Independence of Protein Synthesis and Drug Uptake in Nerve Cell Bodies and Glial Cells isolated by a New Technique

A severe handicap in any study of the cellular biochemistry of the brain is the unavailability of sufficient amounts of pure neurones and glial cells. Many efforts to separate one from the other have been made¹, but only four basic types of isolation procedures have been described for routine use: (1) brain is treated with a mixture of acetone-glycerol and water before mincing and centrifugation^3–5; (2) brain mince is sieved and subjected to gradient centrifugation¹,^6–8—this separates neuronal cell bodies from neuropil⁶ or from intact but rather contaminated glial cells⁸; (3) whole brain is disrupted in a tissue press and zonal centrifugation is used to separate cellular and subcellular components as they leave the zonal centrifuge rotor⁹; and (4) chopped brain is incubated in oxygen at 37° C in the presence of 1 % (w/v) trypsin¹⁰, a treatment which, in our estimation, severely prejudices this technique's general usefulness, because it precludes meaningful comparative investigations of enzymes and non-enzymatic structural and/or soluble cell proteins. Since investigations of the latter type are the aim in our laboratory, we needed a technique in which brain is not subjected to deleterious treatments such as immersion in acetone-glycerol-water mixtures^3–5 or trypsinization¹⁰ and in which no special instrumentation⁹, tissue presses⁹ or homogenizers⁸ are required. We now report a technique which successfully accomplishes this objective, for it makes possible the isolation of highly purified neuronal perikarya and of intact glial cells from a few grams of brain, in yields which permit the subsequent subcellular fractionation of the isolated cells. We also report two applications of the new technique which have enabled us to determine the in vivo time course of protein synthesis in and the uptake of the radioactive convulsant agent methionine sulphoximine^11,12 by, the neurones and the glial cells of the immature rat brain cortex.     

Morphological and Anatomical Studies of Linaria Species from South-west Spain: Seeds

Seeds of 24 taxa of Linaria from south-west Spain were examinedwith a scanning electron microscope and light microscope. Basedon seed features such as shape, surface ornamentation and cellshape, seven morphological types were recognized. Seeds of thedifferent types are described, illustrated and compared. A keyto distinguish the different species or groups of species isprovided. Copyright 1999 Annals of Botany Company Seed-coat, SEM, anatomy, Scrophulariaceae, Antirrhineae, Linaria .     

Primary production dynamics and climate variability: ecological consequences in semiarid Chile

Increase in rainfall variability has important consequences for organisms in arid and semiarid regions around the world. In South American and Australian deserts, the El Niño/Southern Oscillation (ENSO) phenomenon greatly influences rainfall patterns, and therefore the dynamics of plant communities. However, the field data needed to assess the effect of climate change on vegetational patterns is difficult to obtain because of the large spatial scale required for such studies. Normalized Difference Vegetation Index (NDVI) characteristics allow the use of several indexes related to vegetational structure. Due to its direct relationship with primary productivity, it is possible to obtain several measures of annual productivity. These include annual plant yield, annual maximum yield, onset of 'greening-up' and senescence phases, length of the 'green' season, vegetation peak, and therefore, the periods when more or less food is available for herbivores. After verification with ground-truth measures, we used NDVI data from two semiarid localities in north-central Chile (Fray Jorge and Aucó) to determine the relationship between rainfall patterns and vegetation cover and productivity related to El Niño phenomenon. With this information we gauge the influence of climatic processes on primary productivity in western South America, an area subject to strong climate variability. We predict significant variation in Chilean semiarid regions due to climate change, affecting mainly the extent and timing of annual growth season of vegetation, and also including a shorter and delayed greening-up season. Also, we predict that important decreases in rainfall levels will not have strong effects on primary production in these semiarid ecosystems.     

A retrospective,dual‐isotope approach reveals individual predispositions to winter‐drought induced tree dieback in the southernmost distribution limit of Scots pine

Winter‐drought induced forest diebacks in the low‐latitude margins of species' distribution ranges can provide new insights into the mechanisms (carbon starvation, hydraulic failure) underlying contrasting tree reactions. We analysed a winter‐drought induced dieback at the Scots pine's southern edge through a dual‐isotope approach (Δ¹³C and δ¹⁸O in tree‐ring cellulose). We hypothesized that a differential long‐term performance, mediated by the interaction between CO₂ and climate, determined the fates of individuals during dieback. Declining trees showed a stronger coupling between climate, growth and intrinsic water‐use efficiency (WUEi) than non‐declining individuals that was noticeable for 25 years prior to dieback. The rising stomatal control of water losses with time in declining trees, indicated by negative Δ¹³C‐δ¹⁸O relationships, was likely associated with their native aptitude to grow more and take up more water (suggested by larger tracheid lumen widths) than non‐declining trees and, therefore, to exhibit a greater cavitation risk. Freeze‐thaw episodes occurring in winter 2001 unveiled such physiological differences by triggering dieback in those trees more vulnerable to hydraulic failure. Thus, WUEi tightly modulated growth responses to long‐term warming in declining trees, indicating that co‐occurring individuals were differentially predisposed to winter‐drought mortality. These different performances were unconnected to the depletion of stored carbohydrates.     

Electrophoretic characterization of Amaranthus L. seed proteins and its systematic implications

The seed protein profiles of 11 Amaranthus taxa (Amaranthaceae) from Spain were studied. These profiles were evaluated as a chemical character to clarify the taxonomic complexity in the genus. Tricine-sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE) profiles of the Amaranthus seed proteins studied showed a range of peptides varying from 64 to 12 kDa, with a larger number of protein bands observed between 25.1 and 12 kDa. For the taxonomic study, 14 bands, some of them subdivided into several isoforms, were considered. The similarity analysis based on the SDS-PAGE profile is a useful character for the discrimination of species in Amaranthus , except for A. cruentus and A. hypochondriacus , for which a hybrid population was found. © 2007 The Linnean Society of London, Botanical Journal of the Linnean Society , 2007, 155 , 57–63.     

A Soluble Fumarate Reductase in Trypanosoma brucei Procyclic Trypomastigotes

ABSTRACT. The enzyme NADH-fumarate reductase associated with the membrane fraction of Trypanosoma brucei procyclic trypomastigotes, can be solubilized by more than 50% when increasing the ionic strength to the equivalent of 150 mM KCI. The apparent K_Ms for NADH (125 μM) and fumarate (50 μM) remain close to those previously reported for the membrane-bound form of this enzyme. Other electron acceptors (i.e. oxygen or cytochrome c) appear to accept electrons in the absence of fumarate (K_M for cytochrome c = 50 μM). The drug L-092,201 (Merck, Sharp and Dohme Research Laboratories, Rahway, NJ), an inhibitor of the membrane-bound fumarate reductase, also blocked the solubilized enzyme. Given the relatively high ionic strength of the intracellular environment we propose that, in vivo, the enzyme fumarate reductase is in the mitochondrial matrix or in the soluble fraction of another intracellular compartment.     

Chemotaxonomic study of seed glucosinolate composition in Coincya Rouy (Brassicaceae)

A total of 68 populations belonging to the genus Coincya Rouy (Brassicaceae) from the Iberian Peninsula was examined for seed glucosinolate composition. The presence of progoitrin, epiprogoitrin, glucosinalbin and gluconapin was recorded plus a fifth glucosinolate that could not be identified. A geographical clinal variation between progoitrin and epiprogoitrin was observed in the more widespread taxa, C. monensis subsp. orophila (Franco) Aedo, Leadiay & Murioz Garm. and C. moncnsis subsp. cheiranthos var. recurvata (All.) Leadiay. The fewest types of glucosinatc were observed in taxa with morphological characters considered more primitive, viz. subsp. hispida and var. recurvata. On the other hand, more complex and diverse composition was observed in the taxon with more advanced characters, viz. C. rupestris subsp. rupestris Porta & Rigo.     

Demonstration of Various Acid Hydrolases and Preliminary Characterization of Acid Phosphatase in Naegleria fowleri1

ABSTRACT. Extracts of the pathogenic ameba Naegleria fowleri, prepared by freeze-thawing and sonication, were analyzed for their content of various hydrolytic enzymes that have acid pH optima. The organism is rich in acid phosphatase activity as well as a variety of glycosidases which include β-glucosidase, β-galactosidase, β-fucosidase, α-mannosidase, hexosaminidase, arylsulfatase A, and β-glucuronidase. The crude extract contained only negligible levels of sphingomyelinase, neuraminidase, or arylsulfatase B. All of the hydrolases exhibited higher activity at pH 5.5 than at 7.0, indicating that they are truly “acid” hydrolases. In general, after centrifugation (100,000 g, 1 h), except for arylsulfatase B, more than half of the activity of each of the various hydrolases was recovered in the supernatant fraction. The acid phosphatase in the high-speed supernatant was purified 45-fold (32% yield) by chromatography on QAE-Sephadex and Sephadex G-200 and shown to have the following properties: 1) pH optima, 5.5; 2) K_m (4-methylumbelliferyl phosphate), 0.60 mM; 3) molecular weight (estimated by gel filtration chromatography), 92,000; 4) inhibited by heteropolymolybdate complexes but not by L(+) sodium tartrate (0.5 mM) or sodium fluoride (0.5 mM). In addition, unlike the tartrate-resistant acid phosphatase of Leishmania donovani, the major acid phosphatase of N. fowleri is less than 5% as effective in inhibiting superoxide anion production by f-Met-Leu-Phe-stimulated human neutrophils. The finding of high levels of a number of acid hydrolases in Naegleria fowleri raises several questions that merit further study: Do the hydrolases perform a housekeeping function in this single cell eukaryote or do they play some role in the pathogenic process that ensues when the organism infects a suitable host?     

Effect of some heterocyclic herbicides on CO2 assimilation by spinach chloroplasts

We have studied the inhibitory effect of heterocyclic herbicides simazine, paraquat, pyrazon and amitrole on photosynthetic CO₂ fixation and on the level of intermediates of the CO₂ assimilation cycle in isolated spinach (Spinacia oleracea) chloroplasts, as well as their in vitro activities on ribulose-1,5-bisphosphate carboxylase and fructose-1,6-bisphosphatase. The half inhibitory concentrations (I₅₀) of CO₂ assimilation were about 1 μM for simazine and paraquat, and 10 μM for pyrazon. Amitrole, with an I₅₀ 100 μM, gave only a weak inhibition. In the presence of simazine or pyrazon the triose-phosphates/phosphoglycerate ratio diminished because of a decrease of the triose-phosphates percentage from 47% to 19%, which means an inhibition of the phosphoglycerate reduction step by a low NADPH synthesis. However, there was not a parallel increase of phosphoglycerate, because of collateral pathways leading to phospho-enolpyruvate, amino acids and other non-identified compounds. Paraquat did not give such a decreased ratio, which could be explained as an inhibition of some step of the Calvin cycle later than triose-phosphates by the H₂O₂ formed in a Mehler reaction. Amitrole did not show any effect on the pattern of intermediates. Simazine and pyrazon at 10 μM concentration promote a 20–30% activation of ribulose-1,5-bisphosphate carboxylase activity, whereas paraquat, pyrazon and simazine showed an I₅₀ about 100 μM for the inhibition of the photosynthetic fructose-1,6-bisphosphatase.