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Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

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At least 19 records
All sourcesNASA NTRSDOE OSTI
NASA NTRS1992

Dietary protein deficiency reduces lysosomal and nonlysosomal ATP-dependent proteolysis in muscle

When rats are fed a protein deficient (PD) diet for 7 days, rates of proteolysis in skeletal muscle decrease by 40-50% (N. E. Tawa, Jr., and A. L. Goldberg. Am. J. Physiol. 263 (Endocrinol. Metab. 26): E317-325, 1992). To identify the underlying biochemical adaptations, we measured different proteolytic processes in incubated muscles. The capacity for intralysosomal proteolysis, as shown by sensitivity to methylamine or lysosomal protease inhibitors, fell 55-75% in muscles from PD rats. Furthermore, extracts of muscles of PD rats showed 30-70% lower activity of many lysosomal proteases, including cathepsins B, H, and C, and carboxypeptidases A and C, as well as other lysosomal hydrolases. The fall in cathepsin B and proteolysis was evident by 3 days on the PD diet, and both returned to control levels 3 days after refeeding of the normal diet. In muscles maintained under optimal conditions, 80-90% of protein breakdown occurs by nonlysosomal pathways. In muscles of PD rats, this ATP-dependent process was also 40-60% slower. Even though overall proteolysis decreased in muscles of PD rats, their capacity for Ca(2+)-dependent proteolysis increased (by 66%), as did the activity of the calpains (+150-250%). Thus the lysosomal and the ATP-dependent processes decrease coordinately and contribute to the fall in muscle proteolysis in PD animals.

Non-NASA Center↗
NASA NTRS2000

Nitric oxide inhibits calpain-mediated proteolysis of talin in skeletal muscle cells

We tested the hypothesis that nitric oxide can inhibit cytoskeletal breakdown in skeletal muscle cells by inhibiting calpain cleavage of talin. The nitric oxide donor sodium nitroprusside prevented many of the effects of calcium ionophore on C(2)C(12) muscle cells, including preventing talin proteolysis and release into the cytosol and reducing loss of vinculin, cell detachment, and loss of cellular protein. These results indicate that nitric oxide inhibition of calpain protected the cells from ionophore-induced proteolysis. Calpain inhibitor I and a cell-permeable calpastatin peptide also protected the cells from proteolysis, confirming that ionophore-induced proteolysis was primarily calpain mediated. The activity of m-calpain in a casein zymogram was inhibited by sodium nitroprusside, and this inhibition was reversed by dithiothreitol. Previous incubation with the active site-targeted calpain inhibitor I prevented most of the sodium nitroprusside-induced inhibition of m-calpain activity. These data suggest that nitric oxide inhibited m-calpain activity via S-nitrosylation of the active site cysteine. The results of this study indicate that nitric oxide produced endogenously by skeletal muscle and other cell types has the potential to inhibit m-calpain activity and cytoskeletal proteolysis.

NASA Discipline Musculoskeletal↗
NASA NTRS1995

Increase in ubiquitin-protein conjugates concomitant with the increase in proteolysis in rat skeletal muscle during starvation and atrophy denervation

The rapid loss of skeletal-muscle protein during starvation and after denervation occurs primarily through increased rates of protein breakdown and activation of a non-lysosomal ATP-dependent proteolytic process. To investigate whether protein flux through the ubiquitin (Ub)-proteasome pathway is enhanced, as was suggested by related studies, we measured, using specific polyclonal antibodies, the levels of Ub-conjugated proteins in normal and atrophying muscles. The content of these critical intermediates had increased 50-250% after food deprivation in the extensor digitorum longus and soleus muscles 2 days after denervation. Like rates of proteolysis, the amount of Ub-protein conjugates and the fraction of Ub conjugated to proteins increased progressively during food deprivation and returned to normal within 1 day of refeeding. During starvation, muscles of adrenalectomized rats failed to increase protein breakdown, and they showed 50% lower levels of Ub-protein conjugates than those of starved control animals. The changes in the pools of Ub-conjugated proteins (the substrates for the 26S proteasome) thus coincided with and can account for the alterations in overall proteolysis. In this pathway, large multiubiquitinated proteins are preferentially degraded, and the Ub-protein conjugates that accumulated in atrophying muscles were of high molecular mass (> 100 kDa). When innervated and denervated gastrocnemius muscles were fractionated, a significant increase in ubiquitinated proteins was found in the myofibrillar fraction, the proteins of which are preferentially degraded on denervation, but not in the soluble fraction. Thus activation of this proteolytic pathway in atrophying muscles probably occurs initially by increasing Ub conjugation to cell proteins. The resulting accumulation of Ub-protein conjugates suggests that their degradation by the 26S proteasome complex subsequently becomes rate-limiting in these catabolic states.

NASA Discipline Number 40-40↗
NASA NTRS1991

Mechanisms of accelerated proteolysis in rat soleus muscle atrophy induced by unweighting or denervation

A hypothesis proposed by Tischler and coworkers (Henriksen et al., 1986; Tischler et al., 1990) concerning the mechanisms of atrophy induced by unweighting or denervation was tested using rat soleus muscle from animals subjected to hindlimb suspension and denervation of muscles. The procedure included (1) measuring protein degradation in isolated muscles and testing the effects of lysosome inhibitors, (2) analyzing the lysosome permeability and autophagocytosis, (3) testing the effects of altering calcium-dependent proteolysis, and (4) evaluating in vivo the effects of various agents to determine the physiological significance of the hypothesis. The results obtained suggest that there are major differences between the mechanisms of atrophies caused by unweighting and denervation, though slower protein synthesis is an important feature common for both.

Tischler, Marc E.↗
NASA NTRS1995

Oral branched-chain amino acids decrease whole-body proteolysis

BACKGROUND: This study reports the effects of ingesting branched-chain amino acids (leucine, valine, and isoleucine) on protein metabolism in four men. METHODS: To calculate leg protein synthesis and breakdown, we used a new model that utilized the infusion of L-[ring-13C6]phenylalanine and the sampling of the leg arterial-venous difference and muscle biopsies. In addition, protein-bound enrichments provided for the direct calculation of muscle fractional synthetic rate. Four control subjects ingested an equivalent amount of essential amino acids (threonine, methionine, and histidine) to discern the effects of branched-chain amino acid nitrogen vs the effects of essential amino acid nitrogen. Each drink also included 50 g of carbohydrate. RESULTS: Consumption of the branched-chain and the essential amino acid solutions produced significant threefold and fourfold elevations in their respective arterial concentrations. Protein synthesis and breakdown were unaffected by branched-chain amino acids, but they increased by 43% (p < .05) and 36% (p < .03), respectively, in the group consuming the essential amino acids. However, net leg balance of phenylalanine was unchanged by either drink. Direct measurement of protein synthesis by tracer incorporation into muscle protein (fractional synthetic rate) revealed no changes within or between drinks. Whole-body phenylalanine flux was significantly suppressed by each solution but to a greater extent by the branched-chain amino acids (15% and 20%, respectively) (p < .001). CONCLUSIONS: These results suggest that branched-chain amino acid ingestion suppresses whole-body proteolysis in tissues other than skeletal muscle in normal men.

NASA Discipline Number 18-10↗
NASA NTRS1992

Proteolysis, proteasomes and antigen presentation

Proteins presented to the immune system must first be cleaved to small peptides by intracellular proteinases. Proteasomes are proteolytic complexes that degrade cytosolic and nuclear proteins. These particles have been implicated in ATP-ubiquitin-dependent proteolysis and in the processing of intracellular antigens for cytolytic immune responses.

Non-NASA Center↗
NASA NTRS1984

Inhibition of intracellular proteolysis in muscle cultures by multiplication-stimulating activity

The effects of the insulin-like growth factor, multiplication-stimulating activity (MSA), on chick myotube cultures are studied. The results indicate that MSA is an effective anabolic agent regulating protein metabolism and amino acid uptake, but not sugar transport. Similar size effects on protein metabolism and amino acid uptake in serum-free media were observed in parallel studies with insulin, although insulin levels well in excess of the normal physiological range are required to produce significant effects. It is suggested that there is a generally low insulin sensitivity in cultured chick myotubes relative to adult tissues.

Janeczko, Richard A.↗
NASA NTRS1995

Activation of the ATP-ubiquitin-proteasome pathway in skeletal muscle of cachectic rats bearing a hepatoma

Rats implanted with Yoshida ascites hepatoma (YAH) show a rapid and selective loss of muscle protein due mainly to a marked increase (63-95%) in the rate of protein degradation (compared with rates in muscles of pair-fed controls). To define which proteolytic pathways contribute to this increase, epitrochlearis muscles from YAH-bearing and control rats were incubated under conditions that modify different proteolytic systems. Overall proteolysis in either group of rats was not affected by removal of Ca2+ or by blocking the Ca(2+)-dependent proteolytic system. Inhibition of lysosomal function with methylamine reduced proteolysis (-12%) in muscles from YAH-bearing rats, but not in muscles of pair-fed rats. When ATP production was also inhibited, the remaining accelerated proteolysis in muscles of tumor-bearing rats fell to control levels. Muscles of YAH-bearing rats showed increased levels of ubiquitin-conjugated proteins and a 27-kDa proteasome subunit in Western blot analysis. Levels of mRNA encoding components of proteolytic systems were quantitated using Northern hybridization analysis. Although their total RNA content decreased 20-38%, pale muscles of YAH-bearing rats showed increased levels of ubiquitin mRNA (590-880%) and mRNA for multiple subunits of the proteasome (100-215%). Liver, kidney, heart, and brain showed no weight loss and no change in these mRNA species. Muscles of YAH-bearing rats also showed small increases (30-40%) in mRNA for cathepsins B and D, but not for calpain I or heat shock protein 70. Our findings suggest that accelerated muscle proteolysis and muscle wasting in tumor-bearing rats result primarily from activation of the ATP-dependent pathway involving ubiquitin and the proteasome.

NASA Discipline Musculoskeletal↗
NASA NTRS1992

Suppression of muscle protein turnover and amino acid degradation by dietary protein deficiency

To define the adaptations that conserve amino acids and muscle protein when dietary protein intake is inadequate, rats (60-70 g final wt) were fed a normal or protein-deficient (PD) diet (18 or 1% lactalbumin), and their muscles were studied in vitro. After 7 days on the PD diet, both protein degradation and synthesis fell 30-40% in skeletal muscles and atria. This fall in proteolysis did not result from reduced amino acid supply to the muscle and preceded any clear decrease in plasma amino acids. Oxidation of branched-chain amino acids, glutamine and alanine synthesis, and uptake of alpha-aminoisobutyrate also fell by 30-50% in muscles and adipose tissue of PD rats. After 1 day on the PD diet, muscle protein synthesis and amino acid uptake decreased by 25-40%, and after 3 days proteolysis and leucine oxidation fell 30-45%. Upon refeeding with the normal diet, protein synthesis also rose more rapidly (+30% by 1 day) than proteolysis, which increased significantly after 3 days (+60%). These different time courses suggest distinct endocrine signals for these responses. The high rate of protein synthesis and low rate of proteolysis during the first 3 days of refeeding a normal diet to PD rats contributes to the rapid weight gain ("catch-up growth") of such animals.

Non-NASA Center↗
NASA NTRS1999

Pharmacological Inhibitors of the Proteosome in Atrophying Muscles

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.

Goldberg, Alfred↗
NASA NTRS1972

Enzyme activity in terrestrial soil in relation to exploration of the Martian surface

Urease activity in soil is persistent for long periods under low water, low temperature, and sterile regimes, and it was suggested that some form of enzyme-protective mechanism exists in soil. Dublin soil was extracted by sonication in water followed by adding a mixture of salts. Urease activity is associated with the organo-mineral complex thus obtained and is resistant to the activities of proteolytic enzymes. Clay free soil organic matter prepared subsequently by filtration also exhibits urease activity which is resistant to proteolysis. Models consisting of enzymes with bentonite and lignin were found to mimic this resistance to proteolysis. A model system is presented which suggests both the origin and location of soil ureases and a reason for their persistence in nature.

Ardakani, M. S.↗
NASA NTRS1986

Regulation of Ca(2+)-dependent protein turnover in skeletal muscle by thyroxine

Dantrolene, an agent that inhibits Ca(2+) mobilization, improved protein balance in skeletal muscle, as thyroid status was increased, by altering rates of protein synthesis and degradation. Thyroxine (T4) caused increases in protein degradation that were blocked by leupeptin, a proteinase inhibitor previously shown to inhibit Ca(2+)-dependent nonlysosomal proteolysis in these muscles. In addition, T4 abolished sensitivity to the lysosomotropic agent methylamine and the autophagy inhibitor 3-methyladenine, suggesting that T4 inhibits autophagic/lysosomal proteolysis.

Zeman, Richard J.↗
NASA NTRS1993

Skeletal muscle metabolism in hypokinetic rats

This grant focused on the mechanisms of metabolic changes associated with unweighting atrophy and reduced growth of hind limb muscles of juvenile rats. Metabolic studies included a number of different areas. Amino acid metabolic studies placed particular emphasis on glutamine and branched-chain amino acid metabolism. These studies were an outgrowth of understanding stress effects and the role of glucocorticoids in these animals. Investigations on protein metabolism were largely concerned with selective loss of myofibrillar proteins and the role of muscle proteolysis. These investigations lead to finding important differences from denervation and atrophy and to define the roles of cytosolic versus lysosomal proteolysis in these atrophy models. A major outgrowth of these studies was demonstrating an ability to prevent atrophy of the unweighted muscle for at least 24 hours. A large amount of work concentrated on carbohydrate metabolism and its regulation by insulin and catecholamines. Measurements focused on glucose transport, glycogen metabolism, and glucose oxidation. The grant was used to develop an important new in situ approach for studying protein metabolism, glucose transport, and hormonal effects which involves intramuscular injection of various agents for up to 24 hours. Another important consequence of this project was the development and flight of Physiological-Anatomical Rodent Experiment-1 (PARE-1), which was launched aboard Space Shuttle Discovery in September 1991. Detailed descriptions of these studies can be found in the 30 peer-reviewed publications, 15 non-reviewed publications, 4 reviews and 33 abstracts (total 82 publications) which were or are scheduled to be published as a result of this project. A listing of these publications grouped by area (i.e. amino acid metabolism, protein metabolism, carbohydrate metabolism, and space flight studies) are included.

Tischler, Marc E.↗
NASA NTRS1999

Proteolytic dissection of Zab, the Z-DNA-binding domain of human ADAR1

Zalpha is a peptide motif that binds to Z-DNA with high affinity. This motif binds to alternating dC-dG sequences stabilized in the Z-conformation by means of bromination or supercoiling, but not to B-DNA. Zalpha is part of the N-terminal region of double-stranded RNA adenosine deaminase (ADAR1), a candidate enzyme for nuclear pre-mRNA editing in mammals. Zalpha is conserved in ADAR1 from many species; in each case, there is a second similar motif, Zbeta, separated from Zalpha by a more divergent linker. To investigate the structure-function relationship of Zalpha, its domain structure was studied by limited proteolysis. Proteolytic profiles indicated that Zalpha is part of a domain, Zab, of 229 amino acids (residues 133-361 in human ADAR1). This domain contains both Zalpha and Zbeta as well as a tandem repeat of a 49-amino acid linker module. Prolonged proteolysis revealed a minimal core domain of 77 amino acids (positions 133-209), containing only Zalpha, which is sufficient to bind left-handed Z-DNA; however, the substrate binding is strikingly different from that of Zab. The second motif, Zbeta, retains its structural integrity only in the context of Zab and does not bind Z-DNA as a separate entity. These results suggest that Zalpha and Zbeta act as a single bipartite domain. In the presence of substrate DNA, Zab becomes more resistant to proteases, suggesting that it adopts a more rigid structure when bound to its substrate, possibly with conformational changes in parts of the protein.

Non-NASA Center↗
NASA NTRS1998

The N-end rule pathway catalyzes a major fraction of the protein degradation in skeletal muscle

In skeletal muscle, overall protein degradation involves the ubiquitin-proteasome system. One property of a protein that leads to rapid ubiquitin-dependent degradation is the presence of a basic, acidic, or bulky hydrophobic residue at its N terminus. However, in normal cells, substrates for this N-end rule pathway, which involves ubiquitin carrier protein (E2) E214k and ubiquitin-protein ligase (E3) E3alpha, have remained unclear. Surprisingly, in soluble extracts of rabbit muscle, we found that competitive inhibitors of E3alpha markedly inhibited the 125I-ubiquitin conjugation and ATP-dependent degradation of endogenous proteins. These inhibitors appear to selectively inhibit E3alpha, since they blocked degradation of 125I-lysozyme, a model N-end rule substrate, but did not affect the degradation of proteins whose ubiquitination involved other E3s. The addition of several E2s or E3alpha to the muscle extracts stimulated overall proteolysis and ubiquitination, but only the stimulation by E3alpha or E214k was sensitive to these inhibitors. A similar general inhibition of ubiquitin conjugation to endogenous proteins was observed with a dominant negative inhibitor of E214k. Certain substrates of the N-end rule pathway are degraded after their tRNA-dependent arginylation. We found that adding RNase A to muscle extracts reduced the ATP-dependent proteolysis of endogenous proteins, and supplying tRNA partially restored this process. Finally, although in muscle extracts the N-end rule pathway catalyzes most ubiquitin conjugation, it makes only a minor contribution to overall protein ubiquitination in HeLa cell extracts.

Non-NASA Center↗
NASA NTRS1974

Aeromonas proteolyrica bacteria in aerospace environments

Preflight studies on Aeromonas proteolytica are reported to investigate the possibility of genetic alterations resulting in increased proteolysis in spacecraft environments. This organism may be present on human tissue and could pose medical problems if its endopeptidase and a hemolysin were to be produced in ususually high quantities or altered in such a way as to be more effective in their activities. Considered are: (1) Development of a nutrative holding medium for suspension of organisms; (2) the establishment of baseline information for the standardization of the assay for endopeptidase levels and hemolytic titers; (3) formulation of a method by which intracutaneous hemorrhage could be quantitated in guinea pig tissue; and (4) the responses of these organisms to parameters of spaceflight and experimentation.

Foster, B. G.↗
NASA NTRS1983

Prevention of metabolic alterations caused by suspension hypokinesia in leg muscles of rats

Rats were subjected to tail-cast suspension hypokinesia for 6 days with one leg immobilized in dorsal flexion by casting. Control animals were also tail-casted. The soleus, gastrocnemius and plantaris muscles of uncasted hypokinetic legs were smaller than control muscles. Dorsal flexion prevented atrophy of these muscles and caused the soleus to hypertrophy. The anterior muscles were unaffected by hypokinesia. The smaller size of the soleus of the uncasted leg relative to the dorsal flexed and weight bearing limbs correlated with slower protein synthesis and faster proteolysis. The capacity of this muscle to synthesize glutamine (gln), which carries nitrogenous waste from muscle was also measured. Although tissue homogenates showed higher activities of gln synthetase, the rate of de novo synthesis was not altered in intact muscle but the tissue ratio of gln/glutamate was decreased. Glutamate and ATP were not limiting for gln synthesis, but availability of ammonia may be a limiting factor for this process in hypokinesia.

Tischler, M. E.↗