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The identity of pine cell membranes bearing PAL enzyme activity, the isolation of a plasma membrane preparation from pine cells for testing as a regulatory barrier in lignification, and the measurement of the geopotential effect in pine stems are presented. A model to describe and predict the interaction of gravity and lignification of higher plants was developed.
The cellular constituents of extremely halophilic bacteria not only tolerate high salt concentration, but in many cases require it for optical functioning. The characteristics affected by salt include enzyme activity, stability, allosteric regulation, conformation and subunit association. The salt effects are of two major kinds: electrostatic shielding of negative charges by cations at low salt concentration, and hydrophobic stabilization by salting-out type salts at high salt concentration. The composition of halobacterial proteins shows an excess of acidic amino acids and a deficiency of nonpolar amino acids, which accounts for these effects. Since the cohesive forces are weaker and the repulsing forces are stronger in these proteins, preventing aggregation in salt, these structures are no longer suited for functioning in the absence of high salt concentrations. Unlike these nonspecific effects, ribosomes in halobacteria show marked preference for potassium over sodium ions. To ensure the proper intracellular ionic composition, powerful ion transport systems have evolved in the halobacteria, resulting in the extrusion of sodium ions and their replacement by potassium. It is likely that such membrane transport system for ionic movements is a necessary requisite for salt tolerance.
The goal was to assay enzynes in situ in Prochloron, and to prepare active enzyme preparations for future use. In addition, photosynthesis as an indicator of whole cell viability was measured. This indicator was used to monitor extended survival of Prochloron cells after their removal from the host animal.
Cell-free extracts prepared from Paracoccus halodenitrificans catalyzed the reduction of nitrate to ammonia in the presence of dithionite and methyl viologen. Enzyme activity was located in the soluble fraction and was associated with a cytochrome whose spectral properties resembled those of a cd-type cytochrome. Unlike the sissimilatory cd-cytochrome nitrate reductase associated with the membrane fraction of P. halodenitrificans, this soluble cd-cytochrome did not reduce nitrite to nitrous oxide.
The effects are studied of the oral administration of RU38486, a potent selective glucocorticoid antagonist, on muscle weight, non-collagen protein content, and selected enzyme activities (choline acetyltransferase, glucose 6-phosphate dehydrogenase, and glutamine synthetase) following denervation of rat skeletal muscle. Neither decreases in muscle weight, protein content, and choline acetyltransferase activity, nor increases in the activities of glucose 6-phosphate dehydrogernase and glutamine synthetase were affected by RU38486. These data do not support the hypothesis that denervation atrophy results from enhanced sensitivity of muscle to endogenous glucocorticoids.
The effect of transcutaneous electromyostimulation (EMS) on the development of atrophy and the loss of strength in lower limb musculature in humans exposed to microgravity was determined in three subjects who received EMS twice daily in a 3-d on/1-d off cycle on their dominant leg during 30 days of bedrest. The output waveform from the stimulator was sequenced to the knee extensors, knee flexors, ankle extensors, and ankle flexors, and caused three isometric contractions of each muscle group per minute. It was found that, in the dominant leg, EMS acted to attenuate the changes caused by bedrest, such as reductions in the leg volume, muscle compartment size, cross-sectional area of slow- and fast-twitch fibers, strength, and aerobic enzyme activities, and an increase in leg compliance.
The primary purpose of this research is to study the physiological mechanisms associated with the exercise performance of rats subjected to conditions of simulated weightlessness. A secondary purpose is to study related physiological changes associated with other systems. To facilitate these goals, a rodent suspension model was developed (Overton-Tipton) and a VO2 max testing procedure was perfected. Three methodological developments occurred during this past year deserving of mention. The first was the refinement of the tail suspension model so that (1) the heat dissipation functions of the caudal artery can be better utilized, and (2) the blood flow distribution to the tail would have less external constriction. The second was the development on a one-leg weight bearing model for use in simulated weightlessness studies concerned with change in muscle mass, muscle enzyme activity, and hind limb blood flow. The chemical body composition of 30 rats was determined and used to develop a prediction equation for percent fat using underwater weighing procedures to measure carcass specific gravity and to calculate body density, body fat, and fat free mass.
Portable immune-assessment system developed for use in rapidly identifying infections or contaminated environment. System combines few specific fluorescent reagents for identifying immune-cell dysfunction, toxic substances, buildup of microbial antigens or microbial growth, and potential identification of pathogenic microorganisms using fluorescent microplate reader linked to laptop computer. By using few specific dyes for cell metabolism, DNA/RNA conjugation, specific enzyme activity, or cell constituents, one makes immediate, onsite determination of person's health or of contamination of environment.
The major objective of this study was to evaluate light-regulated ethylene production during gravitropic bending in etiolated pea stems. Previous investigations indicated that ethylene production increases after gravistimulation and is associated with the later (counter-reactive) phase of bending. Additionally, changes in the counter-reaction and locus of curvature during gravitropism are greatly influenced by red light and ethylene production. Ethylene production may be regulated by the levels of available precursor (1-aminocyclopropane-l-carboxylic acid, ACC) via its synthesis, conjugation to malonyl-ACC or glutamyl-ACC, or oxidation to ethylene. The regulation of ethylene production by quantifying ACC and conjugated ACC levels in gravistimulated pea stemswas examined. Also measured was the changes in protein and enzyme activity associated with gravitropic curvature by electrophoretic and spectrophotometric techniques. An image analysis system was used to visualize and quantify enzymatic activity and transcriptional products in gravistimulated and red-light treated etiolated pea stem tissues.
Potato explants (leaf, small stem section, and axillary bud), flown on STS-73, developed tubers of 1.5 cm diameter and 1.7 g mass during the 16 day period of spaceflight. The experiment was undertaken in the ASTROCULTURE(Trademark) experiment package under controlled temperature, humidity, lighting, and carbon dioxide concentrations. The tubers formed in the explant system under microgravity had the same gross morphology, the same anatomical configuration of cells and tissues, and the same sizes, shapes, and surface character of starch granules as tubers formed in a 1 g environment. The total accumulation of starch and other energy containing compounds was singular in space flight and ground control tubers. Enzyme activity of starch synthase, starch phosphorylase, and total hydrolase was similar in spaceflight and ground controls but activity of ADP-glucose pyrophosphorylase was reduced in the spaceflight tuber tissue. This experiment documented that potatoes will metabolize and accumulate starch as effectively in spaceflight as on the ground and thus this data provides the potential for effective utilization of potatoes in life support systems of space bases.
Potato explants (leaf, small stem section, and axillary bud) flown on STS-73 developed tubers of 1.5 cm diameter and 1.7 g mass during the 16-day period of space flight. The experiment was undertaken in the ASTROCULTURE(TM) experiment package under controlled temperature, humidity, lighting, and carbon dioxide concentrations. The tubers that formed in the explant system under microgravity had the same gross morphology, the same anatomical configuration of cells and tissues, and the same sizes, shapes, and surface character of starch granules as tubers formed in a 1 g environment. The total accumulation of starch and other energy containing compounds was similar in space flight and ground control tubers. Enzyme activity of starch synthase, starch phosphorylase, and total hydrolase was similar in space flight and ground controls, but activity of ADP-glucose pyrophosphorylase was reduced in the space flight tuber tissue. This experiment documented that potatoes will metabolize and accumulate starch as effectively in space flight as on the ground. Thus, this data provides the potential for effective utilization of potatoes in life support systems of space bases.
During the purification of the "aerobic" acetyl-CoA synthetase (ACS) of Saccharomyces cerevisiae, strain LK2Gl2, it was noted that stronge at 4 C resulted in the loss of enzyme activity within 24 hr. Similar losses were observed during column chromatography. Addition of boiled extracts from either aerobic or anerobic cells completely prevents this. The stabilizing factor (SF) in these extracts is non-dialyzable and organic in nature. SF is excluded on G-25 and G-50 Sephadex columns and is slightly retarded on G-75 columns. On G-100 columns, SF elutes as a peak exactly coincident with that of cytochrome c, indicating a molecular weight of 13,000. SF activity was not destroyed by Pronase treatment, was adsorbed onto Norite, and absorbed in the UV with a single maximum at 260 nm. The action of SF could be replaced by a number of nucleotides. At 0.01 M, the order of effectiveness was: ATP>ADP>AMP>GTP>CTP>/=UTP>XTP. Even at 2 x 10(exp -4) M, ATP and ADP, but not AMP, cyclic AMP, adenosine or adenine, were effective in stabilizing this ACS. The mechanism of stabilization by ATP and AMP appears to be the same, since AMP competitively inhibited the ACS with respect to ATP in in vitro assays, while ADP gave a mixed type of inhibition, thus indicating a different mechanism. ACS from nonaerobic cells is also unstable in the absence of SF but, unlike aerobic ACS, is not affected by ATP or other nucleotides.
It is now clear that the marked loss of muscle mass that occurs with disuse, denervation or in many systemic diseases (cancer cachexia, sepsis, acidosis, various endocrine disorders) is due primarily to accelerated degradation of muscle proteins, especially myofibrillar components. Recent work primarily in Dr. Goldberg's laboratory had suggested that in these diverse conditions, the enhancement of muscle proteolysis results mainly from activation of the Ub-proteasome degradative pathway. In various experimental models of atrophy, rat muscles show a common series of changes indicative of activation of this pathway, including increases in MRNA for Ub and proteasome subunits, content of ubiquitinated proteins, and sensitivity to inhibitors of the proteasome. In order to understand the muscle atrophy seen in weightlessness, Dr. Goldberg's laboratory is collaborating with Dr. Baldwin in studies to define the changes in these parameters upon hind-limb suspension. Related experiments will explore the effects on this degradative system of exercise regimens and also of glucocorticoids, which are known to rise in space personnel and to promote muscle, especially in inactive muscles. The main goals will be: (A) to define the enzymatic changes leading to enhanced activity of the Ub-proteasome pathway in inactive muscles upon hind-limb suspension, and the effects on this system of exposure to glucocorticoids or exercise; and (B) to learn whether inhibitors of the Ub-proteasome pathway may be useful in retarding the excessive proteolysis in atrophying muscles. Using muscle extracts, Dr. Goldberg's group hopes to define the rate-limiting, enzymatic changes that lead to the accelerated Ub-conjugation and protein degradation. They have recently developed cell-free preparations from atrophying rat muscles, in which Ub-conjugation to muscle proteins is increased above control levels. Because these new preparations seem to reproduce the changes occurring in vivo, they will analyze in depth extracts from normal and atrophying muscles to compare the activities of the Ub-activating enzyme (El), the various LTh-carrier proteins (E2s), and Ub-protein ligases (E3s). Recent studies of other types of muscle wasting -suggest a very important role in muscle proteolysis of certain ubiquitination enzymes, E214k and E3-alpha(i.e. components of the "N-end pathway"). Future studies will focus in understanding their role and test whether they are in fact critical for muscle atrophy in vivo. Since weightlessness leads to a specific loss of contractile proteins and to a switching of myosin isotypes, Dr. Goldberg's group will attempt to identify the ubiquitination enzymes specifically involved in myosin degradation both in normal muscle and after hind-limb suspension.
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.
The effects of cold plasma exposure on Deinococcus radiodurans, plasmid DNA and model proteins were assessed using microbiological, spectrometric and biochemical techniques. Exposure of D. radiodurans, an extremely radiation resistant microbe, to O2 plasma (less than or equal to 25 W, approx. 45 mTorr, 90 min) yielded a approx. 99.999 % sterilization and the sterilization rate was increased approx. 10-fold at 100 W and 500 mTorr. AFM images shows that the exposed cells are significantly deformed and possess 50-70 nm concavities. IR analysis indicates the chemical degradation of lipids, proteins and carotenoids of the cell wall and membrane. Intracellular damage was indicated by major absorbance loss at 1245, 1651 and 1538/cm corresponding to degradation of DNA and proteins, respectively. Biochemical experiments demonstrate that plasmas induce strand scissions and crosslinking of plasmid DNA, and reduction of enzyme activity; the degradation is power dependent with total sample loss occurring in 60 s at 200 W and 500 mTorr. Emission spectroscopy shows that D. radiodurans is volatilized into CO2, CO, N2 and H2O confirming the removal of biological matter from contaminated surfaces. The O2 plasma impacts several cellular components predominantly through chemical degradation by atomic oxygen. A CO2, plasma, however, was not effective at degrading D. radiodurans, revealing the importance of plasma composition, which has implications for planetary protection and the contamination of Mars.
The effects of cold plasma on Deinococcus radiodurans, plasmid DNA, and model proteins were assessed using microbiological, spectrometric, and biochemical techniques. In low power O(2) plasma (approximately 25 W, approximately 45 mTorr, 90 min), D. radiodurans, a radiation-resistant bacterium, showed a 99.999% reduction in bioburden. In higher power O(2) plasma (100 W and 500 mTorr), the reduction rate increased about 10-fold and observation by atomic force microscopy showed significant damage to the cell. Damage to cellular lipids, proteins, and chromosome was indicated by losses of infrared spectroscopic peaks at 2930, 1651, 1538, and 1245 cm(-1), respectively. In vitro experiments show that O(2) plasmas induce DNA strand scissions and cross-linking as well as reduction of enzyme activity. The observed degradation and removal of biomolecules was power-dependent. Exposures to 200 W at 500 mTorr removed biomolecules to below detection limits in 60 s. Emission spectroscopy indicated that D. radiodurans cells were volatilized into CO(2), CO, N(2), and H(2)O, confirming that these plasmas were removing complex biological matter from surfaces. A CO(2) plasma was not as effective as the O(2) plasma, indicating the importance of plasma composition and the dominant role of chemical degradation. Together, these findings have implications for NASA planetary protection schemes and for the contamination of Mars.
Space flight is associated with an increase in oxidative stress after return to 1g. The effect is more pronounced after long-duration space flight. The effects lasts for several weeks after landing. In humans there is increased lipid peroxidation in erythrocyte membranes, reduction in some blood antioxidants, and increased urinary excretion of 8-iso-prostaglandin F(2alpha) and 8-oxo-7,8 dihydro-2 deoxyguanosine. Isoprostane 8-iso-prostaglandin F(2alpha) and 8-oxo-7,8 dihydro-2 deoxyguanosine are markers for oxidative damage to lipids and DNA, respectively. The changes have been attributed to a combination of the energy deficiency that occurs during flight and substrate competition for amino acids occurring between repleting muscle and other tissues during the recovery phase. The observations in humans have been complemented by rodent studies. Most rodent studies showed increased production of lipid peroxidation products postflight and decreased antioxidant enzyme activity postflight. The rodent observations were attributed to the stress associated with reentry into Earth's gravity. Decreasing the imbalance between the production of endogenous oxidant defenses and oxidant production by increasing the supply of dietary antioxidants may lessen the severity of the postflight increase in oxidative stress.
We studied a sample from the GISP 2 (Greenland Ice Sheet Project) ice core to determine the diversity and survival of microorganisms trapped in the ice at least 120,000 years ago. Previously, we examined the phylogenetic relationships among 16S ribosomal DNA (rDNA) sequences in a clone library obtained by PCR amplification from genomic DNA extracted from anaerobic enrichments. Here we report the isolation of nearly 800 aerobic organisms that were grouped by morphology and amplified rDNA restriction analysis patterns to select isolates for further study. The phylogenetic analyses of 56 representative rDNA sequences showed that the isolates belonged to four major phylogenetic groups: the high-G+C gram-positives, low-G+C gram-positives, Proteobacteria, and the Cytophaga-Flavobacterium-Bacteroides group. The most abundant and diverse isolates were within the high-G+C gram-positive cluster that had not been represented in the clone library. The Jukes-Cantor evolutionary distance matrix results suggested that at least 7 isolates represent new species within characterized genera and that 49 are different strains of known species. The isolates were further categorized based on the isolation conditions, temperature range for growth, enzyme activity, antibiotic resistance, presence of plasmids, and strain-specific genomic variations. A significant observation with implications for the development of novel and more effective cultivation methods was that preliminary incubation in anaerobic and aerobic liquid prior to plating on agar media greatly increased the recovery of CFU from the ice core sample.