Blockade by Neurotransmitter Antagonists of Veratridine-Activated Ion Channels in Neuronal Cell Lines

Abstract: The voltage-dependent Na⁺ ionophore of various neuronal cells is permeable not only to Na⁺ ions but also to guanidinium ions. Therefore, the veratridine-(or aconitine-) stimulated influx of [¹⁴C]guanidinium in neuroblastoma × glioma hybrid cells was measured to characterize the Na⁺ ionophore of these cells. Half-maximal stimulation of guanidinium uptake was seen at 30 μ M veratridine. At 1 m M guanidinium, the veratridine-stimulated uptake of guanidinium was lowered to 50% by approximately 60 m M Li⁺, Na⁺, or K⁺ and by a few millimolar Mn²⁺, Co²⁺, or Ni²⁺. The basal, as well as the veratridine-stimulated, uptake of guanidinium was inhibited by the cholinergic antagonists (+)-tubocurarine ( K_i = 50 to 500 n M ) and atropine ( K_i = 5 to 30 μ M ) and the adrenergic antagonists phentolamine ( K_i = 5 μ M ) and propranolol ( K_i = 60 μ M ). The specificity of the inhibitory effects of these agents is stressed by the ineffectiveness of various other neurotransmitter antagonists. However, the corresponding ionophore in neuroblastoma cells (clone N1E-115) seems to be regulated differently. While phentolamine and propranolol inhibit the veratridine-activated uptake as in the hybrid cells, (+)-tubocurarine and atropine exert only a slight effect.     

Microevolution within a clonal population of pathogenic bacteria: recombination, gene duplication and horizontal genetic exchange in the opa gene family of Neisseria meningitidis

Opacity (Opa) proteins are a family of antigenically variable outer-membrane proteins of Neisseria meningitidis. Even among clonally related epidemic meningococcal isolates, there is greater variation of Opa protein expression than can be accounted for by the opa gene repertoire of any individual strain. We characterized the opa genes of eight closely related Isolates of serogroup A N. meningitidis (subgroup IV-1) from a recent meningitis epidemic in West Africa. DNA sequence analysis and Southern blot experiments indicated that changes occurred in the opa genes of these bacteria as they spread through the human population, over a relatively short period of time. Such changes in one or a few loci within a clonal population are referred to as microevolution. The distribution of sequences present in hypervariable (HV) regions of the opa genes suggests that duplication of all or part of opa genes into other opa loci changed the repertoire of Opa proteins that could be expressed. Additional variability in this gene family appears to have been introduced by horizontal exchange of opa sequences from other meningococcal strains and from Neisseria gonorrhoeae. These results indicate that processes of recombination and genetic exchange contributed to variability in major surface antigens of this clonal population of pathogenic bacteria.     

Correlation between the activities of five ribosome-inactivating proteins in depurination of tobacco ribosomes and inhibition of tobacco mosaic virus infection

The rRNA depurination activities of five ribosome-inactivating proteins (RIPs) were compared in vitro using yeast and tobacco leaf ribosomes as substrates. All of the RIPs (pokeweed antiviral protein (PAP), dianthin 32, tritin, barley RIP and ricin A-chain) were active on yeast ribosomes. PAP and dianthin 32 were highly active and ricin A-chain weakly active on tobacco ribosomes, whereas tritin and barley RIP were inactive. PAP and dianthin 32 were highly effective in inhibiting the formation of local lesions caused by tobacco mosaic virus (TMV) on tobacco leaves, whereas tritin, barley RIP and ricin A-chain were ineffective. The apparent anomaly between the in vitro rRNA depurination activity, but lack of antiviral activity of ricin A-chain was further investigated by assaying for rRNA depurination in situ following the topical application of the RIP to leaves. No activity was detected, a finding consistent with the apparent lack of antiviral activity of this RIP. Thus, it is concluded that there is a positive correlation between RIP-catalysed depurination of tobacco ribosomes and antiviral activity which gives strong support to the hypothesis that the antiviral activity of RIPs works through ribosome inactivation.     

Short-Term Response of Land Birds to Ponderosa Pine Restoration

We monitored the short‐term (>3 years) response of land birds to forest restoration treatments in Ponderosa pine forests located on the east slope of the North Cascade Range. Restoration treatments were designed to create stand structure and composition similar to pre‐settlement forests, which were influenced by a frequent fire regime. Overall, avian community composition was changed as a result of the treatments. Cassin’s Finch, Chipping Sparrow, and Yellow‐rumped Warbler were found at higher densities in treated stands, whereas Mountain Chickadee, Western Tanager, and Red‐breasted Nuthatch had higher densities in untreated stands. White‐headed Woodpecker and Western Bluebird were only detected in the treated stands. Brown‐headed Cowbird showed no response to treatments. We detected changes in the density of four of five foraging guilds in response to restoration treatments. Tree seedeaters, low understory and ground insectivores, and aerial insectivores all increased in density in treated stands. Overall, bark insectivores showed no density response to treatments. Tree foliage insectivore density was lower in treated than in untreated stands. Overall avian density, density of neotropical migrants, and density of some focal species were higher in treated stands. Monitoring should be continued to understand the longer‐term (5–10 year) responses of land birds and to guide future forest restoration efforts.     

Variation in Aggressiveness Components in the Hemileia vastatrix Population in Brazil

The Pucciniomycete fungus Hemileia vastatrix causes leaf rust on coffee trees. The pathogen is responsible for considerable yield losses in susceptible coffee cultivars if appropriate management strategies are not implemented. Rapid spread and epidemics of rust fungi are usually associated with the emergence of new races of the pathogen that overcome resistance or with the emergence of more aggressive populations of the pathogen. In Brazil, coffee production is dominated by susceptible cultivars of Coffea arabica and Coffea canephora. We assessed aggressiveness in 46 populations of H. vastatrix from Minas Gerais and Espírito Santo, two of the most important coffee‐producing states in Brazil. We observed a significant difference in the incubation period between the populations from Minas Gerais and Espírito Santo when 183 single‐pustule isolates were inoculated onto Catuaí Vermelho IAC 44, a susceptible C. arabica cultivar. Variation in aggressiveness components was observed between and within localities. Isolates with longer incubation periods also tended to have longer latent periods, although there was only a low correlation between these two aggressiveness components (r² = 0.34, P = 2.2 × 10^?16). Low‐sporulating isolates also had significantly longer incubation and latent periods. The H. vastatrix population from Minas Gerais and Espírito Santo is structured by the formation of groups of individuals with differential level of aggressiveness. Our results indicate that the variation in aggressiveness of the Brazilian H. vastatrix population may be associated with the geographic coffee‐producing areas.     

Heterotrimerization of PII-like signalling proteins: implications for PII-mediated signal transduction systems

PII-like signalling molecules are trimeric proteins composed of 12-13 kDa polypeptides encoded by the glnB gene family. Heterologous expression of a cyanobacterial glnB gene in Escherichia coli leads to an inactivation of E. coli's own PII signalling system. In the present work, we show that this effect is caused by the formation of functionally inactive heterotrimers between the cyanobacterial glnB gene product and the E. coli PII paralogues GlnB and GlnK. This led to the discovery that GlnK and GlnB of E. coli also form heterotrimers with each other. The influence of the oligomerization partner on the function of the single subunit was studied using heterotrimerization with the Synechococcus PII protein. Uridylylation of GlnB and GlnK was less efficient but still possible within these heterotrimers. In contrast, the ability of GlnB-UMP to stimulate the adenylyl-removing activity of GlnE (glutamine synthetase adenylyltransferase/removase) was almost completely abolished, confirming that rapid deadenylylation of glutamine synthetase upon nitrogen stepdown requires functional homotrimeric GlnB protein. Remarkably, however, rapid adenylylation of glutamine synthetase upon exposing nitrogen-starved cells to ammonium was shown to occur in the absence of a functional GlnB/GlnK signalling system as efficiently as in its presence.     

Xylem cavitation and hydraulic control of stomatal conductance in Laurel (Laurus nobilis L.)

The possible link between stomatal conductance (g_L), leaf water potential ( Ψ _L) and xylem cavitation was studied in leaves and shoots of detached branches as well as of whole plants of Laurus nobilis L. (Laurel). Shoot cavitation induced complete stomatal closure in air‐dehydrated detached branches in less than 10 min. By contrast, a fine regulation of g_L in whole plants was the consequence of Ψ _L reaching the cavitation threshold ( Ψ _CAV) for shoots. A pulse of xylem cavitation in the shoots was paralleled by a decrease in g_L of about 50%, while Ψ _L stabilized at values preventing further xylem cavitation. In these experiments, no root signals were likely to be sent to the leaves from the roots in response to soil dryness because branches were either detached or whole plants were growing in constantly wet soil. The stomatal response to increasing evaporative demand appeared therefore to be the result of hydraulic signals generated during shoot cavitation. A negative feedback link is proposed between g_L and Ψ _CAV rather than with Ψ _L itself.     

Locomotor Ability and Wariness in Yellow-Bellied Marmots

Animals employ a variety of behaviors to reduce or manage predation risk. Often, these are studied in isolation, but selection may act on packages of behavior that are referred to as behavioral syndromes. We focused on yellow‐bellied marmots (Marmota flaviventris) and examined three commonly studied antipredator behaviors. We fitted general linear models to explain variation in maximum running speed, time allocated to vigilance and foraging during bouts of foraging, and flight initiation distance (FID). Marmot maximum running speed was influenced by the substrate run across; marmots ran fastest across dirt or low vegetation and slowest across stones or talus. Incline and several other variables shown to affect running speed in other marmot species failed to explain significant variation in yellow‐bellied marmots. From these results we expected marmots to be sensitive to substrate while foraging, but insensitive to incline. However, time allocated to foraging was affected by incline but not by substrate. In bouts of foraging observed in different habitats, and on different inclines, more time was allocated to foraging and less to vigilance on steep slopes and less on level ground. Substrate influenced FID. Marmots in tall vegetation were less tolerant of an approaching person than were those in shorter vegetation. Finally, we found significant correlations between the residuals from the maximum running speed model and the residuals from the time allocated to vigilance and foraging models. We found a tendency for marmots that ran slower than predicted to be less vigilant while foraging. We also found that relatively slow marmots engaged in more active foraging and less vigilance during foraging bouts. This finding suggests a ‘locomotor ability‐wariness while foraging’ syndrome. It also suggests that vulnerable individuals minimize their exposure while foraging.     

Seasonal changes in the ion‐mediated increase of xylem hydraulic conductivity in stems of three evergreens: any functional role?

Changes in hydraulic conductivity (K_h) were measured monthly in stems of Laurus nobilis between July 2005 and June 2006 and in March and June 2006 in stems of Prunus laurocerasus and Phillyrea latifolia . Percent enhancement of K_h (ΔK_h) was recorded as KCl injected into stems compared with deionized water. Stems of L. nobilis perfused with increasing [KCl] saturated the hydraulic effect at 50 m M KCl, while P. laurocerasus and Ph. latifolia showed saturation at 17 and less than 5 m M KCl, respectively. Impressive seasonal changes in ΔK_h were recorded in L. nobilis , from +120% in February to +20% during the growth period. ΔK_h was negatively correlated to minimum air temperatures and was highest during winter frost. Negative correlation also existed between ΔK_h and conduit lengths as detected during transition of plants from activity to winter rest and from winter to spring. In the winter, the thermal effect was prevailing. High cavitation-induced K_h loss coincided with high ΔK_h. Similar but much smaller seasonal effects on ΔK_h were recorded in the other two species where, however, also smaller seasonal changes in conduit lengths were recorded. We conclude that (1) the K⁺-induced effect on K_h was because of interference of the cation with pectins of intervessel pit membranes (increasingly crossed by the ionic solution in stems with shorter conduits); (2) winter frost might have an effect on ΔK_h and (3) seasonal changes in ΔK_h because of possible change in pectic composition may represent a mechanism to regulate water flows in planta.