Showing papers by "University of Hertfordshire published in 2000"

Single cell gel/comet assay: guidelines for in vitro and in vivo genetic toxicology testing.

Abstract: Atthe International Workshop on Genotoxicity Test Procedures (IWGTP) held in Washington, DC, March 25-26, 1999, an expert panel met to develop guidelines for the use of the single-cell gel (SCG)/Comet assay in genetic toxicology. The expert panel reached a consensus that the optimal version of the Comet assay for identifying agents with genotoxic activity was the alkaline (pH > 13) version of the assay developed by Singh et al. [1988]. The pH > 13 version is capable of detecting DNA single-strand breaks (SSB), alkali-labile sites (ALS), DNA-DNA/DNA-protein cross-linking, and SSB associated with incomplete excision repair sites. Relative to other genotoxicity tests, the advantages of the SCG assay include its demonstrated sensitivity for detecting low levels of DNA damage, the requirement for small numbers of cells per sample, its flexibility, its low costs, its ease of application, and the short time needed to complete a study. The expert panel decided that no single version of the alkaline (pH > 13) Comet assay was clearly superior. However, critical technical steps within the assay were discussed and guidelines developed for preparing slides with agarose gels, lysing cells to liberate DNA, exposing the liberated DNA to alkali to produce single-stranded DNA and to express ALS as SSB, electrophoresing the DNA using pH > 13 alkaline conditions, alkali neutralization, DNA staining, comet visualization, and data collection. Based on the current state of knowledge, the expert panel developed guidelines for conducting in vitro or in vivo Comet assays. The goal of the expert panel was to identify minimal standards for obtaining reproducible and reliable Comet data deemed suitable for regulatory submission. The expert panel used the current Organization for Economic Co-operation and Development (OECD) guidelines for in vitro and in vivo genetic toxicological studies as guides during the development of the corresponding in vitro and in vivo SCG assay guidelines. Guideline topics considered included initial considerations, principles of the test method, description of the test method, procedure, results, data analysis and reporting. Special consideration was given by the expert panel to the potential adverse effect of DNA degradation associated with cytotoxicity on the interpretation of Comet assay results. The expert panel also discussed related SCG methodologies that might be useful in the interpretation of positive Comet data. The related methodologies discussed included: (1) the use of different pH conditions during electrophoreses to discriminate between DNA strand breaks and ALS; (2) the use of repair enzymes or antibodies to detect specific classes of DNA damage; (3) the use of a neutral diffusion assay to identify apoptotic/necrotic cells; and (4) the use of the acellular SCG assay to evaluate the ability of a test substance to interact directly with DNA. The alkaline (pH > 13) Comet assay guidelines developed by the expert panel represent a work in progress. Additional information is needed before the assay can be critically evaluated for its utility in genetic toxicology. The information needed includes comprehensive data on the different sources of variability (e.g., cell to cell, gel to gel, run to run, culture to culture, animal to animal, experiment to experiment) intrinsic to the alkaline (pH > 3) SCG assay, the generation of a large database based on in vitro and in vivo testing using these guidelines, and the results of appropriately designed multilaboratory international validation studies.

Rhizosphere interactions and the exploitation of microbial agents for the biological control of plant-parasitic nematodes.

Abstract: A range of specialist and generalist microorganisms in the rhizosphere attacks plant-parasitic nematodes. Plants have a profound effect on the impact of this microflora on the regulation of nematode populations by influencing both the dynamics of the nematode host and the structure and dynamics of the community of antagonists and parasites in the rhizosphere. In general, those organisms that have a saprophytic phase in their life cycle are most affected by environmental conditions in the rhizosphere, but effects on obligate parasites have also been recorded. Although nematodes influence the colonization of roots by pathogenic and beneficial microorganisms, little is known of such interactions with the natural enemies of nematodes in the rhizosphere. As nematodes influence the quantity and quality of root exudates, they are likely to affect the physiology of those microorganisms in the rhizosphere; such changes may be used as signals for nematode antagonists and parasites. Successful biological control strategies will depend on a thorough understanding of these interactions at the population, organismal, and molecular scale.

The Association between Direct and Relational Bullying and Behaviour Problems among Primary School Children

Abstract: The prevalence of direct and relational bullying and their differential relationship to behaviour problems in young primary school children was investigated. Individual interviews were conducted with 1982 children aged 6–9 years (mean age 7.6 years) and 1639 parents completed the Strength and Difficulties Questionnaire regarding behaviour problems of their children. Of the 1639 children with both data sets, 4.3% were direct bullies, 39.8% victims, and 10.2% both bullied and were victimised frequently (bully/victims). The rates for relational bullying were 1.1% bullies, 37.9% victims, and 5.9% bully/victims. All children involved in direct bullying had significantly increased total behaviour problems, hyperactivity, conduct problems, and peer problem scores, and lower prosocial behaviour scores compared to those not involved in bullying (neutrals). Findings were similar for relational bullying involvement and behaviour problems for bully/victims and victims but less pronounced. Relational bullies had the lowest behaviour problem scores while being rated the least prosocially inclined children, consistent with the concept of a cool manipulator. Overall, direct bully/victims and children who were involved in both direct and relational bullying behaviour had the highest rates of behaviour problems. No relationship between victimisation and increased emotional problems were found. Those involved in bullying behaviour who show externalising and hyperactivity problems in primary school may be at increased risk for persistent conduct problems. Different interventions may be needed for those involved in relational bullying only, both direct and relational bullying, and those with additional behaviour problems.

New roles for cis-jasmone as an insect semiochemical and in plant defense.

Abstract: cis-Jasmone, or (Z)-jasmone, is well known as a component of plant volatiles, and its release can be induced by damage, for example during insect herbivory. Using the olfactory system of the lettuce aphid to investigate volatiles from plants avoided by this insect, (Z)-jasmone was found to be electrophysiologically active and also to be repellent in laboratory choice tests. In field studies, repellency from traps was demonstrated for the damson-hop aphid, and with cereal aphids numbers were reduced in plots of winter wheat treated with (Z)-jasmone. In contrast, attractant activity was found in laboratory and wind tunnel tests for insects acting antagonistically to aphids, namely the seven-spot ladybird and an aphid parasitoid. When applied in the vapor phase to intact bean plants, (Z)-jasmone induced the production of volatile compounds, including the monoterpene (E)-β-ocimene, which affect plant defense, for example by stimulating the activity of parasitic insects. These plants were more attractive to the aphid parasitoid in the wind tunnel when tested 48 h after exposure to (Z)-jasmone had ceased. This possible signaling role of (Z)-jasmone is qualitatively different from that of the biosynthetically related methyl jasmonate and gives a long-lasting effect after removal of the stimulus. Differential display was used to compare mRNA populations in bean leaves exposed to the vapor of (Z)-jasmone and methyl jasmonate. One differentially displayed fragment was cloned and shown by Northern blotting to be up-regulated in leaf tissue by (Z)-jasmone. This sequence was identified by homology as being derived from a gene encoding an α-tubulin isoform.

Comparing the German versions of the Strengths and Difficulties Questionnaire (SDQ-Deu) and the Child Behavior Checklist.

Abstract: The Strengths and Difficulties Questionnaire (SDQ) is a brief behavioural screening questionnaire that can be completed in about 5 minutes by the parents and teachers of 4-16 year olds. The scores of the English version correlate well with those of the considerably longer Child Behavior Checklist (CBCL). The present study compares the German versions of the questionnaires. Both SDQ and CBCL were completed by the parents of 273 children drawn from psychiatric clinics (N = 163) and from a community sample (N = 110). The children from the community sample also filled in the SDQ self-report and the Youth Self Report (YSR). The children from the clinic sample received an ICD-10 diagnosis if applicable. Scores from the parent and self-rated SDQ and CBCL/YSR were highly correlated and equally able to distinguish between the community and clinic samples, with the SDQ showing significantly better results regarding the total scores. They were also equally able to distinguish between disorders within the clinic sample, the only significant difference being that the SDQ was better able to differentiate between children with and without hyperactivity-inattention. The study shows that like the English originals, the SDQ-Deu and the German CBCL are equally valid for most clinical and research purposes.

Exploiting chemical ecology and species diversity : stem borer and striga control for maize and sorghum in Africa

Abstract: Stem borers, comprising the larvae of a group of lepidopterous insects, and parasitic witchweeds, particularly Striga hermonthica and S asiatica, cause major yield losses in subsistence cereal production throughout sub-Saharan Africa. Studies are described that have led to the development of a 'push-pull' strategy for minimising stem borer damage to maize and sorghum. This involved the selection of plant species that could be employed as trap crops to attract colonisation away from the cereal plants, or as intercrops to repel the pests. The two most successful trap crop plants were Napier grass, Pennisetum purpureum, and Sudan grass, Sorghum sudanensis. The intercrop giving maximum repellent effect was molasses grass, Melinis minutiflora, but two legume species, silverleaf, Desmodium uncinatum, and greenleaf, D intortum, gave good results and had the added advantage of suppressing development of S hermonthica. In terms of stem borer control, the plant chemistry responsible involves release of attractant semiochemicals from the trap plants and repellent semiochemicals from the intercrops. With M minutiflora, parasitism of stem borers was also increased by certain chemicals repellent to ovipositing adults. The mechanism of striga control has not been fully elucidated, but allelopathic effects from the Desmodium species have been shown to involve stimulation of germination and interference with haustorial development. Significant beneficial effects have been obtained with the individual components of these push-pull strategies. However, the most robust crop-protection package is obtained when these components are combined. The trap crop and intercrop plants also provide valuable forage for cattle, often reared in association with subsistence cereal production. There has been considerable take-up of the system within the communities where farmer-managed trials have been carried out, particularly in the Trans Nzoia and Suba districts of Kenya, and the programme is set to expand throughout and beyond Kenya.

Early H2O2 accumulation in mesophyll cells leads to induction of glutathione during the hyper-sensitive response in the Barley-Powdery mildew interaction

Abstract: H(2)O(2) production and changes in glutathione, catalase, and peroxidase were followed in whole-leaf extracts from the susceptible (AlgS [Algerian/4* (F14) Man.(S)]; ml-a1 allele) and resistant (AlgR [Algerian/4* (F14) Man.(R)]; Ml-a1 allele) barley (Hordeum vulgare) isolines between 12 and 24 h after inoculation with powdery mildew (Blumeria graminis [DC]. Speer [syn. Erysiphe graminis DC] f.sp hordei Marchal). Localized papilla responses and cell death hypersensitive responses were not observed within the same cell. In hypersensitive response sites, H(2)O(2) accumulation first occurred in the mesophyll underlying the attacked epidermal cell. Subsequently, H(2)O(2) disappeared from the mesophyll and accumulated around attacked epidermal cells. In AlgR, transient glutathione oxidation coincided with H(2)O(2) accumulation in the mesophyll. Subsequently, total foliar glutathione and catalase activities transiently increased in AlgR. These changes, absent from AlgS, preceded inoculation-dependent increases in peroxidase activity that were observed in both AlgR and AlgS at 18 h. An early intercellular signal precedes H(2)O(2), and this elicits anti-oxidant responses in leaves prior to events leading to death of attacked cells.

A Subarcsecond Resolution Near-infrared Study of Seyfert and Normal Galaxies II. Morphology

Abstract: We present a detailed study of the bar fraction in the CfA sample of Seyfert galaxies and in a carefully selected control sample of nonactive galaxies to investigate the relation between the presence of bars and of nuclear activity. To avoid the problems related to bar classification in the Third Reference Catalogue (RC3), e.g., subjectivity, low resolution, and contamination by dust, we have developed an objective bar classification method, which we conservatively apply to our new subarcsecond resolution near-infrared (NIR) imaging data set discussed in the first paper in this series. We are able to use stringent criteria based on radial profiles of ellipticity and major axis position angle to determine the presence of a bar and its axial ratio. Concentrating on noninteracting galaxies in our sample for which morphological information can be obtained, we find that Seyfert hosts are barred more often (79% ± 7.5%) than the nonactive galaxies in our control sample (59% ± 9%), a result which is at the ~2.5 σ significance level. The fraction of nonaxisymmetric hosts becomes even larger when interacting galaxies are taken into account. We discuss the implications of this result for the fueling of central activity by large-scale bars. This paper improves on previous work by means of imaging at higher spatial resolution and by the use of a set of stringent criteria for bar presence and confirms that the use of NIR is superior to optical imaging for detection of bars in disk galaxies.

Soluble organic nitrogen in agricultural soils

Abstract: The existence of soluble organic forms of N in rain and drainage waters has been known for many years, but these have not been generally regarded as significant pools of N in agricultural soils. We review the size and function of both soluble organic N extracted from soils (SON) and dissolved organic N present in soil solution and drainage waters (DON) in arable agricultural soils. SON is of the same order of magnitude as mineral N and of equal size in many cases; 20–30 kg SON-N ha–1 is present in a wide range of arable agricultural soils from England. Its dynamics are affected by mineralisation, immobilisation, leaching and plant uptake in the same way as those of mineral N, but its pool size is more constant than that of mineral N. DON can be sampled from soil solution using suction cups and collected in drainage waters. Significant amounts of DON are leached, but this comprises only about one-tenth of the SON extracted from the same soil. Leached DON may take with it nutrients, chelated or complexed metals and pesticides. SON/DON is clearly an important pool in N transformations and plant uptake, but there are still many gaps in our understanding.

Zinc hyperaccumulation and cellular distribution in Arabidopsis halleri

Abstract: Although Arabidopsis halleri (=Cardaminopsis halleri) is known as a Zn hyperaccumulator, there have been no detailed studies on Zn accumulation, tolerance and cellular distribution in this species. In a hydroponic experiment, A. halleri grew healthily with Zn concentrations varying from 1 to 1000 μM, without showing phytotoxicity or reduction in root or shoot dry weights. The concentration of Zn in the shoots increased from 300 μg g−1 dry weight in the 1 μM Zn treatment to 32 000 μg g−1 in the 1000 μM Zn treatment. Approximately 60% of the total Zn in the shoots were water-soluble, and there was no evidence of Zn and P co-precipitation. Both citric and malic acid concentrations in the shoots were not significantly affected by the Zn treatments, whereas in the roots there was a positive response in both organic acids to increasing Zn in solution. Cellular distribution of Zn, Ca and K in frozen hydrated leaf tissues was examined using energy-dispersive X-ray microanalysis. Zinc was sequestered in the base of trichomes, whereas the middle and upper parts of trichomes were highly enriched with Ca. Mesophyll cells appeared to have more Zn than the epidermis, probably because the latter were very small in size. Similarities and differences between A. halleri and the other well-known Zn hyperaccumulator, Thlaspi caerulescens, are discussed.

Modulation of KCNQ2/3 potassium channels by the novel anticonvulsant retigabine.

Abstract: Retigabine is a novel anticonvulsant with an unknown mechanism of action. It has recently been reported that retigabine modulates a potassium channel current in nerve growth factor-differentiated PC12 cells (Rundfeldt, 1999), however, to date the molecular correlate of this current has not been identified. In the present study we have examined the effects of retigabine on recombinant human KCNQ2 and KCNQ3 potassium channels, expressed either alone or in combination inXenopus oocytes. Application of 10 μM retigabine to oocytes expressing the KCNQ2/3 heteromeric channel shifted both the activation threshold and voltage for half-activation by approximately 20 mV in the hyperpolarizing direction, leading to an increase in current amplitude at test potentials between −80 mV and +20 mV. Retigabine also had a marked effect on KCNQ current kinetics, increasing the rate of channel activation but slowing deactivation at a given test potential. Similar effects of retigabine were observed in oocytes expressing KCNQ2 alone, suggesting that KCNQ2 may be the molecular target of retigabine. Membrane potential recordings in oocytes expressing the KCNQ2/3 heteromeric channel showed that application of retigabine leads to a concentration-dependent hyperpolarization of the oocyte, from a resting potential of −63 mV under control conditions to −85 mV in the presence of 100 μM retigabine (IC50 = 5.2 μM). In control experiments retigabine had no effect on either resting membrane potential or endogenous oocyte membrane currents. In conclusion, we have shown that retigabine acts as a KCNQ potassium channel opener. Because the heteromeric KCNQ2/3 channel has recently been reported to underlie the M-current, it is likely that M-current modulation can explain the anticonvulsant actions of retigabine in animal models of epilepsy.

A High-Throughput Screening Method for the Determination of Aqueous Drug Solubility Using Laser Nephelometry in Microtiter Plates

Abstract: The determination of aqueous solubility in a high-throughput screening environment is invaluable in the selection of the most promising potential drug candidates. We describe a fast method based on laser nephelometry that can determine the solubility of potential drug candidates (usually from combinatorial chemistry) supplied as dimethyl sulfoxide (DMSO) solutions in 96-well plates. In the sample, the percent DMSO is kept constant allowing direct comparison of results. The nephelometric method has been shown to produce results equivalent to those produced by an HPLC method and to be largely unaffected by colored solutions.

Ontogeny of orientation flight in the honeybee revealed by harmonic radar

Abstract: Cognitive ethology focuses on the study of animals under natural conditions to reveal ecologically adapted modes of learning But biologists can more easily study what an animal learns than how it learns For example, honeybees take repeated ‘orientation’ flights before becoming foragers at about three weeks of age1 These flights are a prerequisite for successful homing2 Little is known2,3 about these flights because orienting bees rapidly fly out of the range of human observation Using harmonic radar, we show for the first time a striking ontogeny to honeybee orientation flights With increased experience, bees hold trip duration constant but fly faster, so later trips cover a larger area than earlier trips In addition, each flight is typically restricted to a narrow sector around the hive Orientation flights provide honeybees with repeated opportunities to view the hive and landscape features from different viewpoints, suggesting that bees learn the local landscape in a progressive fashion We also show that these changes in orientation flight are related to the number of previous flights taken instead of chronological age, suggesting a learning process adapted to changes in weather conditions, flower availability and the needs of bee colonies

A population of very young brown dwarfs and free‐floating planets in Orion

Abstract: We describe the results of a very deep imaging survey of the Trapezium cluster in the IJH bands, using the UKIRT high-resolution camera UFTI. Approximately 32 per cent of the 515 point sources detected are brown dwarf candidates, including several free-floating objects with masses below the deuterium-burning (planetary) threshold at 0.013 M⊙, which are detectable because of their extreme youth. We have confidence that almost all the sources detected are cluster members, since foreground contamination is minimal in the 33-arcmin2 area surveyed, and the dense backdrop of OMC-1 obscures all background stars at these wavelengths. Extinction is calculated from the (J−H) colours, permitting accurate luminosity estimates, and temperatures are derived from the dereddened (I−J) colours. There is some evidence for a cut-off in the luminosity function below the level corresponding to several Jupiter masses, which may represent the bottom end of the initial mass function. Since star formation is complete in the Trapezium, this limit could have wide significance, if confirmed. However, it could well be an effect of the dispersal of the molecular cloud by the central O-type stars, a process for which the time-scale will vary between star formation regions.

Prospective Memory in 2000: Past, Present, and Future Directions

Abstract: During the past four years there has been an explosion of interest in prospective memory research, culminating recently in the success of the First International Conference on Prospective Memory (July, 2000). In this paper we take the opportunity to review progress in the area by identifying some key themes and issues that arose during the conference and that are exemplified in the papers contained in this special issue. Finally, we consider future directions for research and some of the key questions that we believe all researchers in this area will need to address. Copyright © 2000 John Wiley & Sons, Ltd.

Effects of gestation and birth weight on the growth and development of very low birthweight small for gestational age infants: a matched group comparison.

Abstract: AIMS—To investigate the effects of small for gestational age (SGA) in very low birthweight (VLBW) infants on growth and development until the fifth year of life. METHODS—VLBW (< 1500 g) infants, selected from a prospective study, were classified as SGA (n = 115) on the basis of birth weight below the 10th percentile for gestational age and were compared with two groups of appropriate for gestational age (AGA) infants matched according to birth weight (AGA-BW; n = 115) or gestation at birth (AGA-GA; n = 115). Prenatal, perinatal, and postnatal risk factors were recorded, and duration and intensity of treatment were computed from daily assessments. Body weight, length, and head circumference were measured at birth, five and 20 months (corrected for prematurity), and at 56 months. General development was assessed at five and 20 months with the Griffiths scale of babies abilities, and cognitive development at 56 months with the Columbia mental maturity scales, a vocabulary (AWST) and language comprehension test (LSVTA). RESULTS—Significant group differences were found in complications (pregnancy, birth, and neonatal), parity, and multiple birth rate. The AGA-GA group showed most satisfactory growth up to 56 months, with both the AGA-BW and SGA groups lagging behind. The AGA-GA group also scored significantly more highly on all developmental and cognitive tests than the other groups. Developmental test results were similar for the SGA and AGA-BW groups at five and 20 months, but AGA-BW infants (lowest gestation) had lower scores on performance intelligence quotient and language comprehension at 56 months than the SGA group. When prenatal and neonatal complications, parity, and multiple birth were accounted for, group differences in growth remained, but differences in cognitive outcome disappeared after five months. CONCLUSIONS—Being underweight and with a short gestation (SGA and VLBW) leads to poor weight gain and head growth in infancy but does not result in poorer growth than in infants of the same birth weight but shorter gestation (AGA-BW) in the long term. SGA is related to early developmental delay and later language problems; however, neonatal complications may have a larger detrimental effect on long term cognitive development of VLBW infants than whether they are born SGA or AGA.

An emotional business: a guide to understanding the motivations of small business decision takers

Abstract: Argues that the way in which the UK Government, through its various departments and quangos, approaches designed to approve the effectiveness of the small business sector, is based on a flawed understanding of how small businesses actually operate. Argues that this naive, over‐simplistic understanding of the motivation of those in the small business sector means that many government interventions that are made, are blunt instruments destined to fail, given the limited understanding shown of the complexity of the small business market. Presents evidence from two recent studies amongst small firms; a series of large‐scale qualitative studies undertaken for a blue chip company and a mixed study on the Business Link network. The emphasis is based on – using qualitative research – getting to grips with the emotion, ambiguity and complexity that characterises this market.

Immunolocalization of SNS/PN3 and NaN/SNS2 sodium channels in human pain states.

Abstract: The tetrodotoxin-resistant (TTX-R) voltage-gated sodium channel SNS/PN3 and the newly discovered NaN/SNS2 are expressed in sensory neurones, particularly in nociceptors. Using specific antibodies, we have studied, for the first time in humans, the presence of SNS/PN3 and NaN/SNS2 in peripheral nerves, including tissues from patients with chronic neurogenic pain. In brachial plexus injury patients, there was an acute decrease of SNS/PN3- and NaN/SNS2-like immunoreactivity in sensory cell bodies of cervical dorsal root ganglia (DRG) whose central axons had been avulsed from spinal cord, with gradual return of the immunoreactivity to control levels over months. In contrast, there was increased intensity of immunoreactivity to both channels in some peripheral nerve fibers just proximal to the site of injury in brachial plexus trunks, and in neuromas. These findings suggest that the expression of these sodium channels in neuronal cell bodies is reduced after spinal cord root avulsion injury in man, but that pre-synthesized channel proteins may undergo translocation with accumulation at sites of nerve injury, as in animal models of peripheral axotomy. The latter may contribute to positive symptoms, as our patients all showed a positive Tinel's sign. Nerve terminals in distal limb neuromas and skin from patients with chronic local hyperalgesia and allodynia all showed marked increases of SNS/PN3-immunoreactive fibers, but little or no NaN/SNS2-immunoreactivity, suggesting that the former may be related to the persistent hypersensitive state. Axonal immunoreactivity to both channels was similar to control nerves in sural nerve biopsies in a selection of neuropathies, irrespective of nerve inflammation, demyelination or spontaneous pain, including a patient with congenital insensitivity to pain. Our studies suggest that the best target for SNS/PN3 blocking agents is likely to be chronic local hypersensitivity.

Three pathways for trehalose biosynthesis in mycobacteria.

Abstract: Trehalose is present as a free disaccharide in the cytoplasm of mycobacteria and as a component of cell-wall glycolipids implicated in tissue damage associated with mycobacterial infection To obtain an overview of trehalose metabolism, we analysed data from the Mycobacterium tuberculosis genome project and identified ORFs with homology to genes encoding enzymes from three trehalose biosynthesis pathways previously characterized in other bacteria Functional assays using mycobacterial extracts and recombinant enzymes derived from these ORFs demonstrated that mycobacteria can produce trehalose from glucose 6-phosphate and UDP-glucose (the OtsA–OtsB pathway) from glycogen-like α(1→4)-linked glucose polymers (the TreY–TreZ pathway) and from maltose (the TreS pathway) Each of the pathways was found to be active in both rapid-growing Mycobacterium smegmatis and slow-growing Mycobacterium bovis BCG The presence of a disrupted treZ gene in Mycobacterium leprae suggests that this pathway is not functional in this organism The presence of multiple biosynthetic pathways indicates that trehalose plays an important role in mycobacterial physiology

In vivo rodent erythrocyte micronucleus assay. II. Some aspects of protocol design including repeated treatments, integration with toxicity testing, and automated scoring.

Abstract: An expert working group on the in vivo micronucleus assay, formed as part of the International Workshop on Genotoxicity Test Procedures (IWGTP), discussed protocols for the conduct of established and proposed micronucleus assays at a meeting held March 25-26, 1999 in Washington, DC, in conjunction with the annual meeting of the Environmental Mutagen Society. The working group reached consensus on a number issues, including: (1) protocols using repeated dosing in mice and rats; (2) integration of the (rodent erythrocyte) micronucleus assay into general toxicology studies; (3) the possible omission of concurrently-treated positive control animals from the assay; (4) automation of micronucleus scoring by flow cytometry or image analysis; (5) criteria for regulatory acceptance; (6) detection of aneuploidy induction in the micronucleus assay; and (7) micronucleus assays in tissues (germ cells, other organs, neonatal tissue) other than bone marrow. This report summarizes the discussions and recommendations of this working group. In the classic rodent erythrocyte assay, treatment schedules using repeated dosing of mice or rats, and integration of assays using such schedules into short-term toxicology studies, were considered acceptable as long as certain study criteria were met. When the micronucleus assay is integrated into ongoing toxicology studies, relatively short-term repeated-dose studies should be used preferentially because there is not yet sufficient data to demonstrate that conservative dose selection in longer term studies (longer than 1 month) does not reduce the sensitivity of the assay. Additional validation data are needed to resolve this point. In studies with mice, either bone marrow or blood was considered acceptable as the tissue for assessing micronucleus induction, provided that the absence of spleen function has been verified in the animal strains used. In studies with rats, the principal endpoint should be the frequency of micronucleated immature erythrocytes in bone marrow, although scoring of peripheral blood samples gives important supplementary data about the time course of micronucleus induction. When dose concentration and stability are verified appropriately, concurrent treatment with a positive control agent is not necessary. Control of staining and scoring procedures can be obtained by including appropriate reference samples that have been obtained from a separate experiment. For studies in rats or mice, treatment/sampling regimens should include treatment at intervals of no more than 24 hr (unless the test article has a half-life of more than 24 hr) with sampling of bone marrow or blood, respectively, within 24 or 40 hr after the last treatment. The use of a DNA specific stain is recommended for the identification of micronuclei, especially for studies in the rat. In the case of a negative assay result with a non-toxic test article, it is desirable that systemic exposure to the test article is demonstrated. The group concluded that successful application of automated scoring by both flow cytometry and image analysis had been achieved, and defined criteria that should be met if automated scoring is employed. It was not felt appropriate to attempt to define specific recommended protocols for automated scoring at the present time. Other issues reviewed and discussed by the working group included micronucleus assays that have been developed in a number of tissues other than bone marrow. The group felt that these assays were useful research tools that could also be used to elucidate mechanisms in certain regulatory situations, but that these assays had not yet been standardized and validated for routine regulatory application.

Peroxide processing in photosynthesis: antioxidant coupling and redox signalling.

Abstract: Photosynthesis has a high capacity for production of hydrogen peroxide (H2O2), but the intracellular levels of this relatively weak oxidant are controlled by the antioxidant system, comprising a network of enzymatic and non-enzymatic components that notably includes reactions linked to the intracellular ascorbate and glutathione pools. Mutants and transformed plants with specific decreases in key components offer the opportunity to dissect the complex system that maintains redox homeostasis. Since H2O2 is a signal-transducing molecule relaying information on intracellular redox state, the pool size must be rigorously controlled within each compartment of the cell. This review focuses on compartment-specific differences in the stringency of redox coupling between ascorbate and glutathione, and the significance this may have for the flexibility of the control of gene expression that is linked to photosynthetic H2O2 production.

Plant responses to sulphur deficiency and the genetic manipulation of sulphate transporters to improve S‐utilization efficiency

Abstract: Decreased inputs of S have increased the incidence of S-deficiency in crops, resulting in decreased yields and quality. Remediation by fertilizer application is not always successful because this often results in an uneven supply of S. The ability to respond to S-deficiency stress varies between crops and this is a target for the genetic improvement of S-utilization efficiency. Improved capture of resources, the accumulation of greater reserves of S and improved mechanisms for the remobilization of these reserves are required. It is an inability to over-accumulate S and subsequently, effectively remobilize S-reserves, which restricts optimum S-use efficiency. Genetic manipulation of the transporters and their expression will contribute to overcoming these limitations. Control of gene expression limits excess uptake and activity of the assimilatory pathway: the endogenous expression of sulphate transporters is regulated by S-supply, with negative regulation from reduced S-containing compounds and positive regulation by O-acetylserine, the C/N skeleton precursor of cysteine. Constitutive expression of the transporter will remove this control and may enable the accumulation of sulphate reserves. Sulphate in the vacuole and other pools of reduced sulphur, such as glutathione or protein may be remobilized under S-limiting conditions. Low efficiencies of these remobilization processes, particularly the remobilization of vacuolar sulphate, suggest that the transporters involved in the remobilization are a target for modification. Transporters are involved in facilitating the multiple trans-membrane transport steps between uptake of sulphate from the soil solution, and delivery to the site of reduction in the chloroplast or plastid. A gene family has been identified and phylogenetic relationships based on primary sequence information indicate multiple sub-groups. Groups which are expressed in roots, in shoots and in both tissue types are postulated, however, the functional roles for these groups and the identification of transporters involved in recycling remain to be confirmed.

A combined RFLP and AFLP linkage map of upland rice (Oryza sativa L.) used to identify QTLs for root-penetration ability

Abstract: Acombined RFLP and AFLP linkage map of an F6 recombinant inbred population, which was derived from a previously mapped F2 of a cross between the two drought resistant upland rice varieties Bala and Azucena, is presented. The map contains 101 RFLP and 34 AFLP markers on 17 linkage groups covering 1680 cM. Also presented is the approximate mapping position of a further four RFLP and 75 AFLP markers, which either could not be given a unique place on the map or for which the available data is not sufficient to allow confident positioning, and the result of quantitative trait locus (QTL) mapping of traits related to root-penetration ability. Root penetration was assessed by counting the number of root axes that penetrated a 3 mm-thick layer consisting of 80% wax and 20% white soft paraffin. Good root penetration would be expected to increase drought resistance where soil strength is high. Single-marker analysis revealed seven QTLs for the number of roots which penetrate the wax layer. In identical locations were seven QTLs for the ratio of penetrated to the total number of roots. Transgressive inheritance of positive alleles from Bala explained four of these QTLs. Comparison of the QTLs identified here with previous reports of QTLs for root morphology suggest that alleles which improve root penetration ability may also either make the roots longer or thicker.

Interactions between entomopathogenic fungi and other natural enemies: implications for biological control.

Abstract: Pathogens and arthropod natural enemies may contribute to the suppression of insect pest populations either as individual species or as species complexes. However, because natural enemies of insects have evolved and function in a multitrophic context it is important to assess interactions within complexes of natural enemies if they are to be exploited effectively in pest management. Natural enemies can interact either synergistically/additively (e.g. enhanced transmission and dispersal of insect pathogens) or antagonistically (e.g. parasitism/infection, predation and competition). In this paper, studies assessing the potential interactions between insect and fungal natural enemies are reviewed. In general, these studies indicate the positive nature of the interactions between arthropod natural enemies and fungal pathogens with respect to the control of insect populations. More work is required to investigate further the many ways in which the natural enemy community interacts in the agroecosystem