Anatomy, physiology and biochemistry of root nodules of Sprint-2 Fix-, a symbiotically defective mutant of pea (Pisum sativum L.)

A plant-determined pea mutant Sprint-2 Fix^– and the parentalline Sprint-2 were compared for selected physiological and biochemicalparameters. The Fix^– mutation prevented differentiationof Rhizobium leguminosarum bacteria into bacteroids and producedlarge, white, non-fixing nodules. These lacked nitrogenase-linkedrespiration, but had a background rate of CO₂ evolution similarto the normal Fix⁺ nodules. The cortical structure of the ineffectivenodules suggests the existence of an oxygen diffusion barrierand this was supported by a low oxygen concentration in thecentral region (0.5–3.0%), measured using an O₂ sensitivemicro-electrode. Sucrose and starch contents were similar innormal and ineffective nodules while ononitol content was about15 times lower in the Fix^– nodules. The distribution ofstarch was also different in the two nodule types. The activitiesof glutamine synthetase (GS), sucrose synthase (SS), phosphoenolpyruvatecarboxylase (PEPC) and alanine pyruvate aminotransferase (APAT)were markedly higher in Fix⁺ nodules while the activities ofpyruvate decarboxylase (PDC), alcohol dehydrogenase (ADH) andglutamate dehydrogenase (GDH) were higher in Fix^– nodules.The data from immunodetection of host nodule proteins confirmedthe reduced levels of sucrose synthase and the almost completeabsence of glutamine synthetase and leghaemoglobin in mutantnodules. There was no significant difference in the amount ofnitrogenase component 1 extracted from the microsymbiont ofnormal and ineffective nodules, but component 2 was hardly detectablein the Fix^– mutant. Key words: Pisum sativum, Fix^– mutant, nodules     

Loss of Genetic Diversity and Increased Subdivision in an Endemic Alpine Stonefly Threatened by Climate Change

Much remains unknown about the genetic status and population connectivity of high-elevation and high-latitude freshwater invertebrates, which often persist near snow and ice masses that are disappearing due to climate change. Here we report on the conservation genetics of the meltwater stonefly Lednia tumana (Ricker) of Montana, USA, a cold-water obligate species. We sequenced 1530 bp of mtDNA from 116 L. tumana individuals representing “historic” (>10 yr old) and 2010 populations. The dominant haplotype was common in both time periods, while the second-most-common haplotype was found only in historic samples, having been lost in the interim. The 2010 populations also showed reduced gene and nucleotide diversity and increased genetic isolation. We found lower genetic diversity in L. tumana compared to two other North American stonefly species, Amphinemura linda (Ricker) and Pteronarcys californica Newport. Our results imply small effective sizes, increased fragmentation, limited gene flow, and loss of genetic variation among contemporary L. tumana populations, which can lead to reduced adaptive capacity and increased extinction risk. This study reinforces concerns that ongoing glacier loss threatens the persistence of L. tumana, and provides baseline data and analysis of how future environmental change could impact populations of similar organisms.     

Short-term inhibition of legume N2 fixation by nitrate

The hypothesis of NO ₂ ⁻ toxicity as the causative factor of NO ₃ ⁻ inhibition of nitrogenase (N₂ase; EC 1.18.6.1) activity has been evaluated using a short-term exposure (3 d) of several legumes. Treatment of plants with 10 mM NO ₃ ⁻ induced nitrate reductase (NR) from bacteroids (EC 1.7.99.4) and nodule cytosol (EC 1.6.6.1) in most species. Regardless of the levels of both enzymes, significant accumulation of NO ₂ ⁻ did not occur in nodules. Dissection of nodules into cortical and infected regions, and subsequent NO ₂ ⁻ assays in conditions that suppressed enzyme activities, indicated that, in the short-term, bacteroid NR does not generate NO ₂ ⁻ in vivo. This is probably because NO ₃ ⁻ access is restricted to the nodule cortex. Accumulation of NO ₂ ⁻ at levels that are damaging for N₂ase and leghaemoglobin were only observed when a delay occurred between dissection and assaying of nodules. It is concluded that NO ₂ ⁻ is not responsible for the initial NO ₃ ⁻ -induced decline of N₂ase activity, and that toxic amounts of NO ₂ ⁻ only build up in nodules following longer exposures to NO ₃ ⁻ , when this anion is actively reduced by bacteroid and cytosol enzymes.     

FACTORS INFLUENCING THE EFFLUX OF [3H]GAMMAAMINOBUTYRIC ACID FROM SATELLITE GLIAL CELLS IN RAT SENSORY GANGLIA

The spontaneous efflux of [³H]GABA from the satellite glial cells of rat dorsal root ganglia and the efflux evoked by 64 mM-K⁺ were studied in the presence of 10^-5M-amino-oxyacetic acid and found not to be affected by 10^-4M-D 600 or by elevated (9.6mM) Ca²⁺ in the absence of Mg²⁺. [³H]GABA efflux was increased by replacing sodium ions in the washing medium by choline ions and 64 mM-K⁺ failed to increase the efflux further. The drugs veratridine (10^-6 and 10^-4M) and batrachotoxin (10^-8 and 10^-6 M) failed to alter the spontaneous efflux of [³H]GABA from the glial cells. A variety of compounds, including amino acids, a GABA analogue and a GABA antagonist were tested for their ability to affect [³H]GABA efflux. The results indicated that compounds which inhibit GABA uptake into glial cells were also able to stimulate [³H]GABA efflux from these cells. The results are discussed with reference to possible mechanisms involved in the release of GABA from glial cells.     

RELEASE OF [3H]GAMMA-AMINOBUTYRIC ACID FROM GLIAL CELLS IN RAT DORSAL ROOT GANGLIA.

Abstract— Desheathed rat dorsal root ganglia were incubated in a medium containing amino-oxyacetic acid and [³H]GABA. Under these conditions, [³H]GABA is taken up exclusively by the satellite glial cells in the ganglia. Efflux of [³H]GABA from the tissue was measured after passing the ganglia through a series of wash solutions. The spontaneous efflux of radioactivity, mostly [³H]GABA, was more rapid in the absence of amino-oxyacetic acid in the incubation and wash media.
Raising the potassium concentration in the wash media caused an increase in the efflux of [³H]GABA. This increase was sigmoidally related to the potassium concentration in the wash media, reaching a maximum at 64 m m -K⁺. The releasing effect of K⁺ was inhibited by removing calcium from the media. Reducing the calcium and raising the magnesium concentration in the wash solutions inhibited the increased efflux of [³H]GABA due to 64 m m -K⁺ by 48 per cent, while 5 mM-La³⁺ and diphenylhydantoin (0·005 and 0·5 m m ) had no effect on this increase.
Only a small increase in the efflux of [¹⁴C]glutamate was produced by 64 m m -K⁺ and it had no effect upon the effluxes of [³H]glycine, [³H]alanine or [³H]leucine. The efflux of lactate dehydrogenase was similarly unaffected by 64 mM-K⁺. The results suggest that glial cells in spinal ganglia can respond to depolarizing concentrations of potassium by releasing GABA in a calcium-dependent process.     

Effect of Electroconvulsive Shock on the Uptake and Release of Noradrenaline and 5-Hydroxytryptamine in Rat Brain Slices

Abstract: The uptake and release of [³H]noradrenaline and [³H]-5-hydroxytryptamine (5-HT) were studied in cerebral cortex slices from rats 30 min and 24 h after a single electroconvulsive shock (ECS) and 24 h after a series of five shocks given over 10 days. Both the K _m and V _max for 5-HT uptake were lower than controls 24 h after a single ECS, whereas after 5 ECS spread over 10 days both parameters remained depressed, though only the fall in V_max was significant. Noradrenaline uptake was not altered after a single ECS, but the V_max and K _m were elevated following chronic ECS treatment. Neither ECS treatment schedule had any effect on the potassium-stimulated release of either transmitter. It is possible that the changes in monoamine uptake seen following ECS are an adaptive response to alterations in the synaptic cleft concentration of these transmitters.     

Pulmonary alveolar macrophages express a polyamine transport system

Polyamine transport is an important mechanism by which cells regulate their intracellular polyamine content. It is well established that the lung has a high capacity for polyamine transport, and recently the polyamine putrescine has been shown to be selectively accumulated into the type II pneumocyte of rabbit lung slices (Saunders et al.: Lab. Invest., 95:380-386, 1988). In addition, it has been suggested that there may be more than one polyamine transport system in lung tissue (Byers et al.: Am. J. Physiol., 252:C663-C669, 1987). In the present study, we have examined whether there are differences in the distribution of putrescine and spermidine uptake activities in isolated rabbit lung cells. We report that pulmonary alveolar macrophages have a greater rate of uptake of both putrescine and spermidine than the total lung cell population. Kinetic analysis of the polyamine uptake system present in macrophages showed putrescine uptake consisted of a saturable (Km = 2.1 microM) and nonsaturable component whilst spermidine uptake consisted of both a high- and a low-capacity saturable component (Km = 0.16 microM and 1.97 microM, respectively). The rate of polyamine transport was similar to those reported for many proliferative or tumor cell-lines and appears to be greater than any other major lung cell type. Inhibition studies of the transport of polyamines into pulmonary alveolar macrophages suggested that the uptake of both putrescine and spermidine was mediated by the same system, which could not be described by simple Michaelis-Menten kinetics. The transport appears to be reversible due to significant efflux. This is the first study to describe the presence of multiple polyamine transport systems in pulmonary alveolar macrophages.