Plasma catecholamines: effects of footshock level and hormonal modulators of memory storage

Plasma levels of norepinephrine (NE) and epinephrine (EPI) were measured in male Sprague-Dawley rats before and at several times after training injections of agents known to enhance or to impair later retention performance for a one-trial inhibitory (passive) avoidance task. Two days before testing, each animal was surgically prepared with a chronic tail artery catheter that allows for repeated blood sampling in unhandled rats. Exposure to a single, intense training footshock (3.0 mA, 2.0 sec duration) resulted in an immediate but transient increase in plasma levels of EPI and to a lesser extent NE. Plasma levels of both catecholamines did not differ between unshocked controls and animals that received a weak training footshock (0.6 mA, 0.5 sec duration). An injection of EPI at a dose that enhances retention performance (0.1 mg/kg, sc) resulted in increments in plasma EPI levels of 0.8-1.9 ng/ml from 5 to 40 min after injection. An injection of EPI (0.5 mg/kg, sc) at a dose that produces retrograde amnesia resulted in increments in plasma EPI ranging from 3.7 to 4.5 ng/ml during the 40 min after injection. Plasma NE levels were not significantly altered following an EPI injection. A single injection of adrenocorticotropin (ACTH, 0.3 or 3.0 IU per rat) did not alter the plasma catecholamine responses to training with a weak footshock. Similarly, the synthetic ACTH analog, Organon 2766 (125 or 250 mg/Kg) did not affect plasma catecholamines in untrained (unshocked) rats.These results demonstrate that significant increments in plasma levels of NE and EPI occur shortly after inhibitory avoidance training. Furthermore, an injection of EPI that enhances retention of an inhibitory avoidance task mimics the magnitude, though not the temporal characteristics, of the endogenous adrenal medullary response to a training footshock. Other hormonal treatments (ACTH and Organon 2766) which enhance memory storage do not affect plasma levels of NE and EPI.     

Energy-dependent changes in the conformation of the epsilon subunit of the chloroplast ATP synthase

Rabbit antiserum raised against the isolated native epsilon subunit of the chloroplast coupling factor 1 activated the ATPase activity of coupling factor 1 in solution by removing the epsilon subunit. Incubation of thylakoid membranes with the antiserum in the dark had no effect on photophosphorylation or on the dithiothreitol-induced Mg2+-ATPase activity. Incubation with the antiserum during illumination, however, strongly inhibited both activities and caused the membranes to become leaky to protons. The results indicate that the formation of a proton gradient across the thylakoid membrane induces a change in conformation of the epsilon subunit of the ATP synthase such that it becomes susceptible to attack and removal by the antibodies. This change may be a part of the mechanism that results in energy-dependent activation of the ATP synthase.     

Transposable element-induced mutations of the viviparous-1 gene in maize

The viviparous-1 (vp1) locus in maize is a developmental gene that controls diverse aspects of the maturation phase of seed development. Mutations of vp1 alter embryo sensitivity to the hormone abscisic acid and block formation of anthocyanin pigment. Molecular cloning of a Robertson Mutator-induced mutant allele, vp1-mum-1, by transposable element tagging has allowed analysis of several transposon-induced vp1 mutants. In the vp1-Mc mutation, the gene is disrupted by 4.0 kbp insertion, which results in expression of a 3′ truncated mRNA. Phenotypically, this allele is at least partially functional in causing embryo dormancy, but is ineffective in controlling anthocyanin expression. This result suggests that disruption of the C-terminal domain of the Vp1 protein specifically affects regulation of the anthocyanin pathway. A second Mutator- derived allele, vp1-mum2, exhibits an unusual form of somatic mutability in which endosperm cells revert from wild-type vp1 expression to a mutant condition. The vp1-mum2 allele contains a 1.5 kbp Insertion that has no detectable homology to known Mu elements. This element is retained In wild-type germinal revertants derived from vp1-mum2 An apparent DNA modification affecting cleavage at an internal Sstl restriction site in the element correlates with vp1-mum2 states that exhibit wild-type Vp1 expression. A model involving mitotic assortment of modified and unmodified DNA strands during development is proposed for vp1-mum2 somatic mutation.     

Binding sites for atrial natriuretic factor (ANF) in kidneys and adrenal glands of spontaneously hypertensive (SHR) rats

Binding sites for atrial natriuretic factor (ANF) were studied in kidneys and adrenal glands of 17 week old male spontaneously hypertensive (SHR) and Wistar-Kyoto (WKY) normotensive rats by quantitative autoradiography using 125I-ANF-28. In kidney, 125I-ANF-28 binding sites were found in high concentrations in glomeruli and in much lower concentrations in the renal papilla. In adrenal gland, 125I-ANF-28 binding sites were highly localized to the zona glomerulosa and were of moderate density in the inner cortical regions. ANF binding sites did not occur in the adrenal medulla. The maximum binding capacity (Bmax) of 125I-ANF-28 was reduced by 50% in the kidney glomeruli of SHRs compared to WKY controls. In contrast, the affinity constant (Ka) for 125I-ANF-28 was elevated by 100% in kidney glomeruli of SHRs. There were no significant strain differences in values for Bmax or Ka for 125I-ANF-28 binding in the adrenal zona glomerulosa. These findings suggest that the natriuretic and diuretic actions of ANF within kidney glomeruli may be compromised in adult SHR rats and these alterations may contribute to the development and maintenance of hypertension in rats of this strain.     

Energetic and rate effects on methanogenesis of ethanol and propionate in perturbed CSTRs

Energetic and reaction-rate interactions between hydrogenic (hydrogen-producing) and hydrogenotrophic (hydrogen-consuming) bacteria were investigated in five perturbation experiments performed on steady-state, mixed-culture methanogenic CSTRs receiving ethanol, propionate, or both hydrogenic substrates. When a large quantity of propionate was suddenly added to a propionatefed CSTR, P(H(2) ) increased to 10(-4) atm and propionate oxidation remained energetically favorable. When ethanol was added to a CSTR receiving ethanol, P(H(2) ) rose to 6.3 x 10(-3) atm within 5 h. In both perturbations, P(H(2) ) remained at levels such that oxidation of the hydrogenic substrate remained energetically favorable throughout the transient. Sudden increase in ethanol concentration in the ethanol- and propionate-fed CSTR resulted in an increase in P(H(2) ) such that propionate oxidation became energetically unfavorable and was blocked. Propionate utilization resumed when the added ethanol was depleted and P(H(2) ) returned to its previous steady-state levels. Ethanol perturbation of ethanol- and propionate-fed CSTRs led to the formation of reduced products, including n-propanol and four-through seven-carbon n-carboxylic acids, when P(H(2) ) was elevated; these products disappeared after P(H(2) ) returned to previous, steady-state levels. The transformations were consistent with reaction energetics. Reduced product formation may have been a sink for reducing equivalents, as an alternative to oxidation for propionate utilization, as indicated by an electron equivalents balance over the time course of experiments.     

Effects of phenolic acids on ammonia oxidation by terrestrial autotrophic nitrifying microorganisms

Abstract It has been hypothesized that vegetation in certain ecosystems inhibits nitrification in soil by producing phenolic compounds that inhibit oxidation of ammonia by nitrifying microorganisms. This hypothesis is based largely on a report that very low concentrations (10⁻⁶ M–10⁻⁸ M) of several phenolic acids (notably ferulic acid) completely inhibited NO₂⁻ production in an aqueous suspension of soil treated with (NH₄)₂SO₄ and a nutrient solution suitable for growth of Nitrosomonas and other autotrophic nitrifying microorganisms. To evaluate this hypothesis, we determined the effects of three ohenolic acids (ferulic acid, caffeic acid, and p -coumaric on nitrite production by representatives of three genera of terrestrial autotrophic nitrifying microorganisms ( Nitrosospira, Nitrosomonas , or Nitrosolubos ) grown on a defined medium containing NH₄⁺. We found that nitrite production by the Nitrososspira was not inhibited by ferulic acid, caffeic acid, or p -coumaric acid at concentrations of 10⁻⁶ or 10⁻⁵ M and was only slightly inhibited when these acids were at a concentration of 10⁻⁴ M. We also found that ferulic acid did not markedly inhibit nitrite production by the three genera of nitrifying microorganisms studied, even when its concentration was as high as 10⁻³ M. These observations invalidate the hypothesis tested because the phenolic acids studied did not significantly retard ammonia oxidation by autotrophic microorganisms even when their concentration in cultures of these microorganisms greatly exceeded their concentrations in soils.     

The effects of salts and amino group modification on the iron binding domains of transferrin

The origins of the effects of salts on the properties of the iron binding sites of transferrin have been investigated. The chaotropically distinct salts NaCl and NaClO4 each induce characteristic changes in the EPR lineshapes of the N- and C-terminal Fe3+ binding domains, respectively. To a good approximation the perturbed EPR spectrum of diferric transferrin in the presence of salts is the sum of the EPR spectra of the N- and C-terminal monoferric proteins. Acetylation of amino groups causes spectral and kinetic changes in the protein similar to those induced by NaClO4. Thus, both acetylation and NaClO4 cause a loss of structure in the g' = 4.3 EPR signal of the N-terminal domain, and both retard iron removal from this domain. In contrast, iron removal from the C-terminal domain is accelerated by acetylation or the presence of NaClO4. These observations are ascribed to charge effects of lysine residues which are probably in the vicinity of the iron binding sites.     

d-Glycerate Transport by the Pea Chloroplast Glycolate Carrier : Studies on [1-C]d-Glycerate Uptake and d-Glycerate Dependent O(2) Evolution

Rapid direct conversion of exogenously supplied [¹⁴C]aspartate to [¹⁴C] asparagine and to tricarboxylic cycle acids was observed in alfalfa (Medicago sativa L.) nodules. Aspartate aminotransferase activity readily converted carbon from exogenously applied [¹⁴C]aspartate into the tricarboxylic acid cycle with subsequent conversion to the organic acids malate, succinate, and fumarate. Aminooxyacetate, an inhibitor of aminotransferase activity, reduced the flow of carbon from [¹⁴C]aspartate into tricarboxylic cycle acids and decreased ¹⁴CO₂ evolution by 99%. Concurrently, maximum conversion of aspartate to asparagine was observed in aminooxyacetate treated nodules (30 nanomoles asparagine per gram fresh weight per hour. Metabolism of [¹⁴C]aspartate and distribution of nodulefixed ¹⁴CO₂ suggest that two pools of aspartate occur in alfalfa nodules: (a) one involved in asparagine biosynthesis, and (b) another supplying a malate/aspartate shuttle. Conversion of [¹⁴C]aspartate to [¹⁴C]asparagine was not inhibited by methionine sulfoximine, a glutamine synthetase inhibitor, or azaserine, a glutmate synthetase, inhibitor. The data did not indicate that asparagine biosynthesis in alfalfa nodules has an absolute requirement for glutamine. Radioactivity in the xylem sap, derived from nodule ¹⁴CO₂ fixation, was markedly decreased by treating nodulated roots with aminooxyacetate, methionine sulfoximine, and azaserine. Inhibitors decreased the [¹⁴C]aspartate and [¹⁴]asparagine content of xylem sap by greater than 80% and reduced the total amino nitrogen content of xylem sap (including nonradiolabeled amino acids) by 50 to 80%. Asparagine biosynthesis in alfalfa nodules and transport in xylem sap are dependent upon continued aminotransferase activity and an uninterrupted assimilation of ammonia via the glutamine synthetase/glutamate synthase pathway. Continued assimilation of ammonia apparently appears crucial to continued root nodule CO₂ fixation in alfalfa.     

Properties of a Partially Purified Nucleoside Triphosphatase (NTPase) from the Chloroplast Envelope of Pea

The Mg-nucleoside triphosphatase activity associated with the inner envelope membrane of the pea chloroplast is comprised of at least two components, a major activity that is sensitive to vanadate and sodium fluoride and a minor insensitive activity. The vanadate/fluoride sensitive activity has been partially purified (about 35-fold) from Triton X-100 solubilized membranes by DEAE-Sephadex chromatography and sucrose density gradient centrifugation. The partially purified enzyme resembles the membrane-bound activity in requiring either Mg²⁺ or Mn²⁺, having a broad specificity for nucleoside triphosphates, having a K_m for ATP of 0.18 millimolar, and being inhibited by N-ethylmaleimide, but insensitive to sodium azide and dicyclohexylcarbodiimide. The partially purified enzyme obtained after sucrose gradient centrifugation has a markedly increased sensitivity to inhibition by inorganic pyrophosphate compared with the less pure enzyme. Pyrophosphate is not a substrate of either the membrane-bound or partially purified enzyme.     

Reconstitution of the H+-ATPase complex of Rhodospirillum rubrum by the beta subunit of the chloroplast coupling factor 1

A method is described for isolating the beta subunit from spinach chloroplast F1 (CF1). The isolated beta subunit reconstituted an active F1 hybrid with the F1 of Rhodospirillum rubrum chromatophores from which the beta subunit had been removed. The CF1 beta subunit was similar to the isolated beta subunit of Escherichia coli F1 (Gromet-Elhanan, Z., Khananshivili, D., Weiss, S., Kanazawa, H., and Futai, M. (1985) J. Biol. Chem. 260, 12635-12640) in that it restored a substantial rate of ATP hydrolysis and low, but significant light-dependent ATP synthesis to the beta-less chromatophores. The low rate of photophosphorylation observed with the hybrid enzyme probably resulted from a looser coupling of the CF1 beta subunit to proton translocation in the R. rubrum Fo-F1 complex. The hybrid enzyme exhibited a high specificity for Mg2+-ATP as substrate for ATP hydrolysis and both ATP synthesis and hydrolysis were strongly inhibited by the antibiotic tentoxin. In contrast, chromatophores reconstituted with the native R. rubrum beta subunit actively hydrolyzed both Mg2+-ATP and Ca2+-ATP and were insensitive to tentoxin. These results indicate a close functional homology between the beta subunits of the prokaryotic and eukaryotic H+-ATPases and suggest a role for the beta subunit in conferring the different metal ion specificities and inhibitor sensitivities upon the enzymes. They also demonstrate the feasibility of isolating the beta subunit from CF1 in a reconstitutively active form.