Informing the management of multiple stressors on estuarine ecosystems using an expert-based Bayesian Network model.

The approach of applying stressor load limits or thresholds to aid estuarine management is being explored in many global case studies. However, there is growing concern regarding the influence of multiple stressors and their cumulative effects on the functioning of estuarine ecosystems due to the considerable uncertainty around stressor interactions. Recognising that empirical data limitations hinder parameterisation of detailed models of estuarine ecosystem responses to multiple stressors (suspended sediment, sediment mud and metal content, and nitrogen inputs), an expert driven Bayesian network (BN) was developed and validated. Overall, trends in estuarine condition predicted by the BN model were well supported by field observations, including results that were markedly higher than random (71-84% concordance), providing confidence in the overall model dynamics. The general BN framework was then applied to a case study estuary to demonstrate the model's utility for informing management decisions. Results indicated that reductions in suspended sediment loading were likely to result in improvements in estuarine condition, which was further improved by reductions in sediment mud and metal content, with an increased likelihood of high abundance of ecological communities relative to baseline conditions. Notably, reductions in suspended sediment were also associated with an increased probability of high nuisance macroalgae and phytoplankton if nutrient loading was not also reduced (associated with increased water column light penetration). Our results highlight that if stressor limit setting is to be implemented, limits must incorporate ecosystem responses to cumulative stressors, consider the present and desired future condition of the estuary of interest, and account for the likelihood of unexpected ecological outcomes regardless of whether the experts (or empirical data) suggest a threshold has or has not been triggered.

FTIR imaging detects diet and genotype-dependent chemical composition changes in wild type and mutant C. elegans strains.

This study focuses on the use of Fourier Transform Infrared (FTIR) microspectroscopy to determine chemical changes induced in the nematode Caenorhabditis elegans by supplementation of C. elegans maintenance media (CeMM) by Eicosapentaenoic acid (EPA). Wild-type C. elegans (N2) and mutant strains (tub-1 and fat-3) were grown in CeMM alone, and CeMM supplemented with EPA at 25 or 100 µM. Feeding was performed for 72 h. FTIR imaging was performed in transmission mode on individual worms. The FTIR imaging analysis of wild-type animals revealed the presence of vibrations assigned to unsaturated fatty acids, specifically bands at 3008 cm-1 ([double bond, length as m-dash]C-H, olefinic stretch) and 1744 cm-1 (C[double bond, length as m-dash]O, unsaturated fatty acids). It confirmed previously reported synthesis of unsaturated fatty acids in wild-type C. elegans. For the FTIR spectra of mutant strains, these vibrations were absent or present only as very small shoulder, which indicates that tub-1 and fat-3 synthesize essentially saturated fatty acids as indicated by the presence of -CH2 and C[double bond, length as m-dash]O vibrations. These results are in agreement with previous studies which reported that these mutants have altered lipid compositions. Principal component analysis showed differences in chemical composition between wild-type and mutant strains as well as between mutant strains cultured in normal CeMM and those cultured in CeMM supplemented with EPA. This study demonstrated the usefulness of FTIR microspectroscopy to investigate fat metabolism in C. elegans.

Mutant glycosyltransferase and altered glycosylation of alpha-dystroglycan in the myodystrophy mouse.

Spontaneous and engineered mouse mutants have facilitated our understanding of the pathogenesis of muscular dystrophy and they provide models for the development of therapeutic approaches. The mouse myodystrophy (myd) mutation produces an autosomal recessive, neuromuscular phenotype. Homozygotes have an abnormal gait, show abnormal posturing when suspended by the tail and are smaller than littermate controls. Serum creatine kinase is elevated and muscle histology is typical of a progressive myopathy with focal areas of acute necrosis and clusters of regenerating fibers. Additional aspects of the phenotype include sensorineural deafness, reduced lifespan and decreased reproductive fitness. The myd mutation maps to mouse chromosome 8 at approximately 33 centimorgans (cM) (refs. 2, 4-7). Here we show that the gene mutated in myd encodes a glycosyltransferase, Large. The human homolog of this gene (LARGE) maps to chromosome 22q. In myd, an intragenic deletion of exons 4-7 causes a frameshift in the resultant mRNA and a premature termination codon before the first of the two catalytic domains. On immunoblots, a monoclonal antibody to alpha-dystroglycan (a component of the dystrophin-associated glycoprotein complex) shows reduced binding in myd, which we attribute to altered glycosylation of this protein. We speculate that abnormal post-translational modification of alpha-dystroglycan may contribute to the myd phenotype.

Chromosome 4q DNA rearrangements associated with facioscapulohumeral muscular dystrophy.

Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant neuromuscular disorder which maps to chromosome 4qter, distal to the D4S139 locus. The cosmid clone 13E, isolated in a search for homeobox genes, was subsequently mapped to 4q35, also distal to D4S139. A subclone, p13E-11, detects in normal individuals a polymorphic EcoRI fragment usually larger than 28 kilobases (kb). Surprisingly, using the same probe we detected de novo DNA rearrangements, characterized by shorter EcoRI fragments (14-28 kb), in 5 out of 6 new FSHD cases. In 10 Dutch families analysed, a specific shorter fragment between 14-28 kb cosegregates with FSHD. Both observations indicate that FSHD is caused by independent de novo DNA rearrangements in the EcoRI fragment detected by p13E-11.

Interaction networks in coastal soft-sediments highlight the potential for change in ecological resilience.

Recent studies emphasize the role of indirect relationships and feedback loops in maintaining ecosystem resilience. Environmental changes that impact on the organisms involved in these processes have the potential to initiate threshold responses and fundamentally shift the interactions within an ecosystem. However, empirical studies are hindered by the difficulty of designing appropriate manipulative experiments to capture this complexity. Here we employ structural equation modeling to define and test the architecture of ecosystem interaction networks. Using survey data from 19 estuaries we investigate the interactions between biological (abundance of large bioturbating macrofauna, microphytobenthos, and detrital matter) and physical (sediment grain size) processes. We assess the potential for abrupt changes in the architecture of the network and the strength of interactions to occur across environmental gradients. Our analysis identified a potential threshold in the relationship between sediment mud content and benthic chlorophyll a, at -12 microg/g, using quantile regression. Below this threshold, the interaction network involved different variables and fewer feedbacks than above. This approach has potential to improve our empirical understanding of thresholds in ecological systems and our ability to design manipulative experiments that test how and when a threshold will be passed. It can also be used to indicate to resource managers that a particular system has the potential to exhibit threshold responses to environmental change, emphasizing precautionary management and facilitating a better understanding of how persistent multiple stressors threaten the resilience and long-term use of natural ecosystems.

The development of a national approach to monitoring estuarine health based on multivariate analysis.

New Zealand has a complex coastal environment spanning a large latitudinal gradient and three water masses. Here we assess whether multivariate analyses of benthic macrofaunal community composition can be a sensitive approach to assessing relative estuarine health across the country, negating the need for regional indices and reducing reliance on reference sites. Community data were used in separate canonical analyses of principal coordinates to create multivariate models of community responses to gradients in mud content and heavy metal contamination. Both models performed well (R2 = 0.81, 0.71), and were unaffected by regional and estuarine typology differences. The models demonstrate a sensitive and standardized approach to assessing estuarine health that allowed separation of the two stressors. This approach could be applied to other stressors, countries or regions.

FRG2, an FSHD candidate gene, is transcriptionally upregulated in differentiating primary myoblast cultures of FSHD patients.

BACKGROUND: Autosomal dominant facioscapulohumeral muscular dystrophy (FSHD) is associated with partial deletion of the subtelomeric D4Z4 repeat array on chromosome 4qter. This chromosomal rearrangement may result in regional chromatin relaxation and transcriptional deregulation of genes nearby. METHODS AND RESULTS: Here we describe the isolation and characterisation of FRG2, a member of a chromosomally dispersed gene family, mapping only 37 kb proximal to the D4Z4 repeat array. Homology and motif searches yielded no clues to the function of the predicted protein. FRG2 expression is undetectable in all tissues tested except for differentiating myoblasts of FSHD patients, which display low, yet distinct levels of FRG2 expression, partly from chromosome 4 but predominantly originating from its homologue on chromosome 10. However, in non-FSHD myopathy patients only distantly related FRG2 homologues are transcribed, while differentiating myoblasts from healthy controls fail to express any member of this gene family. Moreover, fibroblasts of FSHD patients and control individuals undergoing forced Ad5-MyoD mediated myogenesis show expression of FRG2 mainly originating from chromosome 10. Luciferase reporter assays show that the FRG2 promoter region can direct high levels of expression but is inhibited by increasing numbers of D4Z4 repeat units. Transient transfection experiments with FRG2 fusion-protein constructs reveal nuclear localisation and apparently FRG2 overexpression causes a wide range of morphological changes. CONCLUSION: The localisation of FRG2 genes close to the D4Z4 repeats on chromosome 4 and 10, their transcriptional upregulation specifically in FSHD myoblast cultures, potential involvement in myogenesis, and promoter properties qualify FRG2 as an attractive candidate for FSHD pathogenesis.

Intron loss in the SART1 genes of Fugu rubripes and Tetraodon nigroviridis.

The human SART1 gene was initially identified in a screen for proteins recognised by IgE, which may be implicated in atopic disease. We have examined the genomic structure and cDNA sequence of the SART1 gene in the compact genomes of the pufferfish Fugu rubripes and Tetraodon nigroviridis. The entire coding regions of both the Fugu and Tetraodon SART1 genes are contained within single exons. The Fugu gene contains only one intron located in the 5' untranslated region. Southern blot hybridisation of Fugu genomic DNA confirmed the SART1 gene to be single copy. Partial genomic structures were also determined for the human, mouse, Drosophila and C. elegans SART1 homologues. The human and mouse genes both contain many introns in the coding region, the human gene possessing at least 20 exons. The Drosophila and C. elegans homologues contain 6 and 12 exons, respectively. This is only the second time such a difference in the organization of homologous Fugu and human genes has been reported. The Fugu and Tetraodon SART1 genes encode putative proteins of 772 and 774 aa, respectively, each having 65% amino acid identity to human SART1. Leucine zipper and basic motifs are conserved in the predicted Fugu and Tetraodon proteins.

An alternatively spliced transcript, p65 delta 2, of the gene encoding the p65 subunit of the transcription factor NF-kappa B.

We have identified a second alternatively spliced transcript of the NF-kappa B transcription factor p65 subunit. This transcript, named p65 delta 2, varies from p65 at two functionally important regions.

Recent amplification of the human FRG1 gene during primate evolution.

There is evidence of multiple copies of the FSHD Region Candidate Gene 1 (FRG1) in humans. Analysis of human FRG1 ESTs showed many of them to be non-processed pseudogenes dispersed throughout the genome. To determine when the amplification of FRG1 occurred, we used a PCR-based approach to identify FRG1 sequences from great apes, chimpanzee, gorilla and orang-utan, and an Old World monkey, Macaca mulatta. In common with humans, multiple copies of FRG1 were detected in the great apes. However, in Macaca mulatta, only two FRG1 loci were identified, one presumed to be the homologue of the human chromosome 4q gene. This is strikingly similar to the distribution of a dispersed 3.3-kb repeat family in primates. A member of this family, D4Z4, maps to the subtelomeric region of 4q, in close proximity to FRG1. We propose that an ancestral duplication of distal 4q included FRG1. This duplication is present in Macaca mulatta whose divergence from hominoids is thought to have occurred at least 33 million years ago. We propose that this telomeric region then underwent further amplification and dispersion events in the great ape lineage, with copies of FRG1 and the 3.3-kb repeats being localized in heterochromatic regions.

FRG1, a gene in the FSH muscular dystrophy region on human chromosome 4q35, is highly conserved in vertebrates and invertebrates.

The human FRG1 gene maps to human chromosome 4q35 and was identified as a candidate for facioscapulohumeral muscular dystrophy. However, FRG1 is apparently not causally associated with the disease and as yet, its function remains unclear. We have cloned homologues of FRG1 from two additional vertebrates, the mouse and the Japanese puffer fish Fugu rubripes, and investigated the genomic organization of the genes in the two species. The intron/exon structure of the genes is identical throughout the protein coding region, although the Fugu gene is five times smaller than the mouse gene. We have also identified FRG1 homologues in two nematodes; Caenorhabditis elegans and Brugia malayi. The FRG1 protein is highly conserved and contains a lipocalin sequence motif, suggesting it may function as a transport protein.

Cloning of the murine unconventional myosin gene Myo9b and identification of alternative splicing.

We report the cloning of a cDNA for the mouse unconventional myosin Myo9b, the orthologue of the rat myr5 and human MYOIXb genes. A full-length spleen cDNA of 7087bp encoding a protein of 1961 amino acids was isolated. By RT-PCR, we show that Myo9b is expressed in a wide range of tissues, including heart, brain, muscle and inner ear. In addition, we have identified two alternatively spliced exons. Equivalent exons have not been previously reported for either the human or rat homologues. These exons are located in the Myo9b specific actin-binding site insert of the head domain and in the tail region. A third splice form utilizing an alternative reading frame within the 3'UTR is also described. Several polymorphisms within the coding region were identified; of interest is an in-frame 33bp imperfect duplication within the tail region that was observed only in the C57Bl/6 strain. Myo9b has been previously mapped to mouse chromosome 8 and is a candidate for the mouse mutations myodystrophy and quinky.

Nucleotide sequence of the partially deleted D4Z4 locus in a patient with FSHD identifies a putative gene within each 3.3 kb element.

Facioscapulohumeral muscular dystrophy (FSHD) is linked to the polymorphic D4Z4 locus on chromosome 4q35. In non-affected individuals, this locus comprises 10-100 tandem copies of members of the 3.3kb dispersed repeat family. Deletions leaving 1-8 such repeats have been associated with FSHD, for which no candidate gene has been identified. We have determined the complete nucleotide sequence of a 13.5kb EcoRI genomic fragment comprising the only two 3.3kb elements left in the affected D4Z4 locus of a patient with FSHD. Sequence analyses demonstrated that the two 3.3kb repeats were identical. They contain a putative promoter that was not previously detected, with a TACAA instead of a TATAA box, and a GC box. Transient expression of a luciferase reporter gene fused to 191bp of this promoter, demonstrated strong activity in transfected human rhabdomyosarcoma TE671 cells that was affected by mutations in the TACAA or GC box. In addition, these 3.3kb repeats include an open reading frame (ORF) starting 149bp downstream from the TACAA box and encoding a 391 residue protein with two homeodomains (DUX4). In-vitro transcription/translation of the ORF in a rabbit reticulocyte lysate yielded two (35)S Cys/ (35)S Met labeled products with apparent molecular weights of 38 and 75kDa on SDS-PAGE, corresponding to the DUX4 monomer and dimer, respectively. In conclusion, we propose that each of the 3.3kb elements in the partially deleted D4Z4 locus could include a DUX4 gene encoding a double homeodomain protein.

Molecular genetics of facioscapulohumeral muscular dystrophy.

Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant neuromuscular disorder. The disease affects specific muscles of the face, shoulder-girdle and upper arm. The biochemical defect underlying FSHD is unknown and there are no specific tests that are diagnostic of FSHD. Genetic linkage studies have mapped the FSHD gene to chromosome 4q35. A DNA marker (p13E-11; D4F104S1, formerly D4S810) has been isolated which recognizes two highly polymorphic loci detectable by EcoRI or HindIII; one locus maps to chromosome 4q35 and shows fragments between about 50 and 320 kb. In FSHD patients deletions occur within this EcoRI/HindIII fragment, yielding fragments that are usually smaller than 28 kb. Characterization of the polymorphic fragments demonstrates that they consist of a 3.2 kb tandem repeat; their number can range between approximately 12 and 96 within the 4q35-specific fragments. In FSHD patients, an integral number of these tandem repeats are deleted, leaving at maximum eight copies.

Effective long-term ecological monitoring using spatially and temporally nested sampling.

Effective environmental management requires documentation of ecosystem status and changes to that status. Without long-term data, short-term natural variability can mask chronic and/or cumulative impacts, often until critical levels are reached. However, a trade-off generally occurs between sampling in space and time. This study analyses a spatially and temporally nested long-term (12 years) monitoring programme conducted on benthic macrofauna in a large harbour. Sampling was carried out at six sites for 5.5 years, after which only two sites were sampled for the next 5 years. After this period, all six sites were sampled for another 2 years. While ecology is frequently thought of being highly variable, this design was able to detect trends, and cycles, in abundance, with only around 10% of species at each site exhibiting unpredictable temporal variability. Sites exhibiting similar trends in the abundance of a species over the 12.5-year period were generally spatially contiguous, and the spatial scale of change could be assessed. Continuous sampling at two sites identified whether changes in unsampled sites were related to long-term cycles. Moreover, this sampling provided a long-term background of temporal fluctuations against which to assess the ecological significance of observed changes.

Glycosylation defects in inherited muscle disease.

The gene mutated in the myodystrophy mouse, a model of muscular dystrophy, encodes a putative glycosyltransferase, Large. Mutations in genes encoding proteins thought to be involved in glycosylation have now been identified in six human forms of muscular dystrophy. Hereditary inclusion body myopathy and Nonaka myopathy result from defects in sialic acid production. Two forms of congenital muscular dystrophy, Fukuyama-type and MDC1C, result from mutations in members of the fukutin family. MDC1C and limb girdle muscular dystrophy type 2I are allelic, as they are both associated with mutations in the FKRP gene. Mutations in POMGnT, which encodes an enzyme involved in the synthesis of O-mannosyl glycans, result in muscle-eye-brain disease--another congenital form of muscular dystrophy. Abnormal alpha-dystroglycan has been reported in the myodystrophy mouse, and in the congenital and limb girdle muscular dystrophies. Recent data have shown that there is altered glycosylation of the protein and that this reduces its ability to bind to extracellular matrix ligands such as laminin and agrin.

Thyroid hormone effects on lactase expression by rat enterocytes.

Thyroxine (T4) and triiodothyronine (T3) injected into adult rats causes first an increase and then a decrease in lactase activity measured subsequently in intestinal homogenates of rat jejunum. These changes are not associated with any alteration in intestinal structure or enterocyte migration rate. Quantitative cytochemistry shows T4 stimulation and inhibition of lactase activity to take place in upper villus and crypt cells respectively (O- and C-enterocytes). T3 injected into thyroidectomized rats produces identical stimulatory effects on lactase development to T4 injected into control animals. Radioactive T3 is distributed in all cell types following intraperitoneal injection into thyroidectomized rats. Highest amounts of recovered T3 are found in C- rather than O- enterocytes. Quantitative autoradiography shows intracellular T3 to be located in nuclear and cytoplasmic compartments following intraperitoneal injection. Simultaneous injection of non-radioactive hormone displaces 50-75% of radioactive T3. These results are discussed in relation to what is already known concerning the ability of thyroid hormones to affect intestinal development. The future need to study the physiological effects of T3 at the cellular level in the intestine is also emphasized.

Binding of triiodothyronine by fully differentiated rat enterocytes.

The cellular binding characteristics of triiodothyronine (T3) have been measured in a preparation of isolated fully differentiated enterocytes prepared from thyroidectomized rats. Specific binding of [125I]T3, defined as that portion displaced by the presence of 20 microM-non-radioactive hormone, was shown to become maximal after 100 min incubation with cells at a temperature of 15 degrees C. The cellular release of lactic dehydrogenase measured under these conditions was found to be 18%. Thin layer chromatography of radioactive compounds recovered from enterocytes and from incubation medium showed less than 2% radioactivity to be as free iodide. The amount of [125I]T3 specifically bound to enterocytes showed a curvilinear dependence on the concentration of T3 present in the incubation medium. Scatchard plot analysis of this data revealed the presence of both high (KD 1.2 +/- 1.1 X 10(-9) M) and low (KD 1.9 +/- 0.8 X 10(-7) M) affinity binding sites in these cells. Previous injection of non-radioactive T3 into thyroidectomized rats, at a time chosen to ensure negligible levels of circulating T3 at the time of the experiment, caused a 3-fold reduction in the maximal binding capacity of the low-affinity site (83 compared with 28 pmol/mg protein). Neither the affinity nor the number of high-affinity binding sites for T3 were significantly affected by this treatment. These results are compared with those obtained using other types of cell responsive to T3. The possible physiological importance of these findings is discussed.