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All sourcesNASA NTRSDOE OSTI
NASA NTRS1987

Protection of wheat against leaf and stem rust and powdery mildew diseases by inhibition of polyamine metabolism

In higher plants, polyamines arise from arginine by one of two pathways: via ornithine and ornithine decarboxylase or via agmatine and arginine decarboxylase but in fungi, only the ornithine decarboxylase pathway is present. Since polyamines are required for normal growth of microorganisms and plants and since the ornithine pathway can be irreversibly blocked by alpha-difluoromethylornithine (DFMO) which has no effect on arginine decarboxylase, fungal infection of green plants might be controlled by the site-directed use of such a specific metabolic inhibitor. DFMO at relatively low concentrations provided effective control of the three biotrophic fungal pathogens studied, Puccinia recondita (leaf rust), P. graminis f. sp. tritici (stem rust), and Erysiphe graminis (powdery mildew) on wheat (Triticum aestivum L.) Effective control of infection by leaf or stem rust fungi was obtained with sprays of DFMO that ranged from about 0.01 to 0.20 mM in experiments where the inhibitor was applied after spore inoculation. The powdery mildew fungus was somewhat more tolerant of DFMO, but good control of the pathogen was obtained at less than 1.0 mM. In general, application of DFMO after spore inoculation was more effective than application before inoculation. Less control was obtained following treatment with alpha-difluoromethylarginine (DFMA) but the relatively high degree of control obtained raises the possibility of a DFMA to DFMO conversion by arginase.

NASA Discipline Number 40-10↗
NASA NTRS1996

Oligomerization of uridine phosphorimidazolides on montmorillonite: a model for the prebiotic synthesis of RNA on minerals

The 5'-phosphorimidazolide of uridine reacts on Na(+)-montmorillonite 22A in aqueous solution to give oligomers as long as 7 mers. The maximum chain length increases to 9 mers and the overall oligomer yield increases when 9:1 ImpU, A5' ppA mixtures react under the same conditions. The oligomer yield and maximum chain length decreases with the structure of the added pyrophosphate in the order A5' ppA > A5' ppU > U5' ppU. Structure analysis of individual oligomer fractions was performed by selective enzymatic hydrolyses followed by HPLC analysis of the products. The regioselectivity for 3',5'-bond formation is 80-90% in the 9:1 ImpU, A5' ppA reaction, a percentage comparable to that observed in the 9:1 ImpA, A5' ppA reaction. Oligomerization of ImpU is inhibited by addition of dA5' ppdA, and MeppA. No oligomers containing A5' ppU were products of the 9:1 ImpU,A5' ppA reaction, a finding consistent with the simple addition of the ImpU to the A5' ppA and not the rearrangement of an ImpU-A5' ppA adduct. Concentrations of lysine or arginine which were close to that of the ImpU did not inhibit oligomer formation. Treatment of Na(+)-montmorillonite with 1 M arginine yielded arginine-montmorillonite, an amino acid-mineral adduct which did not catalyze ImpU oligomerization. Neither the 4-9 mers formed in the 9:1 ImpU, A5' ppA reaction nor the 4-9 mers formed by the base hydrolysis of poly(U) served as templates for the formation of oligo(A)s.

Non-NASA Center↗
NASA NTRS1974

On the possible origin and evolution of the genetic code

The genetic code is examined for indications of possible preceding codes that existed during early evolution. Eight of the 20 amino acids are coded by 'quartets' of codons with fourfold degeneracy, and 16 such quartets can exist, so that an earlier code could have provided for 15 or 16 amino acids, rather than 20. If twofold degeneracy is postulated for the first position of the codon, there could have been ten amino acids in the code. It is speculated that these may have been phenylalanine, valine, proline, alanine, histidine, glutamine, glutanic acid, aspartic acid, cysteine and glycine. There is a notable deficiency of arginine in proteins, despite the fact that it has six codons. Simultaneously, there is more lysine in proteins than would be expected from its two codons, if the four bases in mRNA are equiprobable and are arranged randomly. It is speculated that arginine is an 'intruder' into the genetic code, and that it may have displayed another amino acid such as ornithine, or may even have displayed lysine from some of its previous codon assignments. As a result, natural selection has favored lysine against the fact that it has only two codons.

Jukes, T. H.↗
NASA NTRS1994

Role of Nitric Oxide in the Regulation of Renin and Vasopressin Secretion

Research during recent years has established nitric oxide as a unique signaling molecule that plays important roles in the regulation of the cardiovascular, nervous, immune, and other systems. Nitric oxide has also been implicated in the control of the secretion of hormones by the pancreas, hypothalamus, and anterior pituitary gland, and evidence is accumulating that it contributes to the regulation of the secretion of renin and vasopressin, hormones that play key roles in the control of sodium and water balance. Several lines of evidence have implicated nitric oxide in the control of renin secretion. The enzyme nitric oxide synthase is present in vascular and tubular elements of the kidney, particularly in cells of the macula densa, a structure that plays an important role in the control of renin secretion. Guanylyl cyclase, a major target for nitric oxide, is also present in the kidney. Drugs that inhibit nitric oxide synthesis generally suppress renin release in vivo and in vitro, suggesting a stimulatory role for the L-arginine/nitric oxide pathway in the control of renin secretion. Under some conditions, however, blockade of nitric oxide synthesis increases renin secretion. Recent studies indicate that nitric oxide not only contributes to the regulation of basal renin secretion, but also participates in the renin secretory responses to activation of the renal baroreceptor, macula densa, and beta adrenoceptor mechanisms that regulate renin secretion. Histochemical and immunocytochemical studies have revealed the presence of nitric oxide synthase in the supraoptic and paraventricular nuclei of the hypothalamus and in the posterior pituitary gland. Colocalization of nitric oxide synthase and vasopressin has been demonstrated in some hypothalamic neurons. Nitric oxide synthase activity in the hypothalamus and pituitary is increased by maneuvers known to stimulate vasopressin secretion, including salt loading and dehydration, Administration of L-arginine and nitric oxide donors in vitro and in vivo has variable effects on vasopressin secretion, but the most common one is inhibition. Blockade of nitric oxide synthesis has been reported to increase vasopressin secretion, but again variable results have been obtained. An attractive working hypothesis is that nitric oxide serves a neuromodulatory role as an inhibitor of vasopressin secretion.

Reid, Ian A.↗
NASA NTRS1995

Effect of Blockade of Nitric Oxide Synthesis on the Renin Secretory Response to Frusemide in Conscious Rabbits

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.

Reid, Ian A.↗
NASA NTRS1985

Polyamine levels as related to growth, differentiation and senescence in protoplast-derived cultures of Vigna aconitifolia and Avena sativa

We have previously reported that aseptically cultured mesophyll protoplasts of Vigna divide rapidly and regenerate into complete plants, while mesophyll protoplasts of Avena divide only sporadically and senesce rapidly after isolation. We measured polyamine titers in such cultures of Vigna and Avena, to study possible correlations between polyamines and cellular behavior. We also deliberately altered polyamine titer by the use of selective inhibitors of polyamine biosynthesis, noting the effects on internal polyamine titer, cell division activity and regenerative events. In Vigna cultures, levels of free and bound putrescine and spermidine increased dramatically as cell division and differentiation progressed. The increase in bound polyamines was largest in embryoid-forming callus tissue while free polyamine titer was highest in root-forming callus. In Avena cultures, the levels of total polyamines decreased as the protoplast senesced. The presence of the inhibitors alpha-difluoromethyl-arginine (specific inhibitor of arginine decarboxylase), alpha-difluoromethylornithine (specific inhibitor of ornithine decarboxylase) and dicyclohexylamine (inhibitor of spermidine synthase) reduced cell division and organogenesis in Vigna cultures. Addition of low concentration of polyamines to such cultures containing inhibitors or removal of inhibitors from the culture medium restored the progress of growth and differentiation with concomitant increase in polyamine levels.

NASA Program Space Biology↗
NASA NTRS1986

Polyamine metabolism and osmotic stress. I. Relation to protoplast viability

Cereal leaves subjected to the osmotica routinely used for protoplast isolation show a rapid increase in arginine decarboxylase activity, a massive accumulation of putrescine, and slow conversion of putrescine to the higher polyamines, spermidine and spermine (HE Flores, AW Galston 1984 Plant Physiol 75: 102). Mesophyll protoplasts from these leaves, which have a high putrescine:polyamine ratio, do not undergo sustained division. By contrast, in Nicotiana, Capsicum, Datura, Trigonella, and Vigna, dicot genera that readily regenerate plants from mesophyll protoplasts, the response of leaves to osmotic stress is opposite to that in cereals. Putrescine titer as well as arginine and ornithine decarboxylase activities decline in these osmotically stressed dicot leaves, while spermidine and spermine titers increase. Thus, the putrescine:polyamine ratio in Vigna protoplasts, which divide readily, is 4-fold lower than in oat protoplasts, which divide poorly. We suggest that this differing response of polyamine metabolism to osmotic stress may account in part for the failure of cereal mesophyll protoplasts to develop readily in vitro.

Non-NASA Center↗
NASA NTRS1986

Effect of inhibitors of polyamine biosynthesis on gibberellin-induced internode growth in light-grown dwarf peas

When gibberellic acid (GA3) is sprayed on 9-day-old light-brown dwarf Progress pea (Pisum sativum) seedlings, arginine decarboxylase (ADC; EC 4.1.1.9) activity increases within 3 h and peaks at about 9 h after GA3 application. This is followed by a second lower peak at about 30 h; both peaks were higher than the corresponding peaks in the controls. In contrast, no appreciable effect of GA3 on internode length was observed until about 12 h, after which time a dramatic increase in growth rate occurred and persisted for about 12 h. Specific (DL-alpha-difluoromethylarginine) and non-specific (D-arginine and L-canavanine) inhibitors of ADC strongly inhibited ADC activity and to a lesser extent internode growth. The inhibition was reversed only slightly by the addition of polyamines. Actinomycin D and cycloheximide inhibited the rise in ADC activity induced by GA3. The half-life of the enzyme was increased by GA3 treatment. The results suggest that part of the GA3-induced increase in internode growth may result from enhanced polyamine biosynthesis through the ADC pathway. Furthermore, the GA3 induced increase in ADC activity probably requires de novo synthesis of both RNA and protein.

NASA Program Space Biology↗
NASA NTRS1991

Purification and characterization of ornithine transcarbamylase from pea (Pisum sativum L.)

Pea (Pisum sativum) ornithine transcarbamylase (OTC) was purified to homogeneity from leaf homogenates in a single-step procedure, using delta-N-(phosphonacetyl)-L-ornithine-Sepharose 6B affinity chromatography. The 1581-fold purified OTC enzyme exhibited a specific activity of 139 micromoles citrulline per minute per milligram of protein at 37 degrees C, pH 8.5. Pea OTC represents approximately 0.05% of the total soluble protein in the leaf. The molecular weight of the native enzyme was approximately 108,200, as estimated by Sephacryl S-200 gel filtration chromatography. The purified protein ran as a single molecular weight band of 36,500 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These results suggest that the pea OTC is a trimer of identical subunits. The overall amino acid composition of pea OTC is similar to that found in other eukaryotic and prokaryotic OTCs, but the number of arginine residues is approximately twofold higher. The increased number of arginine residues probably accounts for the observed isoelectric point of 7.6 for the pea enzyme, which is considerably more basic than isoelectric point values that have been reported for other OTCs.

Non-NASA Center↗
NASA NTRS1996

Blood vessel adaptation to gravity in a semi-arboreal snake

The effects of vasoactive agonists on systemic blood vessels were examined with respect to anatomical location and gravity acclimation in the semi-arboreal snake, Elaphe Obsoleta. Major blood vessels were reactive to putative neurotransmitters, hormones or local factors in vessel specific patterns. Catecholamines, adenosine triphosphate, histamine and high potassium (80 mM) stimulated significantly greater tension per unit vessel mass in posterior than anterior arteries. Anterior vessels were significantly more sensitive to catecholamines than midbody and posterior vessels. Angiotensin II stimulated significantly greater tension in carotid artery than in midbody and posterior dorsal aorta. Arginine vasotocin strongly contracted the left and right aortic arches and anterior dorsal aorta. Veins were strongly contracted by catecholamines, high potassium and angiotensin II, but less so by adenosine triphosphate, arginine vasotocin and histamine. Precontracted vessel were relaxed by acetylcholine and sodium nitroprusside, but not by atrial natriuretic peptide or bradykinin. Chronic exposure of snakes to intermittent hypergravity stress ( + 1.5 Gz at tail) did not affect the majority of vessel responses. These data demonstrate that in vitro tension correlates with that catecholamines, as well as other agonists, are important in mediating vascular responses to gravitational stresses in snakes.

NASA Discipline Number 14-10↗
NASA NTRS1995

Editing for an AMPA receptor subunit RNA in prefrontal cortex and striatum in Alzheimer's disease, Huntington's disease and schizophrenia

Animal studies and cell culture experiments demonstrated that posttranscriptional editing of the transcript of the GluR-2 gene, resulting in substitution of an arginine for glutamine in the second transmembrane region (TM II) of the expressed protein, is associated with a reduction in Ca2+ permeability of the receptor channel. Thus, disturbances in GluR-2 RNA editing with alteration of intracellular Ca2+ homeostasis could lead to neuronal dysfunction and even neuronal degeneration. The present study determined the proportions of edited and unedited GluR-2 RNA in the prefrontal cortex of brains from patients with Alzheimer's disease, in the striatum of brains from patients with Huntington's disease, and in the same areas of brains from age-matched schizophrenics and controls, by using reverse transcriptase-polymerase chain reaction, restriction endonuclease digestion, gel electrophoresis and scintillation radiometry. In the prefrontal cortex of controls, < 0.1% of all GluR-2 RNA molecules were unedited and > 99.9% were edited; in the prefrontal cortex both of schizophrenics and of Alzheimer's patients approximately 1.0% of all GluR-2 RNA molecules were unedited and 99% were edited. In the striatum of controls and of schizophrenics, approximately 0.5% of GluR-2 RNA molecules were unedited and 99.5% were edited; in the striatum of Huntington's patients nearly 5.0% of GluR-2 RNA was unedited. In the prefrontal white matter of controls, approximately 7.0% of GluR-2 RNA was unedited. In the normal human prefrontal cortex and striatum, the large majority of GluR-2 RNA molecules contains a CGG codon for arginine in the TMII coding region; this implies that the corresponding AMPA receptors have a low Ca2+ permeability, as previously demonstrated for the rat brain. The process of GluR-2 RNA editing is compromised in a region-specific manner in schizophrenia, in Alzheimer's disease and Huntington's Chorea although in each of these disorders there is still a large excess of edited GluR-2 RNA molecules. Disturbances of GluR-2 RNA editing leading to excessive Ca2+ permeability, may contribute to neuronal dysfunction in schizophrenia and to neuronal death in Alzheimer's disease and Huntington's disease.

NASA Discipline Neuroscience↗
NASA NTRS2006

Bacterial Utilization of L-sugars and D-amino Acids

The fact that organotrophic organisms on Earth use L-amino acids and D-sugars as an energy source is recognized as one of the universal features of life. The chirality of organic molecules with asymmetric location of group- radicals was described a relatively long time ago. In 1848, Louis Pasteur discovered chiral molecules when he investigated the way that crystals of sodium ammonium paratartrate rotated the plane of polarization of light. He found that the crystal structures represented the underlying asymmetry of molecules that existed in either lea-handed or right-handed forms (enantiomers). Pasteur observed that abiotic (chemical) processes produced mixtures with equal numbers (racemic) of the two forms but that living organisms possessed a molecular asymmetry that included only one of the enantiomers (homochirality). He speculated that the origin of the asymmetry of chiral biomolecules might hold the key to the nature of life. All of the amino acids in proteins (except for Glycine which is symmetrical) exhibit the same absolute steric configuration as L-glyceraldehyde. D-amino acids are never found in proteins, although they do exist in nature and are often found in polypeptide antibiotics. Constitutional sugars of cells, opposite to the amino acids, are the D-enantiomers, and the appearance of L-sugars in Nature is extremely rare. Notwithstanding this fact, the metabolism of some bacteria does have capability to use amino acids and sugars with alternative chirality. This property may be caused by the function of specific enzymes belonging to the class of isomerases (racemases, epimerases, isomerases, tautomerases). In our laboratory, we have investigated several anaerobic bacterial strains, and have found that some of these bacteria are capable of using D-amino acids and L-sugars. Strain BK1 is capable of growth on D-arginine, but its growth characteristics on L-arginine are approximately twice higher. Another alkaliphilic strain SCAT(sup T) (= ATCC BAA-1084(sup T)= JCM 12857(sup T) = DSM 17722(sup T) = CIP 107910(sup T)) was found to be capable of growth on L-ribose and L-arabinose. It is interesting that this strain was incapable of growth on D-arabinose, which suggests the involvement of some alternative mechanism of enzyme activity. In this paper, we describe the preliminary results of these microbiological studies and discuss some possible implications.

Pikuta, Elena↗
NASA NTRS2001

Altered TNF-Alpha, Glucose, Insulin and Amino Acids in Islets Langerhans Cultured in a Microgravity Model System

The present studies were designed to determine effects of a microgravity model system upon lipopolysaccharide (LPS) stimulated tumor necrosis factor alpha (TNF-alpha) activity and indices of insulin and fuel homeostasis of pancreatic islets of Langerhans. Islets (1726+/-1 17,150 u IEU) from Wistar Furth rats were treated as: 1) HARV (High Aspect Ratio Vessel cell culture) , 2) HARV plus LPS, 3) static culture, 4) static culture plus LPS. TNF-alpha (L929 cytotoxicity assay) was significantly increased in LPS-induced HARV and static cultures, yet the increase was more pronounced in the static culture group (p<0.05). A decrease in insulin concentration was demonstrated in the LPS stimulated HARV culture (p<0.05). We observed a greater glucose concentration and increased disappearance of arginine in islets cultured in HARVs. While nitrogenous compound analysis indicated a ubiquitous reliance upon glutamine in all experimental groups, arginine was converted to ornithine at a two-fold greater rate in the islets cultured in the HARV microgravity model system (p<0.05). These studies demonstrate alterations in LPS induced TNF-alpha production of pancreatic islets of Langerhans, favoring a lesser TNF activity in the HARV. These alterations in fuel homeostasis may be promulgated by gravity averaged cell culture methods or by three dimensional cell assembly.

Tobin, Brian W.↗
NASA NTRS1972

Detection of non-protein amino acids in the presence of protein amino acids. II.

Studies conducted with the JEOL 5AH amino acid analyzer are described. This instrument makes possible the programming of the chromatographic process. Data are presented showing the separations of seventeen non-protein amino acids in the presence of eighteen protein amino acids. It is pointed out that distinct separations could be obtained in the case of a number of chemically similar compounds, such as ornithine and lysine, N-amidino alanine and arginine, and iminodiacetic acid and S-carboxymethyl cysteine and aspartic acid.

Shapshak, P.↗
NASA NTRS1976

Plasma vasopressin and renin activity in women exposed to bed rest and +G/z/ acceleration

To study the effect of prolonged recumbency on plasma vasopressin and renin activity, eight women were subjected to 17 days of absolute bed rest. The tolerance to +3G vertical acceleration of the subjects was tested before and after 14 days of bed rest. From day 2 and through day 17 of bed rest, plasma arginine vasopressin (AVP) levels were reduced 33%. Plasma renin activity (PRA) increased 91% above ambulatory control values from days 10 through 15 of bed rest. When compared to precentrifuge values, exposure to vertical acceleration prior to bed rest provoked a 20-fold rise in mean plasma AVP but resulted in only a slight increase in PRA. After bed rest, acceleration increased plasma AVP 7-fold; however, the magnitude of this increase was less than the post +3G acceleration value obtained prior to bed rest. After bed rest, no significant rise was noted in PRA following +3G acceleration. This study demonstrates that prolonged bed rest leads to a significant rise in the PRA of female subjects, while exposure to positive vertical acceleration provokes a marked rise in plasma AVP.

Keil, L. C.↗
NASA NTRS1975

Compartmentalization of amino acids in surfactant aggregates - Partitioning between water and aqueous micellar sodium dodecanoate and between hexane and dodecylammonium propionate trapped water in hexane

The partitioning of amino acids (glycine, alanine, leucine, phenylalanine, histidine, aspartic acid, glutamic acid, lysine, isoleucine, threonine, serine, valine, proline, arginine) in aqueous and nonaqueous micellar systems was studied experimentally. Partitioning from neat hexane into dodecylammonium propionate trapped water in hexane was found to be dependent on both electrostatic and hydrophobic interactions, which implies that the interior of dodecylammonium propionate aggregates is negatively charged and is capable of hydrogen bonding in addition to providing a hydrophobic environment. Unitary free energies of transfer of amino acid side chains from hexane to water were determined and solubilities of amino acids in neat hexane substantiated the amino acid hydrophobicity scale. The relevance of the experiments to prebiotic chemistry was examined.

Fendler, J. H.↗
NASA NTRS1977

Reduction in plasma vasopressin levels of dehydrated rats following acute stress

Results are presented for an investigation directed to substantiate and extend preliminary findings of stress-induced reduction in plasma arginine vasopressin (pAVP). Since normally hydrated rats have very low levels of pAVP, it is difficult to measure reliably any decrease in pAVP that may result from stress. To overcome this problem, the pAVP levels of the tested rats were raised by dehydration prior to application of stress. A radioimmunoassay for pAVP is described and used to determine the levels of vasopressin in the plasma of nondehydrated and dehydrated rats after exposure to ether or acceleration stress. Plasma pAVP is also determined in rats following nicotine administration. It is shown that exposure of nondehydrated rats to ether or acceleration stress does not elicit any significant alterations in circulating pAVP levels while nicotine injections stimulate a marked increase. In particular, ether and acceleration stress produce a rapid reduction in the pAVP level of dehydrated rats, the decrease being observed in both large and small animals. The mechanism for this reduction in pAVP level following stress is yet unknown.

Keil, L. C.↗