Search

Abstract Background The PTEN tumour suppressor gene and PIK3CA proto-oncogene encode proteins which contribute to regulation and propagation of signal transduction through the PI3K/AKT signalling pathway. This study investigates the prevalence of loss of PTEN expression and mutations in both PTEN and PIK3CA in colorectal cancers (CRC) and their associations with tumour clinicopathological features, lifestyle factors and dietary consumptions. Methods 186 adenocarcinomas and 16 adenomas from the EPIC Norfolk study were tested for PTEN and PIK3CA mutations by DNA sequencing and PTEN expression changes by immunohistochemistry. Dietary and lifestyle data were collected prospectively using seven day food diaries and lifestyle questionnaires. Results Mutations in exons 7 and 8 of PTEN were observed in 2.2% of CRC and PTEN loss of expression was identified in 34.9% CRC. Negative PTEN expression was associated with lower blood low-density lipoprotein concentrations (p = 0.05). PIK3CA mutations were observed in 7% of cancers and were more frequent in CRCs in females (p = 0.04). Analysis of dietary intakes demonstrated no link between PTEN expression status and any specific dietary factor. PTEN expression negative, proximal CRC were of more advanced Dukes' stage (p = 0.02) and poor differentiation (p < 0.01). Testing of the prevalence of PIK3CA mutations and loss of PTEN expression demonstrated that these two events were independent (p = 0.55). Conclusion These data demonstrated the frequent occurrence (34.9%) of PTEN loss of expression in colorectal cancers, for which gene mutations do not appear to be the main cause. Furthermore, dietary factors are not associated with loss of PTEN expression. PTEN expression negative CRC were not homogenous, as proximal cancers were associated with a more advanced Dukes' stage and poor differentiation, whereas distal cancers were associated with earlier Dukes' stage.

Abstract Background Class III Homeodomain Leucine Zipper (HD-Zip III) proteins have been implicated in the regulation of cambium identity, as well as primary and secondary vascular differentiation and patterning in herbaceous plants. They have been proposed to regulate wood formation but relatively little evidence is available to validate such a role. We characterised and compared HD-Zip III gene family in an angiosperm tree, Populus spp. (poplar), and the gymnosperm Picea glauca (white spruce), representing two highly evolutionarily divergent groups. Results Full-length cDNA sequences were isolated from poplar and white spruce. Phylogenetic reconstruction indicated that some of the gymnosperm sequences were derived from lineages that diverged earlier than angiosperm sequences, and seem to have been lost in angiosperm lineages. Transcript accumulation profiles were assessed by RT-qPCR on tissue panels from both species and in poplar trees in response to an inhibitor of polar auxin transport. The overall transcript profiles HD-Zip III complexes in white spruce and poplar exhibited substantial differences, reflecting their evolutionary history. Furthermore, two poplar sequences homologous to HD-Zip III genes involved in xylem development in Arabidopsis and Zinnia were over-expressed in poplar plants. PtaHB1 over-expression produced noticeable effects on petiole and primary shoot fibre development, suggesting that PtaHB1 is involved in primary xylem development. We also obtained evidence indicating that expression of PtaHB1 affected the transcriptome by altering the accumulation of 48 distinct transcripts, many of which are predicted to be involved in growth and cell wall synthesis. Most of them were down-regulated, as was the case for several of the poplar HD-Zip III sequences. No visible physiological effect of over-expression was observed on PtaHB7 transgenic trees, suggesting that PtaHB1 and PtaHB7 likely have distinct roles in tree development, which is in agreement with the functions that have been assigned to close homologs in herbaceous plants. Conclusions This study provides an overview of HD-zip III genes related to woody plant development and identifies sequences putatively involved in secondary vascular growth in angiosperms and in gymnosperms. These gene sequences are candidate regulators of wood formation and could be a source of molecular markers for tree breeding related to wood properties.

Abstract Background BRAF and K-ras proto-oncogenes encode components of the ERK signalling pathway and are frequently mutated in colorectal cancer. This study investigates the associations between BRAF and K-ras mutations and clinicopathological, lifestyle and dietary factors in colorectal cancers. Methods 186 adenocarcinomas and 16 adenomas from the EPIC Norfolk study were tested for BRAF and K-ras mutations. Diet and lifestyle data were collected prospectively using seven day food diaries. Results BRAF V600E mutation was found in 15.6% of colorectal cancers but at higher frequencies in cancers with proximal location, poor differentiation and microsatellite instability (MSI) (all p < 0.001). K-ras mutation (mostly in codons 12 and 13) was found in 22.0% of colorectal cancers but at higher frequencies in cancers of more advanced Dukes' stage (p = 0.001), microsatellite stable (MSS) status (p = 0.002) and in individuals with lower blood high-density lipoprotein concentrations (p = 0.04). Analysis of dietary factors demonstrated no link between BRAF mutation and any specific dietary constituent, however, K-ras mutation was found at higher frequencies in individuals with higher white meat consumption (p < 0.001). Further analysis of specific mutation type demonstrated that G to A transitions in K-ras were observed at higher frequencies in individuals consuming lower amounts of fruit (p = 0.02). Conclusion These data support the model of BRAF and K-ras mutations arising in distinct colorectal cancer subsets associated with different clinicopathological and dietary factors, acting as mutually exclusive mechanisms of activation of the same signalling pathway.

Abstract Background The sequencing and analysis of ESTs is for now the only practical approach for large-scale gene discovery and annotation in conifers because their very large genomes are unlikely to be sequenced in the near future. Our objective was to produce extensive collections of ESTs and cDNA clones to support manufacture of cDNA microarrays and gene discovery in white spruce (Picea glauca [Moench] Voss). Results We produced 16 cDNA libraries from different tissues and a variety of treatments, and partially sequenced 50,000 cDNA clones. High quality 3' and 5' reads were assembled into 16,578 consensus sequences, 45% of which represented full length inserts. Consensus sequences derived from 5' and 3' reads of the same cDNA clone were linked to define 14,471 transcripts. A large proportion (84%) of the spruce sequences matched a pine sequence, but only 68% of the spruce transcripts had homologs in Arabidopsis or rice. Nearly all the sequences that matched the Populus trichocarpa genome (the only sequenced tree genome) also matched rice or Arabidopsis genomes. We used several sequence similarity search approaches for assignment of putative functions, including blast searches against general and specialized databases (transcription factors, cell wall related proteins), Gene Ontology term assignation and Hidden Markov Model searches against PFAM protein families and domains. In total, 70% of the spruce transcripts displayed matches to proteins of known or unknown function in the Uniref100 database (blastx e-value < 1e-10). We identified multigenic families that appeared larger in spruce than in the Arabidopsis or rice genomes. Detailed analysis of translationally controlled tumour proteins and S-adenosylmethionine synthetase families confirmed a twofold size difference. Sequences and annotations were organized in a dedicated database, SpruceDB. Several search tools were developed to mine the data either based on their occurrence in the cDNA libraries or on functional annotations. Conclusion This report illustrates specific approaches for large-scale gene discovery and annotation in an organism that is very distantly related to any of the fully sequenced genomes. The ArboreaSet sequences and cDNA clones represent a valuable resource for investigations ranging from plant comparative genomics to applied conifer genetics.

Prior to use, newly generated induced pluripotent stem cells (iPSC) should be thoroughly validated. While excellent validation and release testing assays designed to evaluate potency, genetic integrity, and sterility exist, they do not have the ability to predict cell type-specific differentiation capacity. Selection of iPSC lines that have limited capacity to produce high-quality transplantable cells, places significant strain on valuable clinical manufacturing resources. The purpose of this study was to determine the degree and root cause of variability in retinal differentiation capacity between cGMP-derived patient iPSC lines. In turn, our goal was to develop a release testing assay that could be used to augment the widely used ScoreCard panel. IPSCs were generated from 15 patients (14-76 years old), differentiated into retinal organoids, and scored based on their retinal differentiation capacity. Despite significant differences in retinal differentiation propensity, RNA-sequencing revealed remarkable similarity between patient-derived iPSC lines prior to differentiation. At 7 days of differentiation, significant differences in gene expression could be detected. Ingenuity pathway analysis revealed perturbations in pathways associated with pluripotency and early cell fate commitment. For example, good and poor producers had noticeably different expressions of OCT4 and SOX2 effector genes. QPCR assays targeting genes identified via RNA sequencing were developed and validated in a masked fashion using iPSCs from 8 independent patients. A subset of 14 genes, which include the retinal cell fate markers RAX, LHX2, VSX2, and SIX6 (all elevated in the good producers), were found to be predictive of retinal differentiation propensity., (© The Author(s) 2023. Published by Oxford University Press.)

Objectives: Primary objectives were outcomes comparison of instrumented surgery used for de-novo spinal infections in terms of infection recurrence, reoperations, primary failure, mortality, and length of stay relative to non-instrumented surgery. Secondary objectives were outcomes for surgical and non-surgical treatment of de-novo spinal infections regarding recurrence of infection, mortality, quality of life, and length-of-stay., Methods: A systematic literature review was performed using the PubMed database. Studies comparing outcome variables of patients with de-novo spinal infections (DNSI) treated with and without instrumentation and surgical versus non-surgical treatment were included. Studies primarily focusing on epidural abscesses or non-de-novo infections were excluded. A meta-analysis was performed for infection recurrence, reoperation, primary treatment failure, mortality, and quality-of-life parameters., Results: A total of 17 retrospective studies with 2.069 patients met the inclusion criteria. 1.378 patients received surgical treatment with or without instrumentation; 676 patients were treated non-surgically. For the comparison of instrumented to non-instrumented surgery Odds-Ratios were .98 (P = .95) for infection recurrence, .83 (P = .92) for primary failure, .53 (P = .02) for mortality and .32 (P = .05) for reoperation. For the comparison of non-surgical to surgical treatment, Odds-Ratios were .98 (P = .95) for infection recurrence, and 1.05 (P = .89) for mortality., Conclusion: Available data support that instrumented surgery can be performed safely without higher rates of infection recurrence or primary failure and lower reoperation and mortality rates compared to nonsurgical treatment for DNSI. Furthermore, spine surgical treatment may generally be performed without higher risk of infection recurrence and mortality and better quality-of-life outcomes compared to generic non-surgical treatment.

Objective: De novo infections of the spine are an increasing healthcare problem. The decision for nonsurgical or surgical treatment is often made case by case on the basis of physician experience, specialty, or practice affiliation rather than evidence-based medicine. To create a more systematic foundation for surgical assessments of de novo spinal infections, the authors applied a formal validation process toward developing a spinal infection scoring system using principles gained from other spine severity scoring systems like the Spine Instability Neoplastic Score, Thoracolumbar Injury Classification and Severity Score, and AO Spine classification of thoracolumbar injuries. They utilized an expert panel and literature reviews to develop a severity scale called the "Spinal Infection Treatment Evaluation Score" (SITE Score)., Methods: The authors conducted an evidence-based process of combining literature reviews, extracting key elements from previous scoring systems, and obtaining iterative expert panel input while following a formal Delphi process. The resulting basic SITE scoring system was tested on selected de novo spinal infection cases and serially refined by an international multidisciplinary expert panel. Intra- and interobserver reliabilities were calculated using the intraclass correlation coefficient (ICC) and Fleiss' and Cohen's kappa, respectively. A receiver operating characteristic analysis was performed for cutoff value analysis. The predictive validity was assessed through cross-tabulation analysis., Results: The conceptual SITE scoring system combines the key variables of neurological symptoms, infection location, radiological variables for instability and impingement of neural elements, pain, and patient comorbidities. Ten patients formed the first cohort of de novo spinal infections, which was used to validate the conceptual scoring system. A second cohort of 30 patients with de novo spinal infections, including the 10 patients from the first cohort, was utilized to validate the SITE Score. Mean scores of 6.73 ± 1.5 and 6.90 ± 3.61 were found in the first and second cohorts, respectively. The ICCs for the total score were 0.989 (95% CI 0.975-0.997, p < 0.01) in the first round of scoring system validation, 0.992 (95% CI 0.981-0.998, p < 0.01) in the second round, and 0.961 (95% CI 0.929-0.980, p < 0.01) in the third round. The mean intraobserver reliability was 0.851 ± 0.089 in the third validation round. The SITE Score yielded a sensitivity of 97.77% ± 3.87% and a specificity of 95.53% ± 3.87% in the last validation round for the panel treatment decision., Conclusions: The SITE scoring concept showed statistically meaningful reliability parameters. Hopefully, this effort will provide a foundation for a future evidence-based decision aid for treating de novo spinal infections. The SITE Score showed promising inter- and intraobserver reliability. It could serve as a helpful tool to guide physicians' therapeutic decisions in managing de novo spinal infections and help in comparison studies to better understand disease severity and outcomes.

Our understanding of inherited retinal disease has benefited immensely from molecular genetic analysis over the past several decades. New technologies that allow for increasingly detailed examination of a patient's DNA have expanded the catalog of genes and specific variants that cause retinal disease. In turn, the identification of pathogenic variants has allowed the development of gene therapies and low-cost, clinically focused genetic testing. Despite this progress, a relatively large fraction (at least 20%) of patients with clinical features suggestive of an inherited retinal disease still do not have a molecular diagnosis today. Variants that are not obviously disruptive to the codon sequence of exons can be difficult to distinguish from the background of benign human genetic variations. Some of these variants exert their pathogenic effect not by altering the primary amino acid sequence, but by modulating gene expression, isoform splicing, or other transcript-level mechanisms. While not discoverable by DNA sequencing methods alone, these variants are excellent targets for studies of the retinal transcriptome. In this review, we present an overview of the current state of pathogenic variant discovery in retinal disease and identify some of the remaining barriers. We also explore the utility of new technologies, specifically patient-derived induced pluripotent stem cell (iPSC)-based modeling, in further expanding the catalog of disease-causing variants using transcriptome-focused methods. Finally, we outline bioinformatic analysis techniques that will allow this new method of variant discovery in retinal disease. As the knowledge gleaned from previous technologies is informing targets for therapies today, we believe that integrating new technologies, such as iPSC-based modeling, into the molecular diagnosis pipeline will enable a new wave of variant discovery and expanded treatment of inherited retinal disease., (Copyright © 2020. Published by Elsevier Ltd.)

Inherited retinal degenerative disorders such as retinitis pigmentosa and Usher syndrome are characterized by progressive death of photoreceptor cells. To restore vision to patients blinded by these diseases, a stem cell-based photoreceptor cell replacement strategy will likely be required. Although retinal stem cell differentiation protocols suitable for generating photoreceptor cells exist, they often yield a rather heterogenous mixture of cell types. To enrich the donor cell population for one or a few cell types, scientists have traditionally relied upon the use of antibody-based selection approaches. However, these strategies are quite labor intensive and require animal derived reagents and equipment that are not well suited to current good manufacturing practices (cGMP). The purpose of this study was to develop and evaluate a microfluidic cell sorting device capable of exploiting the physical and mechanical differences between retinal cell types to enrich specific donor cell populations such as Retinal Pigment Epithelial (RPE) cells and photoreceptor cells. Using this device, we were able to separate a mixture of RPE and iPSC-derived photoreceptor precursor cell lines into two substantially enriched fractions. The enrichment factor of the RPE fraction was 2 and that of the photoreceptor precursor cell fraction was 2.7. Similarly, when human retina, obtained from 3 independent donors, was dissociated and passed through the sorting device, the heterogeneous mixture could be reliably sorted into RPE and photoreceptor cell rich fractions. In summary, microfluidic cell sorting is a promising approach for antibody free enrichment of retinal cell populations., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Given the importance of body composition in maintaining optimal physical and functional capacities, the use of appropriate, field-based assessment tools should be a priority to assist in maintaining the occupational safety of firefighters and the community. For ease, body mass index has often been used to assess these changes. However, it is limited in its accuracy. The purposes of this study were twofold: (a) to compare the validity of different measures of body composition against dual-energy X-ray absorptiometry (DXA) in urban firefighters and (b) to assess these measures in their ability to provide meaningful interpretation of criteria-driven categories of adiposity. A total of 64 male firefighters (age = 44.0 ± 9.5 years) underwent full anthropometric profiling (predictor equations used to determine body fat percentage [BF%]), bioelectrical impedance analysis (BIA), and DXA assessments. Participants' body mass index was calculated, and BF% and lean mass were determined along with criteria-driven categorizations of adiposity. Anthropometric (skinfolds) predictor equations (e.g., mean bias = -4.4% for BF%) were typically closer to DXA measures, compared with BIA (9.4% for BF%). However, when determining categories of criteria-driven adiposity, BIA (42.9% overweight or obese) provided closer estimates to the DXA-determined distribution (44.6%) than anthropometric-based measures (up to 40%). Body mass index appears an inappropriate measure for accurately determining categories of adiposity with 64.1% classified as overweight or obese. Given the logistical constraints of anthropometric profiling, and the closeness of BIA to DXA in adiposity categories, BIA may be a suitable alternative to DXA for assessing body composition in professional urban firefighters.

Enhanced S-cone syndrome (ESCS) is caused by recessive mutations in the photoreceptor cell transcription factor NR2E3 . Loss of NR2E3 is characterized by repression of rod photoreceptor cell gene expression, over-expansion of the S-cone photoreceptor cell population, and varying degrees of M- and L-cone photoreceptor cell development. In this study, we developed a CRISPR-based homology-directed repair strategy and corrected two different disease-causing NR2E3 mutations in patient-derived induced pluripotent stem cells (iPSCs) generated from two affected individuals. In addition, one patient's iPSCs were differentiated into retinal cells and NR2E3 transcription was evaluated in CRISPR corrected and uncorrected clones. The patient's c.119-2A>C mutation caused the inclusion of a portion of intron 1, the creation of a frame shift, and generation of a premature stop codon. In summary, we used a single set of CRISPR reagents to correct different mutations in iPSCs generated from two individuals with ESCS. In doing so we demonstrate the advantage of using retinal cells derived from affected patients over artificial in vitro model systems when attempting to demonstrate pathophysiologic mechanisms of specific mutations.

Gene correction is a valuable strategy for treating inherited retinal degenerative diseases, a major cause of irreversible blindness worldwide. Single gene defects cause the majority of these retinal dystrophies. Gene augmentation holds great promise if delivered early in the course of the disease, however, many patients carry mutations in genes too large to be packaged into adeno-associated viral vectors and some, when overexpressed via heterologous promoters, induce retinal toxicity. In addition to the aforementioned challenges, some patients have sustained significant photoreceptor cell loss at the time of diagnosis, rendering gene replacement therapy insufficient to treat the disease. These patients will require cell replacement to restore useful vision. Fortunately, the advent of induced pluripotent stem cell and CRISPR-Cas9 gene editing technologies affords researchers and clinicians a powerful means by which to develop strategies to treat patients with inherited retinal dystrophies. In this review we will discuss the current developments in CRISPR-Cas9 gene editing in vivo in animal models and in vitro in patient-derived cells to study and treat inherited retinal degenerative diseases., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The absence of a crystal structure of the calcium free state of the cardiac isoform of the troponin complex has hindered our understanding of how the simple binding of Ca 2+ triggers conformational changes in troponin which are then propagated to enable muscle contraction. Here we have used continuous wave (CW) and Double Electron-Electron Resonance (DEER) pulsed EPR spectroscopy to measure distances between TnI and TnC to track the movement of the functionally important regulatory 'switch' region of cardiac Tn. Spin labels were placed on the switch region of Troponin I and distances measured to Troponin C. Under conditions of high Ca 2+ , the interspin distances for one set (TnI151/TnC84) were 'short' (9-10Å) with narrow distance distribution widths (3-8Å) indicating the close interaction of the switch region with the N-lobe of TnC. Additional spin populations representative of longer interspin distances were detected by DEER. These longer distance populations, which were ∼16-19Å longer than the short distance populations, possessed notably broader distance distribution widths (14-29Å). Upon Ca 2+ removal, the interspin population shifted toward the longer distances, indicating the release of the switch region from TnC and an overall increase in disorder for this region. Together, our results suggest that under conditions of low Ca 2+ , the close proximity of the TnI switch region to TnC in the cardiac isoform is necessary for promoting the interaction between the regulatory switch helix with the N-lobe of cardiac Troponin C, which, unlike the skeletal isoform, is largely in a closed conformation., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Site-Directed Spin Labeling Electron Paramagnetic Resonance (SDSL-EPR) offers a powerful method for the structural analysis of protein folds. This method can be used to test and build secondary, tertiary, and quaternary structural models as well as measure protein conformational changes in solution. Insertion of two cysteine residues into the protein backbone using molecular biology methods and the subsequent labeling of the cysteine residues with a paramagnetic spin label enables the technique of EPR to be used as a molecular spectroscopic ruler. EPR measures the dipolar interaction between pairs of paramagnetic spin labels to yield internitroxide distances from which quantitative structural information on a protein fold can then be obtained. Interspin dipolar interaction between two spin labels at less than 25  Å are measured using continuous wave (CW) EPR methods. As for any low-resolution distance methods, the positioning of the spin labels and the number of distance constraints to be measured are dependent on the structural question being asked, thus a pattern approach for using distance sets to decipher structure mapping, including protein folds and conformational changes associated with biological activity, is essential. Practical guidelines and hints for the technique of SDSL-EPR are described in this chapter, including methods for spin labeling the protein backbone, CW-EPR data collection at physiological temperatures and two semiquantitative analysis methods to extract interspin distance information from the CW-EPR spectra.

There is incontrovertible evidence that neural progenitor cells (NPC) are found in the adult brain. The ability to identify and track NPC in the adult brain is of considerable importance if the properties of these cells are to be harnessed as potential therapies for degenerative brain disorders. The most commonly used approach of identifying these NPC in experimental studies, bromodeoxyuridine (BrdU) labelling, is outlined in this chapter. Immunohistochemical protocols for detecting endogenous and exogenous (introduced via transplantation) NPC in fresh-frozen and paraffin wax embedded brain tissue are described. Advice on how to label these NPC is also offered and multi-label fluorescence immunochemical staining approaches to determine the differentiation fate of NPC are described.

The inner cell mass of the preimplantation blastocyst, from which all the cells of the body develop, is a source of embryonic stem cells. These cells can be maintained in their undifferentiated state over long periods in culture and yet retain their pluripotency. The generation of human stem cells capable of differentiating into all the cell types of the human body opens the way for the use of these cells in therapeutic transplantation for a myriad of diseases. However, as discussed here, there are a number of logistical, biological, and clinical hurdles that must be overcome prior to the use of these cells in routine clinical practice., (Copyright 2007 Reproductive Healthcare Ltd, Duck End Farm, Dry Drayton, Cambridge CB23 8DB, UK. Published by Elsevier Ltd. All rights reserved.)

Due to lack of suitable organ donors, future degenerative diseases and traumas could be treated with stem cell engraftment. To do this, large numbers of cells must be grown and maintained in culture. These cells must also be capable to differentiate into all the cells of the body. Embryonic stem cells fulfill many of the necessary criteria for clinical translation for use in therapeutic transplantation for a myriad of diseases. There are still many issues including immunological, cell cycling and differentiation that must be overcome for them to reach their potential use in the clinical arena.

Many revegetated landfills have poor cover including bare areas where plants do not grow. This study, on the Bisasar Road Landfill site in South Africa, assessed grass species preferences to microhabitat conditions in a mosaic of patches of well-established grassed areas and bare, nonvegetated areas. Factors, including soil CO2, CH4, O2, nutrients, and other general soil conditions, were measured in relation to species distribution and grass biomass in the field. Cynodon dactylon was the dominant grass in the established grass areas but was less abundant in the areas bordering the bare areas where Paspalum paspalodes and Sporobolus africanus were common. A number of soil factors measured were significantly correlated with grass biomass and these included Mg, Ca, Zn, Mn, K, temperature, moisture, and CO2. However, a laboratory bioassay using the growth of C. dactylon with soils removed from the landfill indicated that there were no differences in the soils from the bare areas and those that supported high plant biomass. Thus, no nutrient deficiency or chemical toxicity was inherent in the soil in the laboratory. The results of the field investigation and bioassay indicated that soil CO2 as a result of landfill gas infiltration into the root zone was probably the main factor causing bare areas on the landfill where no grass species could colonize and grow and that C. dactylon was more sensitive to elevated soil CO2 than other grass species such as P. paspalodes and S. africanus.

Responses to Al3+ of embryogenic callus cells of an Al-sensitive (Al-S) and Al-resistant (Al-R) Cynodon dactylon genotype were evaluated with regard to Al3+ toxicity and resistance. A chemical equilibrium speciation model (MINTEQA2) was used to ensure the availability of the Al3+ ion in culture media, which was supplied as 0.08-2.3 mM Al3+ for 2-8 weeks. Increasing Al3+ concentration and exposure time had a greater negative impact on the Al-S than on the Al-R genotype, in terms of callus growth rate and frequency of non-embryogenic cells. Exposure to 0.8 mM Al3+ for 2 weeks resulted in an 88% reduction in the Al-S meristematic cell number, whereas that of the Al-R genotype remained unaffected. In addition, the Al-S cells accumulated three times more Al in the nucleus than did the Al-R cells, suggesting that Al interfered with mitosis. The Al-R cells appeared to exclude Al3+ from its cells through an increase in extracellular pH (4.34 in Al-R and 4.08 in Al-S) and by the immobilisation of Al in the cell wall (33% more in Al-R). The results showed that by studying the cellular responses to Al3+ it is possible to discriminate between the Al-S and Al-R C. dactylon genotypes.

Lead, zinc, and cadmium were determined in a range of tissues from wild populations of bank voles (Clethrionomys glareolus) trapped on an abandoned metalliferous mine site and a reference site. Estimated dietary intakes indicated that animals were exposed to elevated levels of all three metals at the mine site, and this was generally reflected in metal residues in body tissues. Lead concentrations were significantly higher in all tissues of animals from the mine compared to the reference site, while Cd was higher only in the kidney. There was evidence of age-accumulation (using total body weight as an index of age) of Cd in both the liver and kidney of mine site animals but no evidence of such accumulation of lead in bone. In contrast to Cd and Pb, Zn was lower in the tissues of mine site animals compared to the reference site. Based on critical tissue concentrations, the ecotoxicological risk to a wild population of bank voles (Clethrionomys glareolus), associated with total substrate levels of 1 microg g(-1) dry weight Cd and 700 microg g(-1) dry weight Zn at this mine site is negligible, but that associated with 4000 microg g(-1) dry weight Pb may be considered significant.

The effect of fluoride on the appearance of the teeth of the short-tailed field vole, Microtus agrestis, was investigated in both wild animals collected from field sites affected by different levels of industrial fluoride contamination and laboratory-reared animals consuming experimental grass diets of known fluoride concentration or with known fluoride concentrations in drinking water. The extent and severity of lesions on the surface and structure of both incisors and molars are described as six lesion types and related to the amount of biologically available orally ingested fluoride. In the incisors of voles consuming relatively low fluoride diets, lesions are mainly confined to those resulting from disruption of enamel pigmentation expressing itself as concentric bands of pigmentation-free areas on incisor surfaces. The visible effects on molars at low fluoride levels are confined to minor alterations in surface appearance. At higher levels of available dietary fluoride, effects on enamel pigmentation are superseded by alterations in the formation, composition, and strength of both enamel and dentine. The incisors exhibit a marked to severe increase in the cutting tip erosion rates with comparable increases in the extent of abnormal surface changes (enamel hypoplasia) and the loss of enamel pigmentation. The grinding surfaces of molars from animals exposed to high levels of dietary fluoride exhibit increasingly severe erosion of outer enamel regions, combined with cavitation and staining of the exposed central dentine. The mechanisms through which fluoride elicits increasingly visible and pathological alterations to the surface and subsurfaces of rodent teeth are discussed.

A laboratory experiment was conducted to compare the toxicological response and metabolism of inorganic fluoride by three species of wild mammals and laboratory white mice (Mus musculus L.). The experimental populations of the wild species--the short-tailed field vole (Microtus agrestis L.), the bank vole (Clethrionomys glareolus L.) and the the wood mouse (Apodemus sylvaticus L.)--were laboratory reared from wild stock, and all test animals were exposed to 0, 40 or 80 microgram F per ml in their drinking water for up to 84 days. The 40 and 80 microgram F per ml treatments induced premature mortalities in M. agrestis and C. glareolus only. Differential intakes, absorption and retention of fluoride were evident between M. musculus and M. agrestis, the two species subject to cage studies of fluoride metabolism budgets. Interspecific variation in accumulation of fluoride with time was also evident between all four species as regards the femur, molars and incisors. Severe dental lesions were apparent in species surviving the 80 microgram ml(-1) treatment for 84 days. Overall, however, there were few clear differences in inherent species sensitivity to fluoride, the interspecific variation in metabolism and accumulation rates being attributable mainly to variation in intake.

Populations of two species of small mammal, the field vole (Microtus agrestis L.) and the bank vole (Clethrionomys glareolus L.), inhabiting grasslands contaminated by industrial sources of fluoride were examined for fluoride concentrations in skeletal tissue and for morphological changes in the teeth. Concentrations of fluoride in teeth and bones were higher for C. glareolus than for M. agrestis at the chemical works and smelter sites. Severe dental lesions were recorded on the incisor and molar teeth of both species at the chemical works and smelter sites, with less marked damage at the mine tailings dam. This is attributed to inter-site differences in fluoride speciation and the consequent effects on the availability of fluoride in the diet for bioassimilation.

Laboratory experiments were undertaken to study uptake and retention of antimony and to investigate whether the elevated organ antimony concentrations found previously in a population of Microtus agrestis at a contaminated site could cause harmful effects. Antimony trioxide in the diet produced elevated organ concentrations, but even in a 60-day experiment no harmful effects were evident. An equilibrium between uptake and excretion of antimony seemed to be rapidly established and progressive increases in organ concentrations did not occur. When dietary intake was terminated antimony was rapidly cleared. Comparison of findings from the laboratory and field suggested that inhalation was an additional route of intake in the field. It seems that present levels of antimony are unlikely to cause toxic effects in the wild population.

Two trials were conducted with 15 wethers surgically equipped with duodenal and ileal cannulas to study the utilization of P, Ca, Mg, Cu, Fe and Zn from swine waste and broiler litter. For each trial, animals were fed a low-P basal diet until serum inorganic P averaged 5.5 mg/dl; then they were allotted at random to the following 50% DM ensiled diets: low-P basal, basal + swine waste, basal + broiler litter, basal + dicalcium phosphate and basal + soybean meal. Each trial consisted of a 7-d preliminary period, a 7-d collection of feces and urine and 6-d sampling of duodenal and ileal digesta and feces. Apparent P absorption was not different (P greater than .05) between sheep fed waste-supplemented diets (37%) and those fed the conventionally supplemented diets (28%). Phosphorus absorption, calculated by difference, tended (P less than .1) to be higher from the waste supplements (59%) than from dicalcium phosphate and soybean meal (37%). Less (P less than .05) Ca was absorbed from the waste diets (.62 g/d) than from the conventional diets (1.28 g/d). More (P less than .05) Cu (mg/d) was absorbed from the waste diets, but no difference was found when absorption was expressed as percentage of intake. Broiler litter and swine waste were good sources of available P and Mg for ruminants.

Concentrations of antimony in invertebrates and small mammals from grasslands in the vicinity of an antimony smelter were significantly elevated compared to a control site. Higher concentrations of antimony were recorded in liver, lung and kidney tissue of herbivorous and insectivorous mammals from the contaminated sites. However, there is little evidence of bioaccumulation of antimony in food chains represented by the soil-vegetation-invertebrate-insectivore pathway of the grasslands, and little indication of significant accumulation by herbivorous mammals despite marked contamination of their diet.

Antimony concentrations in surface soils were found to decrease with increasing distance from an antimony smelter. This pattern was also found in moss bags exposed in the same area. At three sites close to the smelter, antimony concentrations in soil and vegetation were much higher than at a rural control site and published background levels. Maximum soil and plant concentrations on a dry weight basis of 1489 mg kg(-1) and 336 mg kg(-1), respectively, were found, compared to background levels of <1 mg kg(-1). Field exposure of grass in pots of uncontaminated soil and a laboratory experiment using soils from near the smelter suggested that the antimony in vegetation was largely due to continued aerial deposition and not to uptake from soil.

High total soil fluoride (10 000 microg g(-1)) in the metalliferous fluorspar tailings was reflected by elevated concentrations in standing live vegetation (300-1000 microg g(-1)); plant roots (c. 6000 microg g(-1)); plant litter (c. 4000 microg g(-1)); total body concentrations of invertebrates (400-4000 microg g(-1)) and the small mammals Microtus agrestis (120-360 microg g(-1)) and Sorex araneus (140-250 microg g(-1)). Seasonal changes in the standing live vegetation and the availability of soil fluoride to plants are discussed. Seasonal changes in total body concentrations of the small mammals were related to the age structure of the populations as well as dietary levels. In the small mammals, the concentration ratios were < 0.5 at the tailings dam and > 1.1 at the control site, indicating that both species were able to regulate fluoride accumulation at the higher levels of intake. Soft tissue concentrations were, as expected, very low compared to the hard tissues but, still, were generally significantly higher at the tailings dam compared to the control site. Evidence of dental fluorosis was found in Microtus agrestis, but not Sorex araneus.

Concentrations of lead in vegetation, invertebrates and small mammals in a grassland ecosystem evolved from a mine waste revegetation scheme indicated the high concentration of lead in the original tailings. Lead levels in invertebrates reflected dietary concentrations and feeding strategy, a pattern also true of the indigenous small mammals. Total body and tissue concentrations of lead in the herbivorous Microtus agrestis L. (field vole) and insectivorous Sorex araneus L. (common shrew) were significantly higher in the contaminated grassland than in an uncontaminated area, but concentration ratios (body:diet) were less than unity and there was no evidence of age-dependent accumulation of lead.

Concentrations of zinc in vegetation, invertebrates and small mammals in a grassland ecosystem derived from a mine waste vegetation scheme indicated the high concentration of zinc in the original tailings. However, the considerable differences in soil and vegetation zinc concentrations between the tailings and an uncontaminated site were not reflected in the invertebrate and small mammal communities. Invertebrate zinc levels and total body concentrations in Microtus agrestis L. (field vole) and Sorex araneus L. (common shrew) suggested a notable buffering of zinc absorption and tissue accumulation in the presence of substantially elevated dietary zinc. In the small mammals the skeleton appeared to function as the major storage site for zinc, possibly contributing to homeostasis in the soft tissues.

Vegetation and soil surveys were conducted on metalliferous mine wastes in the northern and southern Pennines of England. Analyses of vegetation composition in relation to soil chemical variation were performed. Ordination analysis facilitated the detection of groups of co-occurring species which are characteristic of types of metal-contaminated soil. The results of regression analysis implied the importance of soil pH and concentration of available lead or zinc, depending upon region, in determining species distributions. A strong interactive effect was evident between soil phosphorus and zinc content in influencing species distributions in the southern Pennines sample.Regional similarities in the vegetation of apparently similar metalliferous soils existed. The unusually high species richness of some soils was associated with relatively low concentrations of heavy metals. Many species of relatively floristically-rich wastes were also colonists of surrounding grasslands or woodland. This indicated the potential importance of propagule availability and capability for rapid establishment on bare or unstable ground. Mechanisms of physiological stress avoidance, rather than heavy-metai tolerance, may explain the occurrence of non-metallophytes on soils contaminated with lead and zinc.