The elevated anthocyanin level in the shaded peel of ‘Anjou’ pear enhances its tolerance to high temperature under high light

Pigments, chlorophyll fluorescence, dark respiration, and the antioxidant system in the shaded peel of green ‘Anjou’ pear (Pyrus communis L.) and its bud mutation, red ‘Anjou’, were compared in response to high peel temperature, high light alone or in combination to determine the protective role of anthocyanins under high temperature with or without light. Under high temperature treatment alone, no difference in the maximum quantum yield of PSII (F_V/F_M) was detected between red ‘Anjou’ and green ‘Anjou’; the superoxide dismutase activity and the glutathione pool were up-regulated in green ‘Anjou’ peel but remained unchanged in red ‘Anjou’ peel. Under high temperature coupled with high light, the F_V/F_M of green ‘Anjou’ peel was decreased to a lower value than that of red ‘Anjou’, and significant interaction was detected between temperature and light for both cultivars. Furthermore, the difference in F_V/F_M between red ‘Anjou’ and green ‘Anjou’ under high temperature coupled with high light was significantly larger than that under high light alone, indicating that this larger difference was caused by the interaction between high temperature and high light as no significant difference was detected in F_V/F_M between the two cultivars under high temperature treatment alone at any sampling point. It is concluded that the elevated anthocyanin level in the shaded peel of red ‘Anjou’ does not alter its thermotolerance in the dark, but makes it more tolerant of high temperature under high light.     

Molecular Systematics of Middle American Cichlid Fishes and the Evolution of Trophic-Types in ‘Cichlasoma(Amphilophus)’ and ‘C.(Thorichthys)’

The majority of Middle American cichlids are placed in the informal assemblage ‘Cichlasoma.’ The group is divided into eight sections which appear to be based primarily on trophic morphology. Although several members of ‘Cichlasoma’ have been used in ecomorphological, behavioral, and biogeographic studies, no phylogenetic hypotheses for the group exist. In an attempt to develop a better understanding of the phylogenetic relationships of ‘cichlasomine’ cichlids, we examined the evolution of the trophic specialization, substratum-sifting, in two sections, ‘Cichlasoma(Thorichthys)’ and ‘C.(Amphilophus),’ to determine whether the trait reflects common ancestry. We sequenced the complete mitochondrial cytochromebgene for 19 cichlids representing six sections of ‘Cichlasoma,’ and representatives of other Neotropical Cichlidae. Additional cichlid, and noncichlid outgroup sequences were included for a total of 22 taxa. The molecular phylogeny supports the recognition of the section ‘C.(Thoricthys)’ as a natural group, and we place those cichlids in the genusThorichthys.The phylogeny also depicts ‘C.(Amphilophus)’ as paraphyletic, with substratum-sifters and generalized predators forming separate nonsister clades. We recommend that the substratum-sifting clade of the section ‘C.(Amphilophus)’ be placed in the resurrected genusAstatheros.The generalized predator clade of ‘C.(Amphilophus)’ contains only two species, ‘C. (A.) citrinellum’ and ‘C. (A.) labiatum,’ which we place in the genusAmphilophus.The phylogenetic hypotheses generated indicate that the substratum-sifting generaThorichthysandAmphilophusdo not share a common ancestor. Reconstruction of the evolution of substratum-sifting is equivocal, requiring either the independent evolution of the trait on two separate occasions or its presence in a more inclusive clade and subsequent loss in nonsubstratum sifting species.     

Can elevated CO(2) improve salt tolerance in olive trees?

We compared growth, leaf gas exchange characteristics, water relations, chlorophyll fluorescence, and Na⁺ and Cl⁻ concentration of two cultivars (‘Koroneiki’ and ‘Picual’) of olive (Olea europaea L.) trees in response to high salinity (NaCl 100 mM) and elevated CO₂ (eCO₂) concentration (700 μL L⁻¹). The cultivar ‘Koroneiki’ is considered to be more salt sensitive than the relatively salt-tolerant ‘Picual’. After 3 months of treatment, the 9-month-old cuttings of ‘Koroneiki’ had significantly greater shoot growth, and net CO₂ assimilation (A_CO2) at eCO₂ than at ambient CO₂, but this difference disappeared under salt stress. Growth and A_CO2 of ‘Picual’ did not respond to eCO₂ regardless of salinity treatment. Stomatal conductance (g_s) and leaf transpiration were decreased at eCO₂ such that leaf water use efficiency (WUE) increased in both cultivars regardless of saline treatment. Salt stress increased leaf Na⁺ and Cl⁻ concentration, reduced growth and leaf osmotic potential, but increased leaf turgor compared with non-salinized control plants of both cultivars. Salinity decreased A_CO2, g_s, and WUE, but internal CO₂ concentrations in the mesophyll were not affected. eCO₂ increased the sensitivity of PSII and chlorophyll concentration to salinity. eCO₂ did not affect leaf or root Na⁺ or Cl⁻ concentrations in salt-tolerant ‘Picual’, but eCO₂ decreased leaf and root Na⁺ concentration and root Cl⁻ concentration in the more salt-sensitive ‘Koroneiki’. Na⁺ and Cl⁻ accumulation was associated with the lower water use in ‘Koroneiki’ but not in ‘Picual’. Although eCO₂ increased WUE in salinized leaves and decreased salt ion uptake in the relatively salt-tolerant ‘Koroneiki’, growth of these young olive trees was not affected by eCO₂.     

Concentration and shear rate dependence of viscosity in random coil polysaccharide solutions

The concentration (c) and shear rate (γ) dependence of viscosity (η) has been studied for a wide range of random coil polysaccharide solutions, and the following striking generalities are observed:

1. 1. The transition from dilute to concentrated solution behaviour occurs at a critical concentration , when ‘zero shear’ specific viscosity (η_sp) ≈ 10. η_sp varies as c^1.4 for dilute solutions, and as c^3.3 for concentrated solutions.

2. 2. The shear rate dependence of viscosity, and frequency dependence of dynamic (oscillatory) viscosity are closely superimposable.

3. 3. Double logarithmic plots of against (where η₀ is ‘zero shear’ viscosity, and is the shear rate at which ) are essentially identical for all concentrated solutions studied, and thus the two parameters η₀ and completely define the viscosity at all shear rates of practical importance.

Departures from points 1 and 2, but not 3, are observed for concentrated solutions of locust bean gum, guar gum, and hyaluronate at low pH and high ionic strength and are attributed to specific intermolecular associations (‘hyperentanglements’) of longer timescale than non-specific physical entanglements.     

Effects of free amino acids on the isolated symbiotic algae of the coral Plesiastrea versipora (Lamarck): absence of a host release factor response

Symbiotic algae incubated in host tissue homogenate of the coral Plesiastrea versipora for 2 h in the light released at least four and a half times as much photosynthetically fixed carbon (range 13.8±3.1 to 158±9.5 nmol C/10⁶ algae) as algae incubated in seawater (range 1.4±0.3 to 10.8±0.6 nmol C/10⁶ algae) indicating the presence of ‘host release factor’. When algae were incubated in a low molecular weight fraction of homogenate containing partially purified ‘host release factor’ they also released more carbon (range 62.2±3.7 to 279±11.4 nmol C/10⁶ algae) than algae incubated in seawater. This low molecular weight fraction contained free amino acids. We tested the hypothesis that the free amino acids in this fraction were responsible for ‘host release factor’ activity. Algae incubated in a mixture of free amino acids equivalent to those found in this fraction, released more fixed carbon (range 2.4±0.3 to 25.2±0.2 nmol C/10⁶ algae) than algae incubated in seawater but in each experiment, release was much lower than when algae were incubated in host tissue homogenate. These data indicate that the stimulation of release of photosynthetically fixed carbon from the symbiotic algae of Plesiastrea versipora incubated in partially purified host release factor is not primarily due to the presence of free amino acids. We are continuing further studies to determine the exact nature of the active compound.     

Cytogenetic stability of <Emphasis Type="Italic">Centaurea ragusina</Emphasis> long-term culture

Centaurea ragusina L. (Asteraceae) is an endemic Croatian plant species, which developed xeromorphic characteristics as a consequence of its natural environment – vertical limestone cliffs above the Adriatic sea. Cytogenetic status of C. ragusina long-term culture (94th subculture) and C. ragusina seedlings was analysed and compared after 4 weeks of growth. Cytogenetic stability was investigated in root meristem cells of C. ragusina cultured plants originated from Pen đa (cliffs near Dubrovnik) and seedlings originated from three different localities in south Adriatic (Penđa, Pasjača – cliffs near Dubrovnik and island of Komiža) using mitotic index and mitotic and chromosomal abnormalities as parameters. Mitotic indices of cultured plants and ‘Penđa’ seedlings were similar and showed significant increase compared to mitotic indices of ‘Komiža’ and ‘Pasjača’ seedlings. Although the highest number of mitotic abnormalities was recorded in root meristem cells of cultured plants, it was only a bit higher than in root tips of ‘Pasjača’ and ‘Penđa’ seedlings, while that of ‘Komiža’ was two times lower compared to cultured plants. Pattern of analysed mitotic abnormalities was very similar in root tips of cultured plants and ‘Pasjača’ and ‘Penđa’ seedlings, with exception of ‘Komiža’ seedlings. Presented results suggest that long-term cultivation of C. ragusina has almost no effect on culture ageing considering similar distribution of scored mitotic abnormalities as in ‘Penđa’ and ‘Pasjača’ seedlings.     

Analysis of human disease genes in the context of gene essentiality

The characteristics of human disease genes were investigated through a comparative analysis with mouse mutant phenotype data. Mouse orthologs with mutations that resulted in discernible phenotypes were separated from mutations with no phenotypic defect, listing ‘phenotype’ and ‘no phenotype’ genes. First, we showed that phenotype genes are more likely to be disease genes compared to no phenotype genes. Phenotype genes were further divided into ‘embryonic lethal’, ‘postnatal lethal’, and ‘non-lethal phenotype’ groups. Interestingly, embryonic lethal genes, the most essential genes in mouse, were less likely to be disease genes than postnatal lethal genes. These findings indicate that some extremely essential genes are less likely to be disease genes, although human disease genes tend to display characteristics of essential genes. We also showed that, in lethal groups, non-disease genes tend to evolve slower than disease genes indicating a strong purifying selection on non-disease genes in this group. In addition, phenotype and no phenotype groups showed differing types of disease mutations. Disease genes in the no phenotype group displayed a higher frequency of regulatory mutations while those in the phenotype group had more frequent coding mutations, indicating that the types of disease mutations vary depending on gene essentiality. Furthermore, missense disease mutations in no phenotype genes were found to be more radical amino acid substitutions than those in phenotype genes.     

Interactions of cardiac glycosides with cells and membranes Properties and structural aspects of two receptor sites for ouabain in erythrocytes

The existence of two types of binding sites for ouabain in human erythrocyte membranes is described. Receptor sites designated as ‘type I’, which may be identical to the K⁺-insensitive sites of intact cells, were detected at concentrations of ouabain as low as 10⁻⁷ M. The ‘type II’ receptor sites require the inclusion of Mg²⁺ + P_i to form complexes with ouabain; they may be identical to the K⁺-sensitive sites of intact cells. These sites were saturated at approx. 5 · 10⁻⁷ M ouabain but could not be detected at higher concentrations. The range of ouabain concentrations at which ‘type I’ receptors start to predominate (i.e. 5 · 10⁻⁸–5 · 10⁻⁷ M) was termed ‘critical digitalis concentrations’. The process of binding reached equilibrium within 1 and 4 h for ‘type I’ and ‘type II’ sites, respectively. The dissociation constant for ‘type II’ receptor-ouabain complexes was 7.6 · 10⁻⁹ M.Under similar experimental conditions, rat erythrocyte membranes exhibited only non-saturable sites.Alterations in the proportions of the two types of receptors were demonstrated by preincubation of the membranes, in the presence or absence of Mg²⁺ + P_i, prior to the addition of ouabain. In the first case, ‘type II receptor-ouabain’ complexes were stabilized at about 50% of the untreated membranes and ‘type I-ouabain’ complexes slowly approached equilibrium over a period of 24 h. In the latter instance, ‘type I’ receptors were not detected, and only ‘type II-ouabain’ complexes prevailed.     

Effect of epiphytes on the extent of necrotic injuries of resistant and susceptible poplar clones infected with Dothichiza populea

Poplar cuttings of a resistant clone, Populus ‘Grandis’, and susceptible clones, Populus nigra ‘Italica’ and Populus ‘Robusta’, were infected with the pathogenic fungus Dothichiza populea alone, or with the pathogen and one of five strains of epiphytes antagonistic towards it (in vitro), isolated from poplar bark. The extent of injury was examined for 28 days after infection by determining the length of necrotic patches and their area as expressed in per cent of the total area of a cutting or the area of necrotic injuries caused by the pathogen alone.All the poplar cuttings of both the resistant and susceptible clones became diseased when infected with the pathogen alone. Surprisingly enough, however, the least affected clone was the susceptible P. ‘Robusta’, in which necrotic injuries covered 28% of the total area, as against 40% and 70% in the resistant P. ‘Grandis’ and the susceptible P. nigra ‘Italica’, respectively.When the cuttings were infected simultaneously with Dothichiza populea and its antagonistic epiphytes, the diseased area in the resistant clone diminished by as much as two-thirds, and in the susceptible P. nigra ‘Italica’, by one-third in comparison with the area affected by the pathogen alone. In turn, in the susceptible P. ‘Robusta’ the introduction of three out of five epiphytes stimulated the growth of the pathogenic fungus producing on average a double increase in the necrotic area. The differences in the response of the pathogen to the presence of epiphytes recorded in the susceptible clones indicate a marked influence of the plant on the nature of interactions between its epiphytic microflora and the pathogen.     

Morphological Stasis of Two Species Belonging to the L-morphotype in the <Emphasis Type="Italic">Brachionus plicatilis</Emphasis> Species Complex

Detection and characterization of sibling species complexes in zooplankton are critical to understanding their ecological responses and patterns of evolution. The taxon Brachionus plicatilis is a complex of at least 14 species with three major, deeply diverged clades, which are morphologically distinct. We studied morphometric differences between two species – B. plicatilis sensu stricto and B. ‘Manjavacas’– which belong to the L-(large) morphotype and often co-occur in ponds or lakes. B. plicatilis s.s. was on average 6% longer than B. ‘Manjavacas’. They differed significantly in the measurements related to lorica spines. A significant discriminant function relating spine measurements was found, however, individuals from each species showed extensive overlap. Our morphometric data provide additional evidence for the species status of B. plicatilis s.s. and B. ‘Manjavacas’. Since these are ancient species, our results support that a morphological stasis occurs in these taxa. We identified COI restriction sites for PvuII and KpnI which are diagnostic for B. ‘Manjavacas’ and B. plicatilis s.s., respectively. We conclude that morphometry is not useful in classifying the two species. At present, this can only be done reliably using molecular methods.     

Candidatus Monilibacter spp., common bulking filaments in activated sludge, are members of Cluster III Defluviicoccus

Two alphaproteobacterial Neisser negative ‘Nostocoida limicola’ morphotypes differing slightly in their trichome diameter and filament regularity were dominant populations in the Bendigo, Victoria, Australia activated sludge community removing phosphorus (P). Neither responded to the FISH probes available for any of the other alphaproteobacterial ‘N. limicola’ morphotypes. Instead both fluoresced with the DF988 FISH probe designed originally to target alphaproteobacterial cluster II Defluviicoccus tetrad forming organisms. A 16S rRNA based clone library from this biomass revealed that the alphaproteobacterial clones grouped closely with Candidatus ‘Monilibacter batavus’ and Defluviicoccus clones in a cluster separate from the existing cluster I and II Defluviicoccus. When a FISH probe was designed against these, it only hybridized to the thinner and less abundant ‘N. limicola’ morphotype. Micromanipulation–RT-PCR was used to selectively recover the main ‘N. limicola’ morphotype and a FISH probe designed against the 16S rRNA clones generated from it showed only this filament fluoresced. From FISH based surveys, both ‘N. limicola’ variants occurred frequently in phosphorus removal activated sludge systems in Australia treating domestic waste. The data suggest that they represent two new strains of Candidatus ‘Monilibacter’, which on this evidence are filamentous members of the genus Defluviicoccus, a potential competitor for the polyphosphate accumulating organisms in these communities.     

The distribution and conservation of threatened Sphaeriidae on British grazing marshland

Freshwater sphaeriid bivalves are poorly known ecologically, particularly uncommon species such as Pisidium pseudosphaerium (UK RDB (Red Data Book) staus = ‘rare’). In the UK, this species occupies grazing marsh where conservation opportunities might be shared with other threatened molluscs. We surveyed sphaeriids including P. pseudosphaerium and snails in 106 drainage ditches in SE England in 1999. P. pseudosphaerium occupied over half the ditches surveyed, at slightly elevated BOD (6 ± 7 mg l⁻¹ sd) but reduced calcium (64 ± 31 mg l⁻¹) and nitrate (0.5 ± 1.2 mg l⁻¹). As part of a sphaeriid assemblage comprising Sphaerium corneum, Musculium lacustre and Pisidium obtusale (=Assemblage 1), P. pseudosphaerium occurred in ditches with floating vegetation and abundant snails of conservation importance, co-occurring significantly with either Valvata macrostoma (RDB ‘vulnerable’) or Anisus vorticulus (RDB: ‘vulnerable’). A more diverse sphaeriid assemblage (=Assemblage 2) included species common in wider, deeper ditches with open water. We suggest that traditional ditch management can support both rare and representative snails and bivalves on grazing marsh. Quasi-traditional and rotational ditch clearance will favour common sphaeriids and pioneer snails during early succession; P. pseudosphaerium, V. macrostoma, A. vorticulus during mid-succession; and the snail Segmentina nitida (RDB: ‘Endangered’) in late successional ditches. In common with threatened snails, P. pseudosphaerium will also benefit from reduced eutrophication. We recommend that P. pseudosphaerium retain RDB status to ensure protection and to emphasise the need for improved ecological information.     

Time-activity budgets and energetics of Dipper Cinclus cinclus are dictated by temporal variability of river flow

The white-throated Dipper (Cinclus cinclus) is unique among passerine birds by its reliance on diving to achieve energy gain in fast-flowing waters. Consequently, it should have evolved behavioural adaptations allowing responding directly to runoff patterns (one of the assumptions of the Natural Flow Regime Paradigm—NRFP). In this study (October 1998–August 2001), we investigated how behavioural and energy use strategies in Dippers might vary under the natural flow regime of snowmelt-dominated streams in The Pyrénées (France) where natural flow regime is highly seasonal and predictable. We recorded time spent in each of 5 behavioural activities of ringed birds to estimate time–activity budgets and derive time–energy budgets enabling the modelling of daily energy expenditure (DEE). Annual pattern in ‘foraging’ and ‘resting’ matched perfectly the annual pattern of the natural regime flow and there was a subtle relationship between water stage and time spent ‘diving’ the later increasing with rising discharge up to a point where it fell back. Thus, time–activity budgets meet the main prediction of the NRFP. For males and females Dippers, estimates of feeding rates (ratio E_obs/E_req = observed rate of energy gain / required foraging rate) and energy stress (M = DEE / Basal Metabolic Rate) also partly matched the NFRP. Maximum value for the ratio E_obs/E_req was registered in May whilst M peaked in spring. These ratios indicated that Pyrenean Dippers could face high energy stress during winter but paradoxically none during high snowmelt spates when food is expected to be difficult to obtain in the channel and when individual birds were observed spending ca 75% of the day ‘resting’. Annual pattern in DEE did not match the NFRP ; two phases were clearly identified, the first between January to June (with oscillating values 240–280 kJ d^− 1 ind^− 1) and the second between July and December (200–220 kJ d^− 1 ind^− 1). As total energy expenditure was higher during the most constraining season or life cycle, we suggest that energy management by Dippers in Pyrenean mountain streams may fit the ‘peak total demand’ hypothesis. At this step of the study, it is not possible to tell whether Dippers use an ‘energy-minimisation’ or an ‘energy-maximisation' strategy.     

A physico-chemical study on the polysaccharide ulvan from hot water extraction of the macroalga Ulva

Results of a study on the solution behaviour of the cell-wall polysaccharide named ulvan obtained from hot water extraction of a flour of Ulva ‘rigida’ are reported. In particular the spectroscopic properties and ion binding capacity of this charged polysaccharide were studied by circular dichroism and isothermal microcalorimetric titrations in order to gain information on the potential exploitation of this low cost biomass. A marked tendency of this polysaccharide to uptake water was evidenced by studying the proton spin-lattice relaxation times of the solvent, T₁, embedded in this highly charged polysaccharide.     

The state of water in muscle as studied by pulsed NMR

Spin-lattice relaxation times for the water protons in rat gastronemius muscle are reported over the temperature range +37 to −70°C at six resonance frequencies ranging from 4.5 to 60.0 MHz. From −8 to −70°C, the bulk of the muscle water is frozen. The unfrozen part is termed the hydrated layer and amounts to 7–12% of the total water content. Its correlation time takes teh form of a log-Gaussaian distribution function. From +37 to −8°C, the spin-lattice relaxation time is explained by the exchange of water between the hydration layer and the rest of the water, which behaves like ordinary liquid water. The fact that the observed T₂ values are smaller than the calculated values is attributed to the inner field inhomogeneity of the heterogenous system and/or the modification of T₂ due to non-zero dipolar interaction.In the presence of perdeuterated dimethylsulfoxide, the freezing point of water decreases and the amount of non-freezable water increases. T₁ of water protons for muscle containing 10, 20, and 40% dimethylsulfoxide was calculated.     

NMR spectroscopy of α-crystallin. Insights into the structure, interactions and chaperone action of small heat-shock proteins

The subunit molecular mass of α-crystallin, like many small heat-shock proteins (sHsps), is around 20 kDa although the protein exists as a large aggregate of average mass around 800 kDa. Despite this large size, a well-resolved ¹H NMR spectrum is observed for α-crystallin which arises from short, polar, highly-flexible and solvent-exposed C-terminal extensions in each of the subunits, αA- and αB-crystallin. These extensions are not involved in interactions with other proteins (e.g. β- and γ-crystallins) under non-chaperone conditions. As determined by NMR studies on mutants of αA-crystallin with alterations in its C-terminal extension, the extensions have an important role in acting as solubilising agents for the relatively-hydrophobic α-crystallin molecule and the high-molecular-weight (HMW) complex that forms during the chaperone action. The related sHsp, Hsp25, also exhibits a flexible C-terminal extension. Under chaperone conditions, and in the HMW complex isolated from old lenses, the C-terminal extension of the αA-crystallin subunit maintains its flexibility whereas the αB-crystallin subunit loses, at least partially, its flexibility, implying that it is involved in interaction with the ‘substrate’ protein. The conformation of ‘substrate’ proteins when they interact with α-crystallin has been probed by ¹H NMR spectroscopy and it is concluded that α-crystallin interacts with ‘substrate’ proteins that are in a disordered molten globule state, but only when this state is on its way to large-scale aggregation and precipitation. By monitoring the ¹H and ³¹P NMR spectra of α-crystallin in the presence of increasing concentations of urea, it is proposed that α-crystallin adopts a two-domain structure with the larger C-terminal domain unfolding first in the presence of denaturant. All these data have been combined into a model for the quaternary structure of α-crystallin. The model has two layers each of approximately 40 subunits arranged in an annulus or toroid. A large central cavity is present whose entrance is ringed by the flexible C-terminal extensions. A large hydrophobic region in the aggregate is exposed to solution and is available for interaction with ‘substrate’ proteins during the chaperone action.     

DNA replication in macronuclei of Tetrahymena pyriformis GL

DNA replication in macronuclei of Tetrahymena pyriformis GL has been studied to discriminate between hypotheses developed for the interpretation of intraclonal differentiation in ciliated protozoa (the diploid subnuclear, and the ‘master’-‘slave’ hypotheses). Tetrahymena cells were grown in a heavy ¹⁵N-³H medium and then transferred to a light ¹⁴N-¹⁴C medium. DNA was isolated after various periods following this transfer and studied in equilibrium CsCl density gradient centrifugation. Time-related changes in the DNA buoyant density pattern were investigated. The data obtained are interpreted to mean that all DNA in macronuclei of asynchronously growing Tetrahymena at exponential phase replicates semiconservatively once in a cell cycle. These data are in good agreement with the findings of Andersen & Zeuthen obtained on synchronous Tetrahymena cultures in the presence of BUdR.These results are not consistent with the ‘master’-‘slave’ hypothesis. The diploid subnuclear hypothesis is not in accord with other experimental evidence. An alternative hypothesis has been proposed concerning the nature of the macronuclear units and the process of determination. The two main points of this hypothesis are: (a) macronuclear units are diploid genome fragments (‘nucleosomes’); (b) determination is a process of haploidization by ‘allelic splitting’ at a definite macronuclear fission. Consistency with experimental data is discussed and some predictions of the hypothesis are given.     

Genotoxicity of 2,4-D and dicamba revealed by transgenic Arabidopsis thaliana plants harboring recombination and point mutation markers

The phenoxy herbicides 2,4-D and dicamba are released daily into the environment in large amount. The mechanisms of genotoxicity and mutagenicity of these herbicides are poorly understood, and the available genotoxicity data is controversial. There is a cogent need for a novel genotoxicity monitoring system that could provide both reliable information at the molecular level, and complement existing systems.We employed the transgenic Arabidopsis thaliana ‘point mutation’ and ‘recombination’ plants to monitor the genetic effects of the herbicides 2,4-D and dicamba. We found that both herbicides had a significant effect on the frequency of homologous recombination A→G mutation. Neither herbicides affected the T→G mutation frequency. Interestingly, these transgenic biomonitoring plants were able to detect the presence of phenoxy herbicides at concentrations that were lower than the guideline levels for Drinking Water Quality. The results of our studies suggest that our transgenic system may be ideal for the evaluation of the genotoxicity of herbicide-contaminated water. Moreover, the unique ability of the plants to detect both double-strand breaks (homologous recombination) and point mutations provides tremendous potential in the study of molecular mechanisms of genotoxicity and mutagenicity of phenoxy herbicides.     

Characterization of the solubilization of lipid bilayers by surfactants

This communication addresses the state of aggregation of lipid-detergent mixed dispersions. Analysis of recently published data suggest that for any given detergent-lipid mixture the most important factor in determining the type of aggregates (mixed vesicles or mixed micelles) and the size of the aggregate is the detergent to lipid molar ratio in these aggregates, herein denoted the effective ratio, R_e. For mixed bilayers this effective ratio has been previously shown to be a function of the lipid and detergent concentrations and of an equilibrium partition coefficient, K, which describes the distribution of the detergent between the bilayers and the aqueous phase. We show that, similar to mixed bilayers, the size of mixed micelles is also a function of the effective ratio, but for these dispersions the distribution of detergent between the mixed micelles and the aqueous medium obeys a much higher partition coefficient. In practical terms, the detergent concentration in the mixed micelles is equal to the difference between the total detergent concentration and the critical micelle concentration (cmc). Thus, the effective ratio is equal to this difference divided by the lipid concentration. Transformation of mixed bilayers to mixed micelles, commonly denoted solubilization, occurs when the surfactant to lipid effective ratio reaches a critical value. Experimental evaluation of this critical ratio can be based on the linear dependence of detergent concentration, required for solubilization, on the lipid concentration. According to the ‘equilibrium partition model’, the dependence of the ‘solubilizing detergent concentration’ on the lipid concentration intersects with the lipid axis at −1/K, while the slope of this dependence is the critical effective ratio. On the other hand, assuming that when solubilization occurs the detergent concentration in the aqueous phase is approximately equal to the critical micelle concentration, implies that the above dependence intersects with the detergent axis at the critical micelle concentration, while its slope, again, is equal to the critical effective ratio. Analysis of existing data suggests that within experimental error both these distinctively different approaches are valid, indicating that the critical effective ratio at which solubilization occurs is approximately equal to the product of the critical micelle concentration and the distribution coefficient K. Since the nature of detergent affects K and the critical micelle concentration in opposite directions, the critical (‘solubilizing’) effective ratio depends upon the nature of detergent less than any of these two factors.