all phases of growth and development. A particularly sensitive and critical stage in this cycle is seed germination. The present inflight capability to manipulate seed from a state of dormancy to germination and the performance of such activity under aseptic conditions is extremely limited. An Automated Sowing Mechanism (ASM) has been designed to address this area of science and technology. The self-contained system is readily compatible with the existing Shuttle middeck locker Plant Growth Unit (PGU) and planned Plant Growth Facility (PGF), presenting an opportunity to extend the experimental capability of these systems. The ASM design encompasses the controlled transition of seed from a dry to hydrated state utilizing solid media substrate as the source of water and nutrient support. System activation has been achieved with both photo and timing mechanisms. Controlled induced germination and development of various plant species has been achieved in ground-based trials. The system is presently being prepared for a KC-135 flight test.

Description: Following space flight a diuresis, due to an increase in free water clearance, has been suggested in humans. To assess the effects of space flight on renal function, rats were flown in space for 14 days. Rats were divided into three groups; vivarium controls (V;n=6; housed 2/shoe box cage), flight controls (FC;n=6; group housed in a flight cage), and flight animals (F;n=6). Upon landing all animals were placed into individual metabolic cages. Urine was collected daily for 7 days and every other day for 14 days. Urine output was increased (p less than 0.05; ANOVA) following flight for 3 days. On postflight day 1, flow rates were, V=6.8 plus or minus 0.9, FC=8.711.8 and F=16.6 plus or minus 2.7 microliter/min. Excretion rates of Na+ and K+ were increased, resulting in an increased osmotic excretion rate (V=7.9 plus or minus 0.9, FC=6.1 plus or minus 0.7 and F=13.5 plus or minus 0.7 uOsm/min). Creatinine excretion rate was increased over the first two postflight days. In the absence of changes in plasma creatinine, Na+, or K+ (samples obtained immediately post flight from similar rats compared to Day 14), GFR was increased following space flight. The increased excretion of solute was thus the result of increased delivery and decreased reabsorption. Osmotic clearance was increased (V=28, FC=27 and F=51 microliter/min), while free water clearance was decreased post flight (V=-21,FC=-18 and F=-34 microliter/min). In rats, the postflight diuresis is the result of an increase in solute (osmotic) excretion with an accompanying reduction in free water clearance.

Description: An extremely halophilic bacterium (strain CH-1) was isolated from a saltern adjacent to San Francisco Bay. It grew in a mineral salts medium with ammonium and glucose as sole sources of nitrogen and carbon as well as energy, respectively Cells lysed at less than 10% NaCl and growth was most rapid in medium containing 20% NaCl. Cells were pieomorphic ranging from disc to ovoid-shaved and used a variety of carbohydrates as sole carbon sources. the utilization of certain carbon sources was controlled by temperature with some used at 37 degrees but not 45 C. CH-1 grew between 30 degrees and 50 C with the optimum at 45 C in the presence of 20% NaCl. CH-1 contained 2,3-di-O-isoprenyl glcerol diethers and was sensitive to aphidicofin. The major polar lipid was glucosyl-mannosyl-alucosyl diether, which is diagnostic of the Haloarcula. Thus CH-1 is an extreme halophile and a member of this genus. Among the novel characteristics of this organism was its ability to grow anaerobically in synthetic medium when nitrate was present which was only reduced to nitrous oxide. This organism should prove useful for studying denitrification and carbohydrate metabolism in the extreme halophiles; and to be a valuable resource for generic studies.

Description: In the terrestrial environment multiple time cues exist. Zeitgebers have been identified and studied for their ability to convey temporal information to various physiological systems, In the microgravity experiment it is necessary to define time cues within the flight hardware prior to flight. During flight if changes in the Circadian System (e.g., mean, phase angle, period) occur this would indicate that the gravity vector is important relative to biological timing. This presentation is concerned with the environmental parameters to support rodent experiments in microgravity. The Animal Enclosure Module (AEM) provides solid food bars and water via lixits ad libitum. Flight animals (Sprague-Dawley rats, 60 - 300g) when compared to ground controls show similar growth (mean growth per day, g +/- SD; flight 5.4 +/- 2.0, ground 5.9 +/- 2.1). Current AEMs use incandescent lighting (approx. 5 Lux). Light emitting diode (LED) arrays are being developed that provide a similar light environment as cool-white fluorescent sources (40 Lux). In ground based tests (12L:12D), these arrays show normal circadian entrainment (Tau = 24.0) with respect to the behavioral responses. measured (drinking, eating, gross locomotor activity). A newly developed ultra high efficiency filter system can entrap all feces, urine and odors from 6 rats for 24 days. Maximum sound level exposure limits (per octave band 22 Hz - 179 kHz) have been established. The AEM will effectively support animal experiments in microgravity.

Description: On the Space Shuttle and MIR, mean CO2 levels have been 0.3% which is ten times that of normal air. There have also been extended periods with levels of 0.7% CO2 with peak concentrations at 2.0%. The Space Station program had proposed that CO2 concentration levels be maintained, on average, at 0.7%, and not to exceed 1.0%. To ensure that these levels of CO2 would not compromise the integrity of the science performed on the Space Station, the effects of chronic exposure of rats to 2.0% and 0.7% CO2 were investigated. Ten male rats per group were placed in individual metabolic cages for monitoring of food and water consumption, as well as fecal and urine production. Cages were placed in a large (4W x 10L x 4H ft.) plexiglass chamber with a controlled atmospheric environment. Following 7 days of cage adaptation, animals were exposed to experimental (2.0% or 0.7% CO2) or control (ambient air) conditions for 30 days. Daily body weight, food and water intake, and fecal and urine excretions were measured for the last three days of adaptation and the first ten days of exposure and then every three to four days for the remaining three weeks. Urine was measured for pH and total CO2. During 2.0% and 0.7% CO2 exposures, animal growth, fecal production and food and water consumption were within normal ranges. Urine excretion was significantly (p less than 0.05) higher in both experimental groups compared to controls. Urine pH of animals exposed to 2.0% CO2 was decreased by 0.32 over the first 6 days of exposure, followed by a 0.63 increase by day 30. In animals exposed to 0.7% CO2, urine pH did not decrease early in the exposure period, but did increase by 0.37 by day 30. Urine CO2 excretion did not change the first 6 days of exposure, but significantly increased in both 2.0% and 0.7% CO2 by day 30 (897 and 402 mmol/day, respectively). These results of chronic exposure to 2.0% and 0.7% CO2 are consistent with renal compensation in response to an altered acid-base homeostasis. These findings may impact science conducted on the Space Shuttle or the Space Station if CO2 concentrations reach 0.7%.

Description: This study examined efferent output from the median preoptic nucleus (MnPO) to pontine noradrenergic and serotonergic cell groups using an anterograde tracing technique (Phaseolus vulgaris leucoagglutinin, PHA-L) combined with glucose oxidase immunocytochemistry to scrotonin (5-HT) or to dopamine-(beta)-hydroxylase (DBH). Injections of PHA-L into the ventral MNPO resulted in moderate axonal labeling within the region of the B7 and B8 serotonergic groups in the dorsal raphe. PHA-L labeled fibers and punctate processes were observed in close apposition to many of the 5-HT immunoreactive neurons in these regions, In contrast, sparse terminal labeling was found within the B5 group in the raphe pontis nucleus, and only trace fiber labeling observed in the B3 and B6 groups. Efferents from the MNPO also provided moderate innervation to the A6 and A7 noradrenergic groups. PHA-L labeled punctate processes were found most frequently in close apposition to DBH-immunorcactive neurons at mid- to caudal levels of the locus coeruleus. Some labeled axons were also present within the A7 and A5 groups. Additionally, a close apposition between labeled MNPO efferents and 5-HT fibers within the lateral parabrachial nucleus was observed, The results indicate the MNPO provides a topographic innerva- tion of monoaminergic groups in the upper brainstem.

Description: The purpose of this study was to examine the time course of change in soleus muscle fiber peak force (N), tension (P(sub 0), kN/sq m), elastic modulus (E(sub 0)), and force-pCa and stiffness - pCa relationships. After 1, 2, or 3 wk of Hindlimb Unloading (HU), single fibers were isolated and placed between a motor arm and a transducer, and fiber diameter, peak absolute force, P(sub 0), E(sub 0), and force-pCa and stiffness-pca relationships were characterized. One week of HU resulted in a significant reduction in fiber diameter (68 +/- 2 vs. 57 +/- 1 micrometer), force (3.59 +/- 0.15 vs. 2.19 +/- 0.12 x 10(exp -4) N), P(sub 0) (102 +/- 4 vs. 85 +/- 2 kN/sq m), and E(sub 0) (1.96 +/- 0.12 vs. 1.37 +/- 0.13 X 10(exp 7) N/sq m) and 2 wk of HU caused a further decline in fiber diameter (45 +/- 1 micrometer), force (1.31 +/- 0.06 x 10(exp -4) N), and E(sub 0)(0.96 +/- 0.09 x 10(exp 7) N/sq m). Although the mean fiber diameter and absolute force continued to decline through 3 wk of HU, P(sub 0) recovered to values not significantly different from control. The P(sub 0)/E(sub 0) ratio was significantly increased after 1 (5.5 +/- 0.3 to 7.1 +/- 0.6), 2, and 3 wk of HU, and the 2-wk (9.5 +/- 0.4) and 3-wk (9.4 +/- 0.8) values were significantly greater than the 1-wk values. The force-pCa and stiffness-pCa curves were shifted right- ward after 1, 2, and 3 wk of HU. At 1 wk of HU, the Ca(2+) sensitivity of isometric force, assessed by Ca(2+) concentration required for half-maximal force, was increased from the control value of 1.83 +/- 0.12 to 2.30 +/- 0.10 micrometers. In conclusion, after HU, the decrease in soleus fiber P(sub 0) can be explained by a reduction in the number of myofibrillar cross bridges per cross-sectional area. Our working hypothesis is that the loss of contractile protein reduces the number of cross bridges per cross-sectional area and increases the filament lattice spacing. The increased spacing reduces cross-bridge force and stiffness, but P(sub 0)/E(sub 0) increases because of a quantitatively greater effect on stiffness.

Description: The role of arylphorin as a storage protein was studied using 14C-arylphorin. 14C-arylphorin was produced optimally by incubating one-half fat body from Manduca sexta fifth instar larvae at 22 degrees C for 24 h, in 1 ml of medium containing amino acids at 25% of their physiological concentration with [U-14C]-phenylalanine (phe) provided initially without nonlabeled phenylalanine. Nonlabeled phe was provided after 1 h at 16% of its physiological concentration. The specific activity of 14C-arylphorin produced in vitro was 30 times greater than that generated in vivo. Injection of 14C-arylphorin into pharate adults was used to study the distribution of 14C-phe derived from this protein into 14CO2 and tissues for comparison with injection of free 14C-phe during the middle (days 6 to 12 pharate adult) and late (days 12 to 17 pharate adult) stages of adult development. Appearance of 14CO2 from 14C-arylphorin as compared to 14C-phenylalanine showed a slower time course during both the middle and late stages of development, in keeping with the time needed for degradation of the protein. In accord with faster phe turnover near the end of adult development, total 14CO2 production was greater and the retention of 14C in hemolymph and fat body was less compared to the middle stage of development regardless of whether 14C-arylphorin or 14C-phe was injected. In the middle stage of development, the appearance of 14C in the cuticle and head parts was greater, whereas incorporation into abdomen and thorax was less than during the late stage of development. Since the pattern of 14C distribution from 14C-arylphorin and 14C-phe was similar, one major function of arylphorin must be as a storage protein replenishing the supply of free amino acids used for synthesis of adult tissues. These results also suggest a limited contribution of M. sexta arylphorin to formation of the cuticle subsequent to day-6 pharate adult.

Description: Cysts of brine shrimp attached with a liquid adhesive to 12-mm diameter glass coverslips in a syringe-type fluid processing apparatus were flown aboard the NASA space shuttle Discovery, flight STS-60, from 3-11 February 1994, and were allowed to undergo postencystment embryogenesis and to hatch in microgravity. The shuttle flight and the ground-based control coverslips with attached cysts were parallel to the earth's surface during incubation in salt water. Based on the position of the cyst shell crack in the attached cyst population, the ground-control nauplii emerged mostly upward. On the shuttle in microgravity, although our method of detection of orientation would not reveal emergence toward the coverslip, the ratio of the position of the cyst shell crack in the population after hatching best fit the predicted values of a random direction for nauplii emergence. Centrifugation on earth was then used to create hypergravity forces of up to 73 g during postencystment embryogenesis and hatching. The upward orientation of emerging nauplii showed a high degree of correlation (r(2) =98.8%) with a linear relationship to the log of g, with 78.2% of the total hatching upward at 1 g and 91.0% hatching upward at 73 g.

Description: A new growth subroutine was developed for CERES-Wheat, a computer model of wheat (Triticum aestivum) growth and development. The new subroutine simulates canopy photosynthetic response to CO2 concentrations and light levels, and includes the effects of temperature on canopy light-use efficiency. Its performance was compared to the original CERES-Wheat V-2 10 in 30 different cases. Biomass and yield predictions of the two models were well correlated (correlation coefficient r 〉 0.95). As an application, summer growth of spring wheat was simulated at one site. Modeled crop responses to higher mean temperatures, different amounts of minimum and maximum warming, and doubled CO2 concentrations were compared to observations. The importance of irrigation and nitrogen fertilization in modulating the wheat crop climatic responses were also analyzed. Specifically, in agreement with observations, rainfed crops were found to be more sensitive to CO2 increases than irrigated ones. On the other hand, low nitrogen applications depressed the ability of the wheat crop to respond positively to CO2 increases. In general, the positive effects of high CO2 on grain yield were found to be almost completely counterbalanced by the negative effects of high temperatures. Depending on how temperature minima and maxima were increased, yield changes averaged across management practices ranged from -4% to 8%.

Description: The cytoskeleton of columella cells is believed to be involved in maintaining the developmental polarity of cells observed as a reproducible positioning of cellular organelles. It is also implicated in the transduction of gravitropic signals. Roots of sweet clover (Melilotus alba L.) seedlings were treated with a microfilament disrupter, cytochalasin D, on a slowly rotating horizontal clinostat (2 rpm). Electron micrographs of treated columella cells revealed several ultrastructural effects including repositioning of the nucleus and the amyloplasts and the formation of endoplasmic reticulum (ER) whorls. However, experiments performed during fast clinorotation (55 rpm) showed an accumulation (but no whorling) of a disorganized ER network at the proximal and distal pole and a random distribution of the amyloplasts. Therefore, formation of whorls depends upon the speed of clinorotation, and the overall impact of cytochalasin D suggests the necessity of microfilaments in organelle positioning. Interestingly, a similar drug treatment performed in microgravity aboard the US Space Shuttle Endeavour (STS-54, January 1993) caused a displacement of ER membranes and amyloplasts away from the distal plasma membrane. In the present study, we discuss the role of microfilaments in maintaining columella cell polarity and the utility of clinostats to simulate microgravity.

Description: In contrast to higher plants, the alga Chara has rhizoids with single membrane-bound compartments that function as statoliths in gravity perception. Previous work has demonstrated that these statoliths contain barium sulfate crystals. In this study, we show that statoliths in Chara rhizoids react with a Coomassie Brilliant Blue cytochemical stain for proteins. While statoliths did not react with silver methenamine carbohydrate cytochemistry, the monoclonal antibody CCRC-M2, which is against a carbohydrate (sycamore-maple rhamnogalacturonan I), labeled the statolith compartment. These results demonstrate that in addition to barium sulfate, statoliths in Chara rhizoids have an organic matrix that consists of protein and carbohydrate moieties. Since the statoliths were silver methenamine negative, the carbohydrate in this compartment could be a 3-linked polysaccharide. CCRC-M2 also labeled Golgi cisternae, Golgi-associated vesicles, apical vesicles, and cell walls in the rhizoids. The specificity of CCRC-M2 immunolabeling was verified by several control experiments, including the demonstration that labeling was abolished when the antibody was preabsorbed with its antigen. Since in this and a previous study (John Z. Kiss and L. Andrew Staehelin, American Journal of Botany 80: 273-282, 1993) antibodies against higher plant carbohydrates crossreacted with cell walls of Chara in a specific manner, Characean algae may be a useful model system in biochemical and molecular studies of cell walls.

Description: The recent biomedical investigations conducted on the Space Shuttle and Spacelab have provided a wealth of biomedical information, including the ability to test the efficacy of proposed countermeasures. This achievement was made possible by the ability to conduct mechanistic and control-interventive studies simultaneously with a large number of individuals over a relatively brief period and to compare these data with results obtained from the Skylab missions. Comparisons between short- and long-duration results were limited to establishing trends or extrapolating from short-duration missions. To date, we have evaluated several protocols involving the lower-body negative pressure (LBNP) device, the bicycle-ergometer, the treadmill and preparations for body-fluid replenishment. In many instances, the traditional means of applying these protocols were not sufficient to protect against space-related deconditioning. This paper will review current countermeasures and compare their efficacy to that of existing protocols. Results from in-flight and ground-based experiments will be presented to illuminate the recommended protocols and procedures.

Description: Mobile phase additives, or modulators, are used in gradient elution chromatography to facilitate separation and reduce separation time. The modulators are usually assumed to be linearly adsorbed or unadsorbed. Here, the consequences of nonlinear modulator adsorption are examined for ion-exchange gradient elution through a series of simulations. Even when the buffer salt is identical to the modulator salt, gradient deformation is observed; the extent of deformation increases as the volume of the feed is increased. When the modulator salt is different from the buffer salt, unusual effects are observed, and the chromatograms are quite different from those predicted by classical gradient elution theory. In particular, local increases in the buffer concentration are found between feed bands, and serve to improve the separation. These effects become more pronounced as the feed volume increases, and could therefore prove valuable in preparative applications.

Description: The kinetics of MT [microtubule] reorientation in primary roots of Zea mays cv. Merit, were examined 15, 30, 45, and 60 min after horizontal positioning. Confocal microscopy of longitudinal tissue sections showed no change in MT orientation 15 and 30 min after horizontal placement. However, after 45 and 60 min, MTs of the outer 4-5 cortical cell layers along the lower side were reoriented. In order to test whether MT reorientation during graviresponse is caused by an auxin gradient, we examined the organization of MTs in roots that were incubated for 1 h in solutions containing 10(-9) to 10(-6) M IAA. IAA treatment at 10(-8) M or less showed no major or consistent changes but 10(-7) M IAA resulted in MT reorientation in the cortex. The auxin effect does not appear to be acid-induced since benzoic acid (10(-5) M) did not cause MT reorientation. The region closest to the maturation zone was most sensitive to IAA. The data indicate that early stages of gravity induced curvature occur in the absence of MT reorientation but sustained curvature leads to reoriented MTs in the outer cortex. Growth inhibition along the lower side of graviresponding roots appears to result from asymmetric distribution of auxin following gravistimulation.

Description: We have developed a modified electrochromatography system which minimizes Joule heating at electric field strengths up to 125 V/cm. A non-linear equilibrium model is described which incorporates electrophoretic mobility, hydrodynamic flow velocity, and an electrically induced concentration polarization at the surface of the stationary phase. This model is able to provide useful estimates of protein retention time and velocity in a column packed with Sephadex gel and subjected to an electric field. A correlation of electrophoretic mobility of peptide and proteins with respect to their charge, molecular mass, and asymmetry enables the selection of solute target molecules for electrochromatographic separations. Good separation of protein mixtures have been obtained.

Description: Structurally altered capsular polysaccharides are produced by mutant bacteria. These polysaccharides are isolated by selecting a wild type bacterial strain and a phage producing degradative enzymes that have substrate specificity for the capsular polysaccharides produced by the wild type bacteria. Phage-resistant mutants producing capsular polysaccharides are selected and the structurally altered capsular polysaccharide is isolated therefrom.

Description: A biocalorimeter and waste management system is provided for making metabolic heat release measurements of animals or humans in a calorimeter (enclosure) using ambient air as a low velocity source of ventilating air through the enclosure. A shroud forces ventilating air to pass over the enclosure from an end open to ambient air at the end of the enclosure opposite its ventilating air inlet end and closed around the inlet end of the enclosure in order to obviate the need for regulating ambient air temperature. Psychrometers for measuring dry- and wet-bulb temperature of ventilating air make it possible to account for the sensible and latent heat additions to the ventilating air. A waste removal system momentarily recirculates high velocity air in a closed circuit through the calorimeter wherein a sudden rise in moisture is detected in the ventilating air from the outlet.

Description: Novel methods of retroviral-mediated gene transfer for the in vivo corporation and stable expression of eukaryotic or prokaryotic foreign genes in tissues of living animals is described. More specifically, methods of incorporating foreign genes into mitotically active cells are disclosed. The constitutive and stable expression of E. coli .beta.-galactosidase gene under the promoter control of the Moloney murine leukemia virus long terminal repeat is employed as a particularly preferred embodiment, by way of example, establishes the model upon which the incorporation of a foreign gene into a mitotically-active living eukaryotic tissue is based. Use of the described methods in therapeutic treatments for genetic diseases, such as those muscular degenerative diseases, is also presented. In muscle tissue, the described processes result in genetically-altered satellite cells which proliferate daughter myoblasts which preferentially fuse to form a single undamaged muscle fiber replacing damaged muscle tissue in a treated animal. The retroviral vector, by way of example, includes a dystrophin gene construct for use in treating muscular dystrophy. The present invention also comprises an experimental model utilizable in the study of the physiological regulation of skeletal muscle gene expression in intact animals.

Description: Light, through the mediation of the pigment phytochrome, modulates the gravitropic response of the shoots and roots of many plants. The transduction of both light and gravity stimuli appears to involve Ca(2+)-regulated steps, one or more of which may represent points of intersection between the two transduction chains. To be confident that Ca2+ plays a critical role in stimulus-response coupling for gravitropism, it will be important to identify specific targets of Ca2+ action whose function can be clearly linked to the regulation of growth. Calcium typically exerts its influence on cell metabolism through binding to and activating key regulatory proteins. The three best characterized of these proteins in plants are the calmodulins, calcium-dependent protein kinases, and annexins. In this review we summarize what is known about the structure and function of these proteins and speculate on how their activation by Ca2+ could influence the differential growth response of gravitropism.

Description: RNA molecules that are assembled from the four standard nucleotides contain a limited number of chemical functional groups, a characteristic that is generally thought to restrict the potential for catalysis by ribozymes. Although polypeptides carry a wider range of functional groups, many contemporary protein-based enzymes employ coenzymes to augment their capabilities. The coenzymes possess additional chemical moieties that can participate directly in catalysis and thereby enhance catalytic function. In this work, we demonstrate a mechanism by which ribozymes can supplement their limited repertoire of functional groups through RNAcatalyzed incorporation of various coenzymes and coenzyme analogues. The group I ribozyme of Tetrahymena thermophila normally mediates a phosphoester transfer reaction that results in the covalent attachment of guanosine to the ribozyme. Here, a shortened version of the ribozyme is shown to catalyze the self-incorporation of coenzymes and coenzyme analogues, such as NAD+ and dephosphorylated CoA-SH. Similar ribozyme activities may have played an important role in the "RNA world," when RNA enzymes are thought to have maintained a complex metabolism in the absence of proteins and would have benefited from the inclusion of additional functional groups.

Description: The theory of evolution through natural selection was proposed by Darwin and Wallace to explain how the characteristics of populations of animals change with time. An examination of their assumptions shows that the theory has much broader application than they originally envisaged. We now know that in appropriate environments RNA molecules or computer viruses, for example, can evolve. The adventure with which we are concerned is the quest for chemical systems that undergo processes analogous to Darwinian selection in the test tube. The search is not restricted to systems that are closely related to nucleic acids, although most of the available experimental evidence concerns such systems. A population of molecules satisfies all the requirements of the theory is there are different kinds of molecules in the population and if each individual molecule can direct the formation of copies of itself, then a population of molecules will adapt to a varying environment by changing its composition so as to maintain as high as possible a rate of replication. Sol Spiegelman is the inventor of 'unnatural selection'. He showed clearly that populations of RNA molecules evolve when replicated repeatedly by Q beta RNA polymerase under a chosen set of adverse reaction conditions. In the systems that he studied, the initial population was fairly homogeneous and much of the variation was created during the course of the experiment by mutation, that is, error of replication. The term 'unnatural selection' will be used loosely to describe evolution of nucleic acids or other replicatable polymers in vitro. The term 'Natural Selection' will be reserved for the evolution of living organisms and their viruses. Natural Section usually involves the coevolution of nucleic acids and proteins, while 'unnatural selection', as practiced so far, allows replicating nucleic acids to evolve but hold constant the enzymes that catalyze replication. It is widely believed that biology based on DNA, RNA, and proteins was preceded by the biology of an 'RNA world' in which enzymes were composed of RNA alone. The origin of RNA replication is thus the central puzzle of the origins of life. Consequently, RNA-catalyzed RNA replication is presently one of the main goals of experimental work on unnatural selection. However, there is also a more distant goal, namely, to achieve replication and selection in systems unrelated to RNA. These different systems are discussed in this article.

Description: This study examined efferent output from the median preoptic nucleus (MNPO) to pontine noradrenergic and serotonergic cell groups using an anterograde tracing technique (Phaseolus vulgaris leucoagglutinin, PHA-L) combined with glucose oxidase immunocytochemistry to serotonin (5-HT) or to dopamine-beta-hydroxylase (DBH). Injections of PHA-L into the ventral MNPO resulted in moderate axonal labeling within the region of the B7 and B8 serotonergic groups in the dorsal raphe. PHA-L labeled fibers and punctate processes were observed in close apposition to many of the 5-HT immunoreactive neurons in these regions. In contrast, sparse terminal labeling was found within the B5 group in the raphe pontis nucleus, and only trace fiber labeling observed in the B3 and B6 groups. Efferents from the MNPO also provided moderate innervation to the A6 and A7 noradrenergic groups. PHA-L labeled punctate processes were found most frequently in close apposition to DBH-immunoreactive neurons at mid- to caudal levels of the locus coeruleus. Some labeled axons were also present within the A7 and A5 groups. Additionally, a close apposition between labeled MNPO efferents and 5-HT fibers within the lateral parabrachial nucleus was observed. The results indicate the MNPO provides a topographic innervation of monoaminergic groups in the upper brainstem.

Description: An autotrophic theory of the origin of metabolism and life has been proposed in which carbon dioxide is reduced by ferrous sulfide and hydrogen sulfide by means of a reversed citric acid cycle, leading to the production of amino acids. Similar processes have been proposed for purine synthesis. Ferrous sulfide is a strong reducing agent in the presence of hydrogen sulfide and can produce hydrogen as well as reduce alkenes, alkynes, and thiols to saturated hydrocarbons and reduce ketones to thiols. However, the reduction of carbon dioxide has not been demonstrated. We show here that no amino acids, purines, or pyrimidines are produced from carbon dioxide with the ferrous sulfide and hydrogen sulfide system. Furthermore, this system does not produce amino acids from carboxylic acids by reductive amination and carboxylation. Thus, the proposed autotrophic theory, using carbon dioxide, ferrous sulfide, and hydrogen sulfide, lacks the robustness needed to be a geological process and is, therefore, unlikely to have played a role in the origin of metabolism or the origin of life.

Description: The acromiodeltoid shoulder muscle was demonstrated to be a suitable model for spaceflight studies. The muscle contains a mixture of fast and slow fibers, permitting analysis of muscle fiber type specific changes. Two biopsy sites per muscle were identified that provided samples not degraded by the biopsy procedure. Both sites contained sufficient numbers fibers for determining changes in fiber type percentages and size. There was adequate bilateral symmetry regarding fiber type composition in the left and right muscles such that a total of four times points can be compared. The ESOP cage did not cause atrophy of deltoid muscle fibers; this means that microgravity-induced atrophy should be detectable. As expected, muscle excision stimulated muscle IgM and IgG muscle autoantibody production. Nonrestrained control animals suppressed this response whereas restrained monkeys showed an abnormally pronounced response indicative a compromised immune system. The presence of ESOP cage-induced changes in the immune response may mask spaceflight-induced effects. The ESOP cage modified the dominant hand operation of the PTS. These results demonstrate the importance of high fidelity ground based controls.

Description: This paper will present the Space Station Biological Research Project (SSBRP) User Operations Facility (UOF) architecture and development strategy. A major element of the UOF at NASA Ames Research Center, the Communication and Data System (CDS) will be the primary focus of the discussions. CDS operational, telescience, security, and development objectives will be discussed along with CDS implementation strategy. The implementation strategy discussions will include: Object Oriented Analysis & Design, System & Software Prototyping, and Technology Utilization. A CDS design overview that includes: CDS Context Diagram, CDS Architecture, Object Models, Use Cases, and User Interfaces will also be presented. CDS development brings together "cutting edge" technologies and techniques such as: object oriented development, network security, multimedia networking, web-based data distribution, JAVA, and graphical user interfaces. Use of these "cutting edge" technologies and techniques translates directly to lower development and operations costs.

Description: A closed pyrolysis system has been developed as a tool for studying the reactions of organic compounds under extreme hydrothermal conditions. Small high pressure stainless steel vessels in which the ratio of sediment or sample to water has been adjusted to eliminate the headspace at peak experimental conditions confines the organic components to the bulk solid matrix and eliminates the partitioning of the organic compounds away from the inorganic components during the experiment. Confined pyrolysis experiments were performed to simulate thermally driven catagenetic changes in sedimentary organic matter using a solids to water ratio of 3.4 to 1. The extent of alteration was measured by monitoring the steroid and triterpenoid biomarkers and polycyclic aromatic hydrocarbon distributions. These pyrolysis experiments duplicated the hydrothermal transformations observed in nature. Molecular probe experiments using alkadienes, alkenes and alkanes in H2O and D2O elucidated the isomerization and hydrogenation reactions of aliphatic and the competing oxidative reactions occurring under hydrothermal conditions. This confined pyrolysis technique is being applied to test experiments on organic synthesis of relevance to chemical evolution for the origin of life.

Description: The Physiology Research Branch of Brooks AFB conducts both human and non-human primate experiments to determine the effects of microgravity and hypergravity on the cardiovascular system and to indentify the particular mechanisms that invoke these responses. Primary investigative research efforts in a non-human primate model require the calculation of total peripheral resistance (TPR), systemic arterial compliance (SAC), and pressure-volume loop characteristics. These calculations require beat-to-beat measurement of aortic flow. We have evaluated commercially available electromagnetic (EMF) and transit-time flow measurement techniques. In vivo and in vitro experiments demonstrated that the average error of these techniques is less than 25 percent for EMF and less than 10 percent for transit-time.

Description: Game-like computer tasks offer many benefits for psychological research. In this paper, the usefulness of such tasks to bridge population differences (e.g., age, intelligence, species) is discussed and illustrated. A task called ALVIN was used to assess humans' and monkeys' working memory for sequences of colors with or without tones. Humans repeated longer lists than did the monkeys, and only humans benefited when the visual stimuli were accompanied by auditory cues. However, the monkeys did recall sequences at levels comparable to those reported elsewhere for children. Comparison of similarities and differences between the species is possible because the two groups were tested with exactly the same game-like paradigm.

Description: Dark-grown wheat coleoptiles developed strong curvatures within 5 h of being transferred in orbit from a 1 g centrifuge to microgravity during an experiment flown on the IML-1 shuttle mission. The curving tendency was strongest in seedlings that were immature, with coleoptiles shorter than 10 mm at the time of transfer. The curvature direction was non-random, and directed away from the caryopsis (the coleptile face adjacent to the caryopsis becoming convex). The curvatures were most marked in the basal third of the coleoptiles, contrasting with phototropic responses, which occur in the apical third. We interpret these curvatures as being nastic, and related to the curvatures commonly reported to occur during clinostat rotation treatments.

Description: A distinctive feature of tip-growing plant cells is that cell components are distributed differentially along the length of the cell, although most ultrastructural analyses have been qualitative. The longtitudinal distribution of cell components was studied both qualitatively and quantitatively in the apical cell of dark-grown protonemata of the moss Ceratodon. The first 35 micrometers of the apical cell was analyzed stereologically using transmission electron microscopy. There were four types of distributions along the cell's axis, three of them differential: (1) tubular endoplasmic reticulum was evenly distributed, (2) cisternal endoplasmic reticulum and Golgi vesicles were distributed in a tip-to-base gradient, (3) plastids, vacuoles, and Golgi stacks were enriched in specific areas, although the locations of the enrichments varied, and (4) mitochondria were excluded in the tip-most 5 micrometers and evenly distributed throughout the remaining 30 micrometers. This study provides one of the most comprehensive quantitative, ultrastructural analyses of the distribution of cell components in the apex of any tip-growing plant cell. The finding that almost every component had its own spatial arrangement demonstrates the complexity of the organization and regulation of the distribution of components in tip-growing cells.

Description: The development of pollen and ovules in Arabidopsis thaliana on the space shuttle 'Endeavour' (STS-54) was investigated. Plants were grown on nutrient agar for 14 days prior to loading into closed plant growth chambers that received light and temperature control inside the Plant Growth Unit flight hardware on the shuttle middeck. After 6 days in spaceflight the plants were retrieved and immediately dissected and processed for light and electron microscope observation. Reproductive development aborted at an early stage. Pistils were collapsed and ovules inside were seen to he empty. No viable pollen was observed from STS-54 plants; young microspores were deformed and empty. At a late stage, the cytoplasm of the pollen contracted and became disorganized, but the pollen wall developed and the exine appeared normal. The tapetum in the flight flowers degenerated at early stages. Ovules from STS-54 flight plants stopped growing and the integuments and nucellus collapsed and degenerated. The megasporocytes appeared abnormal and rarely underwent meiosis. Apparently they enlarged, or occasionally produced a dyad or tetrad, to assume the form of a female gametophyte with the single nucleus located in an egglike cell that lacks a cell wall. Synergids, polar nuclei, and antipodals were not observed. The results demonstrate the types of lesions occurring in plant reproductive material under spaceflight conditions.

Description: Wheat seedlings grown with roots exposed to constant red light (300-500 micromoles m-2 s-1) did not accumulate chlorophyll in the leaves. In contrast, seedlings grown with their roots shielded from light accumulated chlorophylls. Chlorophyll biosynthesis could be induced in red-light-grown chlorophyll-deficient yellow plants by either reducing the red-light intensity at the root surface to 100 micromoles m-1 s-1 or supplementing with 6% blue light. The inhibition of chlorophyll biosynthesis was due to impairment of the Mg-chelatase enzyme working at the origin of the Mg-tetrapyrrole pathway. The root-perceived photomorphogenic inhibition of shoot greening demonstrates root-shoot interaction in the greening process.

Description: An improved method of flow injection analysis for aqueous nitrite ion exploits the sensitivity and selectivity of the nitric oxide (NO) chemilluminescence detector. Trace analysis of nitrite ion in a small sample (5-160 microL) is accomplished by conversion of nitrite ion to NO by aqueous iodide in acid. The resulting NO is transported to the gas phase through a semipermeable membrane and subsequently detected by monitoring the photoemission of the reaction between NO and ozone (O3). Chemiluminescence detection is selective for measurement of NO, and, since the detection occurs in the gas-phase, neither sample coloration nor turbidity interfere. The detection limit for a 100-microL sample is 0.04 ppb of nitrite ion. The precision at the 10 ppb level is 2% relative standard deviation, and 60-180 samples can be analyzed per hour. Samples of human saliva and food extracts were analyzed; the results from a standard colorimetric measurement are compared with those from the new chemiluminescence method in order to further validate the latter method. A high degree of selectivity is obtained due to the three discriminating steps in the process: (1) the nitrite ion to NO conversion conditions are virtually specific for nitrite ion, (2) only volatile products of the conversion will be swept to the gas phase (avoiding turbidity or color in spectrophotometric methods), and (3) the NO chemiluminescence detector selectively detects the emission from the NO + O3 reaction. The method is free of interferences, offers detection limits of low parts per billion of nitrite ion, and allows the analysis of up to 180 microL-sized samples per hour, with little sample preparation and no chromatographic separation. Much smaller samples can be analyzed by this method than in previously reported batch analysis methods, which typically require 5 mL or more of sample and often need chromatographic separations as well.

Description: A new route for the formation of gammacerane from tetrahymanol is proposed; in addition to dehydration and hydrogenation, sulphurisation and early C-S cleavage are shown to be important in the pathway of formation, especially in marine sediments. Evidence is twofold. First, relatively large amounts of the gammacerane skeleton are sequestered in S-rich macromolecular aggregates formed by natural sulphurisation of functionalised lipids. Selective cleavage of polysulphide linkages with MeLi/MeI led to formation of 3-methylthiogammacerane, indicating that the gammacerane skeleton is primarily bound via sulphur at position 3, consistent with the idea that tetrahymanol (or the corresponding ketone) is the precursor for gammacerane. Second, upon mild artificial maturation of two sediments using hydrous pyrolysis, gammacerane is released from S-rich macromolecular aggregates by cleavage of the relatively weak C-S bonds. The stable carbon isotopic compositions of gammacerane and lipids derived from primary producers and green sulphur bacteria in both the Miocene Gessoso-solfifera and Upper Jurassic Allgau Formations indicate that gammacerane is derived from bacterivorous ciliates which were partially feeding on green sulphur bacteria. This demonstrates that anaerobic ciliates living at or below the chemocline are important sources for gammacerane, consistent with the fact that ciliates only biosynthesize tetrahymanol if their diet is deprived of sterols. This leads to the conclusion that gammacerane is an indicator for water column stratification, which solves two current enigmas in gammacerane geochemistry. Firstly, it explains why gammacerane is often found in sediments deposited under hypersaline conditions but is not necessarily restricted to this type of deposits. Secondly, it explains why lacustrine deposits may contain abundant gammacerane since most lakes in the temperate climatic zones are stratified during summer.

Description: Caulonemata of the moss Funaria were examined to determine whether they are gravitropic. Funaria and Physcomitrella were also evaluated to compare amyloplast sedimentation with that of Ceratodon. Protonemata were either chemically fixed in place or examined alive using infrared timelapse videomicroscopy. Funaria caulonemata were found to be negatively gravitropic, i.e., they grew upwards in the dark. Upward curvature reversed temporarily before cytokinesis in Funaria, a phenomenon already known for Ceratodon and Physcomitrella. Most horizontal and upward-curving Funaria tip cells contained a broad subapical zone where plastid sedimentation occurred. In dark-grown Physcomitrella caulonemata, sedimentation was detected by the presence of a thin, amyloplast-free strip of cytoplasm at the top of the cell. These results suggest that gravitropism and subapical amyloplast sedimentation may be relatively common in moss caulonemata.

Description: Soluble protein expression in Arabidopsis thaliana L. (Heynh.) leaf and stem tissue was examined after chronic clinorotation. Seeds of Arabidopsis were germinated and plants grown to maturity on horizontal or vertical slow-rotating clinostats (1 rpm) or in stationary vertical control units. Total soluble proteins and in vivo-labeled soluble proteins isolated from these plants were analyzed by two-dimensional SDS PAGE and subsequent fluorography. Visual and computer analysis of the resulting protein patterns showed no significant differences in either total protein expression or in active protein synthesis between horizontal clinorotation and vertical controls in the Arabidopsis leaf and stem tissue. These results show chronic clinorotation does not cause gross changes in protein expression in Arabidopsis.

Description: The authors introduce the chapter with a discussion of lessons from nature, agriculture, and landscapes; terms and definitions; and an historical perspective of mechanical stress regulation of plant growth and development. Topics include developmental responses to mechanical stress; mechanical stress-environment interactions; metabolic, productivity, and compositional changes; hormonal involvement; mechanoperception and early transduction mechanisms; applications in agriculture; and research implications. The discussion of hormonal involvement in mechanical stress physiology includes ethylene, auxin, gibberellins, and other phytohormones. The discussion of applications in agriculture examines windbreaks, nursery practices, height control and conditioning, and enhancement of growth and productivity. Implications for research are related to handling plant materials, space biology, and future research needs.

Description: This minireview focuses on the application of fluorogenic compounds in the detection of bacteria with particular emphasis on the assessment of physiological activity using epifluorescence microscopy. Microbiological applications of several related methods will also be reviewed.

Description: During the gravitropic response, auxin-sensitivity of the lower flanks of leaf-sheath pulvini of Avena sativa (oat) is at least 1000-fold higher than those of the upper flanks and non-gravistimulated pulvini. When the pulvini are treated with 1 mM Ca2+, a 10-fold increase in auxin-sensitivity of the pulvini is observed. Related to this difference in auxin-sensitivity, in vitro activation of the vanadate-sensitive H(-)-ATPase by IAA was observed. Results show that the activation of the H(+)-ATPase by IAA is probably mediated by soluble protein factors and that the H(+)-ATPase prepared from the lower flanks is activated by IAA with a 1000-fold higher auxin-sensitivity as compared with that from the upper flanks of the graviresponding pulvini. Ammonium sulfate fractionation experiments show that these soluble protein factors are in the 30 to 60% fraction. Auxin-binding assays reveal that lower flanks contain more high-affinity soluble auxin-binding sites (kD; on the order of 10(-9) M) and less low-affinity soluble auxin-binding sites (kD; on the order of 10(-6) M) than upper flanks. It is concluded that differential auxin-sensitivity of graviresponding oat-shoot pulvini is achieved by the modulation of affinities of auxin-binding sites in upper and lower flanks of the pulvini, that Ca2+ is involved in such modulation, and that one of the probable cellular functions of these auxin binding sites is the activation of the proton pump on the plasma membranes.

Description: The extent to which gravity, and especially the lack thereof, can affect normal development in higher organisms is poorly understood. Underlying this question is the assumption that normal development depends on the embryo's ability to maintain a programmed temporal and spatial coordination of morphogenetic events. There are several reports documenting the apparently normal development of several vertebrate species, including mammals, under conditions of exposure to space flight during various periods of the development process. Evidence to the contrary also exists and it is therefore likely that some alterations in morphology do occur in a microgravity environment. Although subsequent development may appear overtly normal, more subtle abnormalities result. In all studies, the evaluation is restricted by the few numbers of specimens that can be examined and the relatively insensitive techniques for assessing potentially subtle effects. In the present discussion, we summarize some observations of mammalian development made in microgravity and consider which stages might be expected to be differentially sensitive to altered gravity conditions. While we emphasize mammalian development, we discuss the suitability of another model system for examining such effects in a cross-species context. Furthermore, we consider recent developments in our understanding of the molecular genetic program regulating embryogenesis that could serve as markers for assessing perturbations of development.

Description: Light-emitting diodes (LEDs) are a potential irradiation source for intensive plant culture systems and photobiological research. They have small size, low mass, a long functional life, and narrow spectral output. In this study, we measured the growth and dry matter partitioning of 'Hungarian Wax' pepper (Capsicum annuum L.) plants grown under red LEDs compared with similar plants grown under red LEDs with supplemental blue or far-red radiation or under broad spectrum metal halide (MH) lamps. Additionally, we describe the thermal and spectral characteristics of these sources. The LEDs used in this study had a narrow bandwidth at half peak height (25 nm) and a focused maximum spectral output at 660 nm for the red and 735 nm for the far-red. Near infrared radiation (800 to 3000 nm) was below detection and thermal infrared radiation (3000 to 50,000 nm) was lower in the LEDs compared to the MH source. Although the red to far-red ratio varied considerably, the calculated phytochrome photostationary state (phi) was only slightly different between the radiation sources. Plant biomass was reduced when peppers were grown under red LEDs in the absence of blue wavelengths compared to plants grown under supplemental blue fluorescent lamps or MH lamps. The addition of far-red radiation resulted in taller plants with greater stem mass than red LEDs alone. There were fewer leaves under red or red plus far-red radiation than with lamps producing blue wavelengths. These results indicate that red LEDs may be suitable, in proper combination with other wavelengths of light, for the culture of plants in tightly controlled environments such as space-based plant culture systems.

Description: 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.

Description: Previous studies have indicated that acid-optimal cysteine proteinase(s) in the endosomal-lysosomal compartments, cathepsins, play a critical role in the proteolytic processing of endocytosed proteins to generate the antigenic peptides presented to the immune system on major histocompatibility complex (MHC) class II molecules. The presentation of these peptides and the expression of MHC class II molecules by macrophages and lymphocytes are stimulated by gamma-interferon (gamma-IFN). We found that treatment of human U-937 monocytes with gamma-IFN increased the activities and the content of the two major lysosomal cysteine proteinases, cathepsins B and L. Assays of protease activity, enzyme-linked immunosorbant assays (ELISA) and immunoblotting showed that this cytokine increased the amount of cathepsin B 5-fold and cathepsin L 3-fold in the lysosomal fraction. By contrast, the aspartic proteinase, cathepsin D, in this fraction was not significantly altered by gamma-IFN treatment. An induction of cathepsins B and L was also observed in mouse macrophages, but not in HeLa cells. These results suggest coordinate regulation in monocytes of the expression of cathepsins B and L and MHC class II molecules. Presumably, this induction of cysteine proteases contributes to the enhancement of antigen presentation by gamma-IFN.

Description: High-voltage electron-microscopic tomographic (3D) studies of the ultrastructural interaction between mineral and organic matrix in a variety of calcified tissues reveal different crystal structural and organizational features in association with their respective organic matrices. In brittle or weak pathologic or ectopic calcifications, including examples of osteogenesis imperfecta, calciphylaxis, calcergy, and dermatomyositis, hydroxyapatite crystals occur in various sizes and shapes and are oriented and aligned with respect to collagen in a manner which is distinct from that found in normal calcified tissues. A model of collagen-mineral interaction is proposed which may account for the observed crystal structures and organization. The results indicate that the ultimate strength, support, and other mechanical properties provided by a calcified tissue are dependent in part upon the molecular structure and arrangement of its constituent mineral crystals within their organic matrix.

Description: The purpose of the present study was to examine the effects of acute (3 days) unilateral diaphragm denervation (DNV) on 1) levels of alpha 1(I) and alpha 1(III) procollagen mRNA; 2) collagen concentration [hydroxyproline (HYP)]; 3) amount of the nonreducible collagen cross-link hydroxylysylpyridinoline (HP); and 4) the passive force-length relationship of the muscle. The levels of alpha 1(I) and alpha 1(III) procollagen mRNA, HYP concentration, and amount of HP were measured in muscle segments from the midcostal region of DNV and intact (INT) hemidiaphragms of adult male Fischer 344 rats (250-300 g). The in vitro passive force-length relationship of DNV and INT hemidiaphragm was determined by lengthening and shortening the diaphragm muscle segments from 85 to 115% of optimal length at a constant velocity (0.6 optimal length/s). Three days after DNV, the level of alpha 1(I) procollagen mRNA was increased over 15-fold in the DNV hemidiaphragm compared with INT (P 〈 0.05), whereas the level of alpha 1(III) procollagen mRNA was increased by approximately sixfold in the DNV hemidiaphragm compared with INT (P 〈 0.05). Collagen (HYP) concentration did not differ between groups, averaging 8.7 and 8.9 micrograms/mg dry wt for the DNV and INT hemidiaphragms, respectively. In addition, there was no difference in the amount of the mature nonreducible collagen cross-link HP between the DNV and INT hemidiaphragms (0.66 vs. 0.76 mole HP/mole collagen, respectively). The amount of passive force developed during lengthening did not differ between DNV and INT hemidiaphragms. These data indicate that acute DNV of the hemidiaphragm is associated with an increase in the mRNA level of the two principal fibrillar collagen phenotypes in skeletal muscle. However, despite extensive muscle remodeling, the passive force-length relationship of the DNV hemidiaphragm is unaffected compared with the INT muscle.

Description: 1. The effect of the putative neural messenger carbon monoxide (CO) and the role of the cGMP second-messenger system for olfactory signal generation was examined in isolated olfactory receptor neurons (ORNs) of the tiger salamander. 2. With the use of whole cell voltage-clamp recordings in combination with a series of ionic and pharmological tests, it is demonstrated that exogenously applied CO is a potent activator (K1/2 = 2.9 microM) of cyclic nucleotide-gated (CNG) channels previously described to mediate odor transduction. 3. Several lines of evidence suggest that CO mediates its effect through stimulation of a soluble guanylyl cyclase (sGC) leading to formation of the second-messenger cGMP. This conclusion is based on the findings that CO responses show an absolute requirement for guanosine 5'-triphosphate (GTP) in the internal solution, that no direct effect of CO on CNG currents in the absence of GTP is detectable, and that a blocker of sGC activation, LY85383 (10 microM), completely inhibits the CO response. 4. The dose-response curve for cGMP at CNG channels is used as a calibration to provide a quantitative estimate of the CO-stimulated cGMP formation. This analysis implies that CO is a potent activator of olfactory sGC. 5. Perforated patch recordings using amphotericin B demonstrate that low micromolar doses of CO effectively depolarize the membrane potential of ORNs through tonic activation of CNG channels. This effect in turn regulates excitable and adaptive properties of ORNs and modulates neuronal responsiveness. 6. These data argue for an important role of the cGMP pathway in olfactory signaling and support the idea that CO may function as a diffusible messenger in the olfactory system.

Description: Projections of the entorhinal cortex to the hippocampus are well known from the classical studies of Cajal (Ramon y Cajal, 1904) and Lorente de No (1933). Projections from the entorhinal cortex to neocortical areas are less well understood. Such connectivity is likely to underlie the consolidation of long-term declarative memory in neocortical sites. In the present study, a projection arising in layer V of the entorhinal cortex and terminating in a polymodal association area of the superior temporal gyrus has been identified with the use of retrograde tracing. The dendritic arbors of neurons giving rise to this projection were further investigated by cell filling and confocal microscopy with computer reconstruction. This analysis demonstrated that the dendritic arbor of identified projection neurons was largely confined to layer V, with the exception of a solitary, simple apical dendrite occasionally ascending to superficial laminae but often confined to the lamina dissecans (layer IV). Finally, immunoreactivity for glutamate-receptor subunit proteins GluR 5/6/7 of the dendritic arbor of identified entorhinal projection neurons was examined. The solitary apical dendrite of identified entorhinal projection neurons was prominently immunolabeled for GluR 5/6/7, as was the dendritic arbor of basilar dendrites of these neurons. The restriction of the large bulk of the dendritic arbor of identified entorhinal projection neurons to layer V implies that these neurons are likely to be heavily influenced by hippocampal output arriving in the deep layers of the entorhinal cortex. Immunoreactivity for GluR 5/6/7 throughout the dendritic arbor of such neurons indicates that this class of glutamate receptor is in a position to play a prominent role in mediating excitatory neurotransmission within hippocampal-entorhinal circuits.

Description: A new surface-source model has been developed to account for the bioelectrical potential on the body surface. A single-layer surface-charge model on the body surface has been developed to equivalently represent bioelectrical sources inside the body. The boundary conditions on the body surface are discussed in relation to the surface-charge in a half-space conductive medium. The equivalent body surface-charge is shown to be proportional to the normal component of the electric field on the body surface just outside the body. The spatial resolution of the equivalent surface-charge distribution appears intermediate between those of the body surface potential distribution and the body surface Laplacian distribution. An analytic relationship between the equivalent surface-charge and the surface Laplacian of the potential was found for a half-space conductive medium. The effects of finite spatial sampling and noise on the reconstruction of the equivalent surface-charge were evaluated by computer simulations. It was found through computer simulations that the reconstruction of the equivalent body surface-charge from the body surface Laplacian distribution is very stable against noise and finite spatial sampling. The present results suggest that the equivalent body surface-charge model may provide an additional insight to our understanding of bioelectric phenomena.

Description: We screened a panel of monoclonal antibodies against selected macrophage cell surface molecules for their ability to inhibit enterotoxin binding to major histocompatibility complex class II-negative C2D (H-2b) macrophages. Two monoclonal antibodies, HB36 and TIB126, that are specific for the alpha 2 domain of major histocompatibility complex class I, blocked staphylococcal enterotoxins A and B (SEA and SEB, respectively) binding to C2D macrophages in a specific and concentration-dependent manner. Inhibitory activities were haplotype-specific in that SEA and SEB binding to H-2k or H-2d macrophages was not inhibited by either monoclonal antibody. HB36, but not TIB126, inhibited enterotoxin-induced secretion of cytokines by H-2b macrophages. Lastly, passive protection of D-galactosamine-sensitized C2D mice by injection with HB36 antibody prevented SEB-induced death. Therefore, SEA and SEB binding to the alpha 2 domain of the H-2Db molecule induces biological activity and has physiological consequences.

Description: Cultured adult rabbit cardiac myocytes treated with recombinant growth factors display enhanced rates of protein accumulation (ie, growth) in response to insulin and insulin-like growth factors (IGFs), but epidermal growth factor, acidic or basic fibroblast growth factor, and platelet-derived growth factor failed to increase contractile protein synthesis or growth of the heart cells. Insulin and IGF-1 increased growth rates by stimulating anabolic while simultaneously inhibiting catabolic pathways, whereas IGF-2 elevated growth modestly by apparently inhibiting lysosomal proteolysis. Neutralizing antibodies directed against either IGF-1 or IGF-2 or IGF binding protein 3 blocked protein accumulation. A monoclonal antibody directed against the IGF-1 receptor also inhibited changes in protein turnover provoked by recombinant human IGF-1 but not IGF-2. Of the other growth factors tested, only transforming growth factor-beta 1 increased the fractional rate of myosin heavy chain (MHC) synthesis, with beta-MHC synthesis being elevated and alpha-MHC synthesis being suppressed. However, the other growth factors were able to modestly stimulate the rate of DNA synthesis in this preparation. Bromodeoxyuridine labeling revealed that these growth factors increased DNA synthesis in myocytes and nonmyocytes alike, but the heart cells displayed neither karyokinesis or cytokinesis. In contrast, cocultures of cardiac myocytes and nonmyocytes and nonmyocyte-conditioned culture medium failed to enhance the rate of cardiac MHC synthesis or its accumulation, implying that quiescent heart cells do not respond to "conditioning" by cardiac nonmyocytes. These findings demonstrated that insulin and the IGFs promote passively loaded cultured adult rabbit heart cells to hypertrophy but suggest that other growth factors tested may be limited in this regard.

Description: Tobacco and Arabidopsis plants, expressing a transgene for the calcium-sensitive luminescent protein apoaequorin, revealed circadian oscillations in free cytosolic calcium that can be phase-shifted by light-dark signals. When apoaequorin was targeted to the chloroplast, circadian chloroplast calcium rhythms were likewise observed after transfer of the seedlings to constant darkness. Circadian oscillations in free calcium concentrations can be expected to control many calcium-dependent enzymes and processes accounting for circadian outputs. Regulation of calcium flux is therefore fundamental to the organization of circadian systems.

Description: PURPOSE. Lumican is a major proteoglycan of vertebrate cornea. This study characterizes mouse lumican, its molecular form, cDNA sequence, and chromosomal localization. METHODS. Lumican sequence was determined from cDNA clones selected from a mouse corneal cDNA expression library using a bovine lumican cDNA probe. Tissue expression and size of lumican mRNA were determined using Northern hybridization. Glycosidase digestion followed by Western blot analysis provided characterization of molecular properties of purified mouse corneal lumican. Chromosomal mapping of the lumican gene (Lcn) used Southern hybridization of a panel of genomic DNAs from an interspecific murine backcross. RESULTS. Mouse lumican is a 338-amino acid protein with high-sequence identity to bovine and chicken lumican proteins. The N-terminus of the lumican protein contains consensus sequences for tyrosine sulfation. A 1.9-kb lumican mRNA is present in cornea and several other tissues. Antibody against bovine lumican reacted with recombinant mouse lumican expressed in Escherichia coli and also detected high molecular weight proteoglycans in extracts of mouse cornea. Keratanase digestion of corneal proteoglycans released lumican protein, demonstrating the presence of sulfated keratan sulfate chains on mouse corneal lumican in vivo. The lumican gene (Lcn) was mapped to the distal region of mouse chromosome 10. The Lcn map site is in the region of a previously identified developmental mutant, eye blebs, affecting corneal morphology. CONCLUSIONS. This study demonstrates sulfated keratan sulfate proteoglycan in mouse cornea and describes the tools (antibodies and cDNA) necessary to investigate the functional role of this important corneal molecule using naturally occurring and induced mutants of the murine lumican gene.

Description: Previous deletion studies using a series of COL1A1-CAT fusion genes have indicated that the 625 bp region of the COL1A1 upstream promoter between -2295 and -1670 bp is required for high levels of expression in bone, tendon, and skin of transgenic mice. To further define the important sequences within this region, a new series of deletion constructs extending to -1997, -1794, -1763, and -1719 bp has been analyzed in transgenic mice. Transgene activity, determined by measuring CAT activity in tissue extracts of 6- to 8-day-old transgenic mouse calvariae, remains high for all the new deletion constructs and drops to undetectable levels in calvariae containing the -1670 bp construct. These results indicate that the 49 bp region of the COL1A1 promoter between -1719 and -1670 bp is required for high COL1A1 expression in bone. Although deletion of the same region caused a substantial reduction of promoter activity in tail tendon, the construct extending to -1670 bp is still expressed in this tissue. However, further deletion of the promoter to -944 bp abolished activity in tendon. Gel mobility shift studies identified a protein in calvarial nuclear extracts that is not found in tendon nuclear extracts, which binds within this 49 bp region. Our study has delineated sequences in the COL1A1 promoter required for expression of the COL1A1 gene in high type I collagen-producing tissues, and suggests that different cis elements control expression of the COL1A1 gene in bone and tendon.

Description: Anti-dopamine beta-hydroxylase immunotoxin (DHIT) is an antibody-targeted noradrenergic lesioning tool comprised of a monoclonal antibody against the noradrenergic enzyme, dopamine beta-hydroxylase, conjugated to saporin, a ribosome-inactivating protein. Noradrenergic-neuron specificity and completeness and functionality of sympathectomy were assessed. Adult, male Sprague-Dawley rats were given 28.5, 85.7, 142 or 285 micrograms/kg DHIT i.v. Three days after injection, a 6% to 73% decrease in the neurons was found in the superior cervical ganglia of the animals. No loss of sensory, nodose and dorsal root ganglia, neurons was observed at the highest dose of DHIT. In contrast, the immunotoxin, 192-saporin (142 micrograms/kg), lesioned all three ganglia. To assess the sympathectomy, 2 wk after treatment (285 micrograms/kg), rats were anesthetized with urethane (1 g/kg) and cannulated in the femoral artery and vein. DHIT-treated animals' basal systolic blood pressure and heart rate were significantly lower than controls. Basal plasma norepinephrine levels were 41% lower in DHIT-treated animals than controls. Tyramine-stimulated release of norepinephrine in DHIT-treated rats was 27% of controls. Plasma epinephrine levels of DHIT animals were not reduced. DHIT-treated animals exhibited a 2-fold hypersensitivity to the alpha-adrenergic agonist phenylephrine. We conclude that DHIT selectively delivered saporin to noradrenergic neurons resulting in destruction of these neurons. Anti-dopamine beta-hydroxylase immunotoxin administration produces a rapid, irreversible sympathectomy.

Description: To examine the physiological deficit to adrenergic stimulation with aging, five younger adult (3 +/- 1 yr old) and nine older adult (17 +/- 1 yr old) healthy monkeys were studied after instrumentation with a left ventricular (LV) pressure gauge, aortic and left atrial catheters, and aortic flow probes to measure cardiac output directly. There were no significant changes in baseline hemodynamics in conscious older monkeys. For example, an index of contractility, the first derivative of LV pressure (LV dP/dt) was similar (3,191 +/- 240, young vs. 3,225 +/- 71 mmHg/s, old) as well as in isovolumic relaxation, tau (24.3 +/- 1.7 ms, young vs. 23.0 +/- 1.0 ms, old) was similar. However, inotropic, lusitropic, and chronotropic responses to isoproterenol (Iso; 0.1 micrograms/kg), norepinephrine (NE; 0.4 micrograms/kg), and forskolin (For; 75 nmol/kg) were significantly (P 〈 0.05) depressed in older monkeys. For example. Iso increased LV dP/dt by by 146 +/- 14% in younger monkeys and by only 70 +/- 5% in older monkeys. Iso also reduced tau more in younger monkeys (-28 +/- 7%) compared with older monkeys (-13 +/- 3%). Furthermore, peripheral vascular responsiveness to Iso, NE, For, and phenylephrine (PE; 5 micrograms/kg) was significantly (P 〈 0.05) reduced in older monkeys. For example, phenylephrine (5 micrograms/kg) increased total peripheral resistence by 69 +/- 4% in younger monkeys and by only 45 +/- 3% in older monkeys. Thus in older monkeys without associated cardiovascular disease, baseline hemodynamics are preserved, but adrenergic receptor responsiveness is reduced systemically, not just in the heart.

Description: In order to establish guidelines for exposure of astronauts to iodine, used as a water disinfectant in space, we studied the usefulness of hair, saliva, and urine for biological monitoring in humans and in the human hair/nude mouse model. The monitoring of iodine in patients that received 150 mCi of Na131I (carrier-free) showed similar patterns of elimination for blood, saliva, and urine. The mean correlation coefficient (r) between iodine elimination for blood/saliva was 0.99, for blood/urine, 0.95, and for saliva/urine, 0.97. The absolute value of iodine concentrations in urine revealed marked variability, which was corrected by adjusting for creatinine levels. The autoradiographic studies of human hair demonstrated that iodine is rapidly incorporated into external layers of the hair root and can be removed easily during washing. These data were confirmed after iodine exposure using the human hair/nude mouse model. Hair does not provide satisfactory information about exposure due to unstable incorporation of iodine. The most useful medium for biological monitoring of astronauts exposed to high doses of iodine in drinking water is urine, when adjusted for creatinine, and saliva, if quantitative evaluation of flow rate is provided.

Description: The neurochemical characteristics of the neuronal subsets that furnish different types of corticocortical connections have been only partially determined. In recent years, several cytoskeletal proteins have emerged as reliable markers to distinguish subsets of pyramidal neurons in the cerebral cortex of primates. In particular, previous studies using an antibody to nonphosphorylated neurofilament protein (SMI-32) have revealed a consistent degree of regional and laminar specificity in the distribution of a subpopulation of pyramidal cells in the primate cerebral cortex. The density of neurofilament protein-immunoreactive neurons was shown to vary across corticocortical pathways in macaque monkeys. In the present study, we have used the antibody SMI-32 to examine further and to quantify the distribution of a subset of corticocortically projecting neurons in a series of long ipsilateral corticocortical pathways in comparison to short corticocortical, commissural, and limbic connections. The results demonstrate that the long association pathways interconnecting the frontal, parietal, and temporal neocortex have a high representation of neurofilament protein-enriched pyramidal neurons (45-90%), whereas short corticocortical, callosal, and limbic pathways are characterized by much lower numbers of such neurons (4-35%). These data suggest that different types of corticocortical connections have differential representation of highly specific neuronal subsets that share common neurochemical characteristics, thereby determining regional and laminar cortical patterns of morphological and molecular heterogeneity. These differences in neuronal neurochemical phenotype among corticocortical circuits may have considerable influence on cortical processing and may be directly related to the type of integrative function subserved by each cortical pathway. Finally, it is worth noting that neurofilament protein-immunoreactive neurons are dramatically affected in the course of Alzheimer's disease. The present results support the hypothesis that neurofilament protein may be crucially linked to the development of selective neuronal vulnerability and subsequent disruption of corticocortical pathways that lead to the severe impairment of cognitive function commonly observed in age-related dementing disorders.

Description: 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.

Description: A set of cytological studies performed in the utricle and saccule of Astronotus ocellatus (Teleostei, Percomorphi, Cichlidae) identified two basic types of hair cells and others with some intermediate characteristics. This paper reports on applying the same techniques to the saccule of Carassius auratus (Teleostei, Otophysi, Cyprinidae) and demonstrates similar types of hair cells to those found in Astronotus. Since Carassius and Astronous are species of extreme taxonomic distance within the Euteteostei, two classes of mechanoreceptive hair cells are likely to represent the primitive condition for sensory receptors in the euteleost inner ear and perhaps in all bony fish ears.

Description: A novel organ culture system has been developed to study the regulation of statoconia production in the gravity sensing organ in Aplysia californica. Statocysts were cultured in Leibovitz (LI5) medium supplemented with salts and Aplysia haemolymph for four days at 17 C. The viability of the system was evaluated by examining four parameters: statocyst morphology, the activity of the mechanosensory cilia in the statocyst, production of new statoconia during culture and change in statoconia volume after culture. There were no morphological differences in statocysts before and after culture when ciliary beating was maintained. There was a 29% increase in the number of statoconia after four days in culture. Mean statocyst, statolith and statoconia volumes were not affected by culture conditions. The presence of carbonic anhydrase in the statocysts was shown using immunohistochemistry. When statocysts were cultured in the presence of 4.0 x 10(exp -4) M acetazolamide to inhibit the enzyme activity, there was a decrease in statoconia production and statoconia volume, indicating a role for this enzyme in statoconia homeostasis, potentially, via pH regulation. These studies are the first to report a novel system for the culture of statocysts and show that carbonic anhydrase is involved in the regulation of statoconia volume and production.

Description: Analysis of the structure of the ears in teleost fishes has led to the tentative suggestion that otolithic endorgans may function differently, in different species. Recently, evidence has demonstrated different 'types' of sensory hair cells can be found in the ears of teleost fishes, and individual hair cell types are found in discrete regions of individual sensory, epithelia. The presence of multiple hair cell types in fishes provides strong support to the hypothesis of regional differences in the responses of individual otolithic sensory epithelia. The finding of hair cell types in fishes that closely resemble those found in amniote vestibular endorgans also suggests that hair cell heterogeneity arose earlier in the evolution of the vertebrate ear than previously thought.

Description: Stretch-induced skeletal muscle growth may involve increased autocrine secretion of insulin-like growth factor-1 (IGF-1) since IGF-1 is a potent growth factor for skeletal muscle hypertrophy, and stretch elevates IGF-1 mRNA levels in vivo. In tissue cultures of differentiated avian pectoralis skeletal muscle cells, nanomolar concentrations of exogenous IGF-1 stimulated growth in mechanically stretched but not static cultures. These cultures released up to 100 pg of endogenously produced IGF-1/micro-g of protein/day, as well as three major IGF binding proteins of 31, 36, and 43 kilodaltons (kDa). IGF-1 was secreted from both myofibers and fibroblasts coexisting in the muscle cultures. Repetitive stretch/relaxation of the differentiated skeletal muscle cells stimulated the acute release of IGF-1 during the first 4 h after initiating mechanical activity, but caused no increase in the long-term secretion over 24-72 h of IGF-1, or its binding proteins. Varying the intensity and frequency of stretch had no effect on the long-term efflux of IGF-1. In contrast to stretch, embedding the differentiated muscle cells in a three-dimensional collagen (Type I) matrix resulted in a 2-5-fold increase in long-term IGF-1 efflux over 24-72 h. Collagen also caused a 2-5-fold increase in the release of the IGF binding proteins. Thus, both the extracellular matrix protein type I collagen and stretch stimulate the autocrine secretion of IGF-1, but with different time kinetics. This endogenously produced growth factor may be important for the growth response of skeletal myofibers to both types of external stimuli.

Description: Electronic dimming of high-intensity discharge lamps offers control of Photosynthetic Photon Flux (PPF) but is often characterized as causing significant spectral changes. Growth chambers with 400-W Metal Halide (MH) and High-Pressure Sodium (HPS) lamps were equipped with a dimmer system using Silicon-Controlled Rectifiers (SCR) as high-speed switches. Phase control operation turned the line power off for some period of the alternating current cycle. At full power, the electrical input to HPS and MH lamps was 480 W (root mean squared) and could be decreased to 267 W and 428 W, respectively, before the arc was extinguished. Concomitant with this decrease in input power, PPF decreased by 60% in HPS and 50% in MH. The HPS lamp has characteristic spectral peaks at 589 and 595 nm. As power to the HPS lamps was decreased, the 589-nm peak remained constant while the 595-nm peak decreased, equaling the 589-nm peak at 345-W input, and 589-nm peak was almost absent at 270-W input. The MH lamp has a broader spectral output but also has a peak at 589 nm and another smaller peak at 545 nm. As input power approached 428 W, the 589-nm peak shifted to 570 nm. While the spectrum changed as input power was decreased in the MH and HPS lamps, the phytochrome equilibrium ratio (P(sub ft):P(sub tot)) remains unchanged for both lamp types.

Description: A body of literature suggests that the Mauthner cell startle response can be elicited by stimulation of the ear. While we know that there are projections to the M-cell from the ear, the specific endorgan(s) of the ear projecting to the M-cell are not known. Moreover, there are many reasons to question whether there is one pattern of inner ear to M-cell connection or whether the endorgan(s) projection to the M-cell varies in species that have different hearing capabilities of hearing structures. In this paper, we briefly review the structure of fish ears, with an emphasis on structural regionalization within the ear. We also review the central projections of the ear, along with a discussion of the limited data on projections to the M-cell.

Description: Intravenous glucose infusion into ambulatory dogs results in attenuation of exercise-induced increase of both rectal and thigh muscle temperatures. That glucose (Glu) infusion attenuates excessive increase in body temperature from restricted activity during confinement deconditioning. Intravenous glucose infusion attenuates the rise in exercise core temperature in deconditioned dogs by a yet undefined mechanism.

Description: The objective of this study is to assess whether a non-invasive determination of long bone cross-sectional areal properties using bone densitometry accurately estimates true long bone flexural properties. In this study, section properties of two pairs of human female embalmed tibiae were compared using two methods: special analysis of bone densitometry data, and experimental determination of flexural regidities from bone surface strain measurements during controlled loading.

Description: A lipid analysis of the tissues of a cold-seep mytilid mussel collected from the Louisiana slope of the Gulf of Mexico was used in conjunction with a compound-specific isotope analysis to demonstrate the presence of methanotrophic symbionts in the mussel gill tissue and to demonstrate the host's dependence on bacterially synthesized metabolic intermediates. The gill tissue contained large amounts of group-specific methanotrophic biomarkers, bacteriohopanoids, 4-methylsterols, lipopolysaccharide-associated hydroxy fatty acids, and type I-specific 16:1 fatty acid isomers with bond positions at delta-8, delta-10, and delta-ll. Only small amounts of these compounds were detected in the mantle or other tissues of the host animal. A variety of cholesterol and 4-methylsterol isomers were identified as both free and steryl esters, and the sterol double bond positions suggested that the major bacterially derived gill sterol(11.0% 4(alpha)-methyl-cholesta-8(14), 24-dien-3(beta)-ol) was converted to host cholesterol (64.2% of the gill sterol was cholest-5-en-3(beta)-ol). The stable carbon isotope values for gill and mantle preparations were, respectively, -59.0 and -60.4 per thousand for total tissue, -60.6 and -62.4 per thousand for total lipids, -60.2 and -63.9 per thousand for phospholipid fatty acids, and -71.8 and -73.8 per thousand for sterols. These stable carbon isotope values revealed that the relative fractionation pattern was similar to the patterns obtained in pure culture experiments with methanotrophic bacteria further supporting the conversion of the bacterial methyl-sterol pool.

Description: The effect of human cell immersed in culture liquid under a micro-gravity environment has been investigated. The study is based on the numerical simulation of the configuration of human cell affected by the time dependent variation of gravity acceleration ranging from 10(exp -3) to 2 g(sub o) (g(sub o) = 9.81 m/s(exp 2)) in 15 seconds. Both the free floating cell and the cell contacted to the upper and lower inclined walls imposed by the time-dependent reduced gravity acceleration are considered in this study. The results show that the cell configuration changes from spherical to horizontally elongated ellipsoid for both the free floating cell and the cell sitting on the lower inclined wall while the cell configuration varies from spherical to vertically elongated ellipsoid for the cell hanging to the upper inclined wall when the gravity acceleration increases. Experimental observations, carried out of human cells exposed to the variation of gravity levels, show that the results of experimental observations agree exactly with the theoretical model computation described in this paper. These results sre significant for humans exposed to the micro-gravity environment.

Description: USRA's Division of Space Life Sciences (DSLS) was established in 1983 as the Division of Space Biomedicine to facilitate participation of the university community in biomedical research programs at the NASA Johnson Space Center (JSC). The DSLS is currently housed in the Center for Advanced Space Studies (CASS), sharing quarters with the Division of Educational Programs and the Lunar and Planetary Institute. The DSLS provides visiting scientists for the Johnson Space Center; organizes conferences, workshops, meetings, and seminars; and, through subcontracts with outside institutions, supports NASA-related research at more than 25 such entities. The DSLS has considerable experience providing visiting scientists, experts, and consultants to work in concert with NASA Life Sciences researchers to define research missions and goals and to perform a wide variety of research administration and program management tasks. The basic objectives of this contract have been to stimulate, encourage, and assist research and education in the NASA life sciences. Scientists and experts from a number of academic and research institutions in this country and abroad have been recruited to support NASA's need to find a solution to human physiological problems associated with living and working in space and on extraterrestrial bodies in the solar system.

Description: The goal of these experiments was to determine the effect of microgravity on the early development of the fish medaka. There were two objectives for this flight series. The primary objective was to assess the effects of microgravity on different stages of development and to ascertain whether the relevant developmental questions can be addressed at the gross morphological level or if the issues involve more subtle questions about regulation at the molecular and cellular levels. The secondary objective was the assessment of the utility of flight hardware with the capabilities to perform embryological studies. We have been able to take advantage of the flight testing phase of the STL-B hardware to also study the effects of microgravity on the early development of the fish, Medaka. Our initial studies involved monitoring the early Medaka development and raising flight embryos for breeding. Images of the developing embryos were collected either via video which was either taken by the astronauts or broadcast to Earth. Sample video images were digitized and stored on a hard drive resident within the on-board STL-B unit. Embryos were fixed at specific intervals, returned to Earth and are being analyzed for the timing and location of molecular events associated with controlling the morphological pattern for the onset of adult structures.

Description: This paper is an overview of some of the major points to arise in the accompanying contributions of this special symposium issue. The symposium papers arose out of discussions among investigators interested in the inner ear and Mauthner cell, with the focus on hydrodynamic components that activate the Mauthner cell through the octavolateralis system. The intention of the symposium was to investigate the possibility of using our knowledge of the Mauthner system to help understand acoustic processing by the ear, and of using, our knowledge of fish hearing to better understand Mauthner cell function. This is the first attempt to take a broad look at both systems to see how they might function together. As such, these proceedings can serve as a mini-tutorial for investiaators interested in one system or the other. In this summary paper we also identify some of the major uncertainties in our understanding of the ear-Mauthner connection. These include questions about: (1) the identity of the acoustic stimuli that are neuroethologically relevant to the Mauthner system; (2) the relative importance of the various octavolateralis inputs (acoustic, vestibular, or lateral line); (3) the contribution of the different various acoustic endorgans to the Mauthner system; (4) whether the Mauthner system can distinguish sound source location; and (5) whether Mauthner neurobiology is compatible with the prevailing model (the phase model) for determining sound source location by fishes. We believe these issues provide potentially useful avenues of future investigation that should give important insights into both acoustic processing by fish and the function of the Mauthner system.

Description: Eight genomic clones of potato calmodulin (PCM1 to 8) were isolated and characterized. Sequence comparisons of different genes revealed that the deduced amino acid sequence of PCM1 had several unique substitutions, especially in the fourth Ca(2+)-binding area. The expression patterns of different genes were studied by northern analysis using the 3'-untranslated regions as probes. The expression of PCM1, 5, and 8 was highest in the stolon tip and it decreased during tuber development. The expression of PCM6 did not vary much in the tissues tested, except in the leaves, where the expression was lower; whereas, the expression of PCM4 was very low in all the tissues. The expression of PCM2 and PCM3 was not detected in any of the tissues tested. Among these genes, only PCM1 showed increased expression following touch stimulation. To study the regulation of PCM1, transgenic potato plants carrying the PCM1 promoter fused to the beta-glucuronidase (GUS) reporter gene were produced. GUS expression was found to be developmentally regulated and touch-responsive, indicating a positive correlation between the expression of PCM1 and GUS mRNAs. These results suggest that the 5'-flanking region of PCM1 controls developmental and touch-induced expression. X-Gluc staining patterns revealed that GUS localization is high in meristematic tissues such as the stem apex, stolon tip, and vascular regions.

Description: Spacelab Life Sciences-3 (SLS-3) was scheduled to be the first United States man-tended microgravity flight containing Rhesus monkeys. The goal of this flight as in the five untended Russian COSMOS Bion flights and an earlier American Biosatellite flight, was to understand the biomedical and biological effects of a microgravity environment using the non-human primate as human surrogate. The SLS-3/Rhesus Project and COSMOS Primate-BIOS flights all utilized the rhesus monkey, Macaca mulatta. The ultimate objective of all flights with an animal surrogate has been to evaluate and understand biological mechanisms at both the system and cellular level, thus enabling rational effective countermeasures for future long duration human activity under microgravity conditions and enabling technical application to correction of common human physiological problems within earth's gravity, e.g., muscle strength and reloading, osteoporosis, immune deficiency diseases. Hardware developed for the SLS-3/Rhesus Project was the result of a joint effort with the French Centre National d'Etudes Spatiales (CNES) and the United States National Aeronautics and Space Administration (NASA) extending over the last decade. The flight hardware design and development required implementation of sufficient automation to insure flight crew and animal bio-isolation and maintenance with minimal impact to crew activities. A variety of hardware of varying functional capabilities was developed to support the scientific objectives of the original 22 combined French and American experiments, along with 5 Russian co-investigations, including musculoskeletal, metabolic, and behavioral studies. Unique elements of the Rhesus Research Facility (RRF) included separation of waste for daily delivery of urine and fecal samples for metabolic studies and a psychomotor test system for behavioral studies along with monitored food measurement. As in untended flights, telemetry measurements would allow monitoring of thermoregulation, muscular, and cardiac responses to weightlessness. In contrast, the five completed Cosmos/Bion flights, lacked the metabolic samples and behavioral task monitoring, but did facilitate studies of the neurovestibular system during several of the flights. The RRF accommodated two adult 8-11 kg rhesus monkeys, while the Russian experiments and hardware were configured for a younger animal in the 44 kg range. Both the American and Russian hardware maintained a controlled environmental system, specifically temperature, humidity, a timed lighting cycle, and had means for providing food and fluids to the animal(s). Crew availability during a Shuttle mission was to be an optimal condition for retrieval and refrigeration of the animal urine samples along with a manual calcein injection which could lead to greater understanding of bone calcium incorporation. A special portable bioisolation glove box was under development to support this aspect of the experiment profile along with the capability of any contingency human intervention. As a result of recent U.S./Russian negotiations, funding for Space Station, and a series of other events, the SLS-3 mission was cancelled and applicable Rhesus Project experiments incorporated into the Russian Bion 11 and 12 missions. A presentation of the RRF and COSMOS/Bion rhesus hardware is presented along with current plans for the hardware.

Description: The objective of this study was to determine the ability of a single bout of resistance exercise alone or in combination with recombinant human growth hormone (rhGH) to stimulate myofibrillar protein synthesis (Ks) in hindlimb suspended (HLS) adult female rats. Plantar flexor muscles were stimulated with resistance exercise, consisting of 10 repetitions of ladder climbing on a 1 m grid (85 deg.), carrying an additional 50% of their body weight attached to their tails. Saline or rhGH (1 mg/kg) was administered 30' prior to exercise, and Ks was determined with a constant infusion of H-3-Leucine at 15', 60', 180', and 360' following exercise. Three days of HLS depressed Ks is approx. equal to 65% and 30-40% in the soleus and gastrocnemius muscles, respectively (p is less than or equal to 0.05). Exercise increased soleus Ks in saline-treated rats 149% 60' following exercise (p less than or equal to 0.05), decaying to that of non-exercised animals during the next 5 hours. Relative to suspended, non-exercised rats rhGH + exercise increased soleus Ks 84%, 108%, and 72% at 15', 60' and 360' following exercise (p is less than or equal to 0.05). Gastrocnemius Ks was not significantly increased by exercise or the combination of rhGH and exercise up to 360' post-exercise. Results from this study indicate that resistance exercise stimulated Ks 60' post-exercise in the soleus of HLS rats, with no apparent effect of rhGH to enhance or prolong exercise-induced stimulation. Results suggests that exercise frequency may be important to maintenance of the slow-twitch soleus during non-weightbearing, but that the ability of resistance exercise to maintain myofibrillar protein content in the gastrocnemius of hindlimb suspended rats cannot be explained by acute stimulation of synthesis.

Description: Calcium, a universal second messenger, regulates diverse cellular processes in eukaryotes. Ca-2(+) and Ca-2(+)/calmodulin-regulated protein phosphorylation play a pivotal role in amplifying and diversifying the action of Ca-2(+)- mediated signals. A chimeric Ca-2(+)/calmodulin-dependent protein kinase (CCaMK) gene with a visinin-like Ca-2(+)- binding domain was cloned and characterized from lily. The cDNA clone contains an open reading frame coding for a protein of 520 amino acids. The predicted structure of CCaMK contains a catalytic domain followed by two regulatory domains, a calmodulin-binding domain and a visinin-like Ca-2(+)-binding domain. The amino-terminal region of CCaMK contains all 11 conserved subdomains characteristic of serine/threonine protein kinases. The calmodulin-binding region of CCaMK has high homology (79%) to alpha subunit of mammalian Ca-2(+)/calmodulin-dependent protein kinase. The calmodulin-binding region is fused to a neural visinin-like domain that contains three Ca-2(+)-binding EF-hand motifs and a biotin-binding site. The Escherichia coli-expressed protein (approx. 56 kDa) binds calmodulin in a Ca-2(+)-dependent manner. Furthermore, Ca-45-binding assays revealed that CCaMK directly binds Ca-2(+). The CCaMK gene is preferentially expressed in developing anthers. Southern blot analysis revealed that CCaMK is encoded by a single gene. The structural features of the gene suggest that it has multiple regulatory controls and could play a unique role in Ca-2(+) signaling in plants.

Description: Effects of fluid ingestion on CD4+/CD8+ T-lymphocyte cell ratios were measured in four dehydrated men (ages 30-46 yr) before and after 70 min of supine submaximal (71 % VO(sub 2max) lower extremity cycle exercise. Just before exercise, Evans blue dye was injected for measurement of plasma volume. The subjects then drank one of six fluid formulations (12 ml/kg) in 3-4 min. All six mean post-hydration (pre-exercise) CD4+/CD8+ ratios (Becton-Dickinson Fluorescence Activated Cell Sorter and FACScan Consort-30 software program were below the normal range of 1.2-1.5; mean (+/- SE) and range were 0.77 +/- 0.12 and 0.39-1.15, respectively. The post-exercise ratios increased: mean = 1.36 =/- 0.15 (P less than 0.05) and range = 0.98-1.98. Regression of mean CD4+/CD8+ ratios on mean plasma osmolality resulted in pre- and post-exercise correlation coefficients of -0.76 (P less than 0.10) and -0.92 (P less than 0.01), respectively. The decreased pre-exercise ratios (after drinking) were probably not caused by the Evans blue dye but appeared to be associated more with the stress (osmotic) of dehydration. The increased post-exercise ratios to normal levels accompanied the rehydration and were not due to the varied electrolyte and osmotic concentrations of the ingested fluids or to the varied vascular volume shifts during exercise. Thus, the level of subject hydration and plasma osmotality may be factors involved in the mechanism of immune system modulation induced by exercise.

Description: To test whether gravity is required for normal amphibian development, Xenopus laevis females were induced to ovulate aboard the orbiting Space Shuttle. Eggs were fertilized in vitro, and although early embryonic stages showed some abnormalities, the embryos were able to regulate and produce nearly normal larvae. These results demonstrate that a vertebrate can ovulate in the virtual absence of gravity and that the eggs can develop to a free-living stage.

Description: To explore the role of microgravity on cytoskeletal organization and skeletal calcium deposition during fertilization, cell division, and early development, the sea urchin was chosen as a model developmental system. Methods were developed to employ light, immunofluorescence, and electron microscopy on cultures being prepared for flight on the Space Shuttle. For analysis of microfilaments, microtubules, centrosomes, and calcium-requiring events, our standard laboratory protocols had to be modified substantially for experimentation on the Space Shuttle. All manipulations were carried out in a closed culture chamber containing 35 ml artificial sea water as a culture fluid. Unfertilized eggs stored for 24 hours in these chambers were fertilized with sperm diluted in sea water and fixed with concentrated fixatives for final fixation in formaldehyde, taxol, EGTA, and MgCl2(exp -6)H2O for 1 cell to 16 cell stages to preserve cytoskeletal structures for simultaneous analysis with light, immunofluorescence, and electron microscopy, and 1.5 percent glutaraldehyde and 0.4 percent formaldehyde for blastula and plueus stages. The fixed samples wre maintained in chambers without degradation for up to two weeks after which the specimens were processed and analyzed with routine methods. Since complex manipulations are not possible in the closed chambers, the fertilization coat was removed from fixation using 0.5 percent freshly prepared sodium thioglycolate solution at pH 10.0 which provided reliable immunofluorescence staining for microtubules. Sperm/egg fusion, mitosis, cytokinesis, and calcium deposition during spicule formatin in early embryogenesis were found to be without artificial alterations when compared to cells fixed fresh and processed with conventional methods.

Description: Three 4-liter packed bed anaerobic digesters were fabricated and operated at 35 degrees C, pH around 7, and hydraulic retention time (HRT) of 20, 10 and 5 days to study the resource recovery and epidemiology in a controlled ecological life support system (CELSS). A simulated wastewater, consisted of shower water, clothwash water, dishwasher water, handwash water, and urine flush water was used as the feeding solution. Under steady-state operation, chemical oxygen demand (COD), total organic carbon (TOC), pH, nitrogen, phosphorus, and potassium wer monitored in the digester input and output solutions. The volume and the CH4/CO2 ratios in the biogas produced from the anaerobic digesters were measured. The results indicate about 90 percent of TOC is converted while only 5-8 percent of N-P-K are consumed in the digester. A multi-drug resistant strain of Salmonella choleraesuis was used as the indicator bacterium in the epidemiology study. The levels of Salmonella choleraesuis in the influent and the effluent wer determined and decimal decay rate constants, k(d), were estimated. The k(d) values were greater at higher initial doses than lower doses for the same HR, and greater for batch digestion (7.89/d) than for continuous digestion (4.28, 3.82, and 3.82/d for 20, 10, and 5 d HRT, respectively).

Description: The photochromic property of bacteriorhodopsin films is exploited in the application of a focusing schlieren optical system for the visualization of optical phase information. By encoding an image on the film with light of one wavelength and reading out with a different wavelength, the readout beam can effectively see the photographic negative of the original image. The potential advantage of this system over previous focusing schlieren systems is that the updatable nature of the bacteriorhodopsin film allows system adaptation. I discuss two image encoding and readout techniques for the bacteriorhodopsin and use film transmission characteristics to choose the more appropriate method. I demonstrate the system principle with experimental results using argon-ion and He-Cd lasers as the two light sources of different wavelengths, and I discuss current limitations to implementation with a white-light source.