FAM deubiquitylating enzyme is essential for preimplantation mouse embryo development

FAM is a developmentally regulated substrate-specific deubiquitylating enzyme. It binds the cell adhesion and signalling molecules beta -catenin and A-F-6 in vitro, and stabilises both in mammalian cell culture. To determine if FAM is required at the earliest stages of mouse development we examined its expression and function in preimplantation mouse embryos. FAM is expressed at all stages of preimplantation development from ovulation to implantation. Exposure of two-cell embryos to FAM-specific antisense, but not sense, oligodeoxynucleotides resulted in depletion of the FAM protein and failure Of the embryos to develop to blastocysts. Loss of FAM had two physiological effects, namely, a decrease in cleavage rate and an inhibition of cell adhesive events. Depletion of FAM protein was mirrored by a loss of beta -catenin such that very little of either protein remained following 72 h culture. The residual beta -catenin was localised to sites of cell-cell contact suggesting that the cytoplasmic pool of beta -catenin is stabilised by FAM. Although AF-6 levels initially decreased they returned to normal. However, the nascent protein was mislocalised at the apical surface of blastomeres. Therefore FAM is required for preimplantation mouse embryo development and regulates beta -catenin and AF-6 in vivo. (C) 2001 Elsevier Science Ireland Lid. All rights reserved.

EphB2 and two of its ligands have dynamic protein expression patterns in the developing olfactory system

Primary olfactory neurons are located in the olfactory neuroepithelium lining the nasal cavity. Their axons converge and form glomeruli with the dendrites of second-order neurons in the olfactory bulb. The molecular basis of primary olfactory axon guidance, targeting and subsequent arborisation is largely unknown. In this study we examined the spatio-temporal expression of the Eph receptor EphB2 and its ligands, ephrin-B1 and ephrin-B2, during development of the rat primary olfactory system. Unlike in other regions of the nervous system where receptor and ligand expression patterns are usually non-overlapping, EphB2, ephrin-B1 and ephrin-B2 were all expressed by primary and second-order olfactory neurons. In the embryonic animal we found that these three proteins had distinct and different expression patterns. EphB2 was first expressed at E18.5 by the perikarya of primary olfactory neurons. In contrast, ephrin-B1 was expressed from E13.5 and was localised to the axons of these cells up to E18.5 but was then restricted to the perikarya. Ephrin-B2, however, was expressed by olfactory ensheathing cells. EphB2, ephrin-B1 and ephrin-B2 were also expressed in the prenatal olfactory bulb and were restricted to the perikarya of mitral cells. In the post-natal olfactory bulb there was a shift in the localisation of both EphB2 and ephrin-B1 to the dendritic arborisations of mitral cells. The dynamic and tightly regulated spatio-temporal expression patterns of EphB2, ephrin-B1 and ephrin-B2 by specific olfactory cell populations suggest that these molecules have the potential to regulate important developmental events in the olfactory system. (C) 2001 Elsevier Science B.V. All rights reserved.

Expression and purification of enzymatically active recombinant RNA-dependent RNA polymerase (NS5) of the flavivirus Kunjin

The NS5 protein of the flavivirus Kunjin (KUN) contains conserved sequence motifs characteristic of RNA-dependent RNA polymerase (RdRp) activity. To investigate this activity in vitro, recombinant NS5 proteins with C-terminal (NS5CHis) and N-terminal (NS5NHis) hexahistidine tags were produced in baculovirus-infected insect cells and purified to near homogeneity by nickel affinity chromatography. Purified NS5CHis exhibited RdRp activity with both specific (9 kb KUN replicon) and non-specific (8.3 kb Semliki Forest virus replicon) RNA templates; this activity did not require the presence of additional viral and/or cellular cofactors. RdRp activity of purified NS5NHis protein was reduced in comparison to NS5CHis, while purified NS5NHis incorporating a GDD -> GVD mutation within the polymerase active site (NS5GVD) lacked RdRp activity. RNase A digestion of the RdRp reaction products indicated that they were double-stranded and of a similar size to the KUN replicative form produced in Vero cells, thus demonstrating that the KUN NS5 protein has an intrinsic, albeit low and non-specific RdRp activity in vitro, similar to that reported for recombinant RdRp of other flaviviruses. However, in contrast to RNA polymerases of other Flavivirus species, purified KUN NS5 polymerase produced a single, full-length replicon RNA product, thus demonstrating efficient processivity. (C) 2001 Elsevier Science B.V. All rights reserved.

Development of disabled, replication-defective gene transfer vectors from the Jembrana disease virus, a new infectious agent of cattle

Jembrana disease virus (JDV) is a newly isolated and characterised bovine lentivirus. It causes an acute disease in Ball cattle (Bos javanicus). which can be readily transmitted to susceptible cattle with 17% mortality. There is as yet no treatment or preventive vaccine. We have developed a gene transfer vector system based on JDV that has three components. The first of the components is a bicistronic transfer vector plasmid that was constructed to contain cis-sequences from the JDV genome, including 5 '- and 3 ' -long terminal repeats (LTRs), 0.4 kb of truncated gag and 1.1 kb of 3 ' -env, a multiple cloning site to accommodate the gene(s) of interest for transfer, and an internal ribosome entry site plus the neomycin phosphotransferase (Neo) gene cassette for antibiotic selection. The second element is a packaging plasmid that contains trans-sequences. including gag, pol. vif, tar and rev: but without the env and packaging signals. The third is a plasmid encoding the G glycoprotein of vesicular stomatitis virus (VSV-G) to supply the vector an envelope for pseudotyping. Cotransfection of 293T cells with these three plasmid components produced VSV-G pseudotyped. disabled, replication defective, bicistronic JDV vectors encoding the green fluorescent protein (EGFP) and the Neo resistance selection maker simultaneously with a titre range of (0.4-1.2) x 10(6) CFU/ml. Transduction of several replicating primary and transformed cells from cattle, primate and human sources and importantly growth-arrested cells with the JDV vectors showed high efficiency of EGFP gene transfer at 35-75%, which was stable and the expression of EGFP was long term. Furthermore, these JDV vectors were designed to suit the inclusion and expression of genes corresponding to JDV specific proteins, such as gag or env, for the development of vaccines for Jembrana disease. This strategy should also be applicable to other bovine diseases as wall. The design and construction of the JDV vector system should facilitate the study of the lentivirology and pathogenesis of the diseases associated with JDV or other bovine virus infections. To our knowledge, this is the first such vector system developed from a cattle virus. (C) 2001 Elsevier Science B.V. All rights reserved.

Anxious futures: Magpie2 and 'new generationalism' in Australian youth-specific theatre

The field of contemporary youth-specific theatre in Australia is one of change and, in some cases, anxiety. While Drama Studies continue to grow in popularity in schools, previously conventional developmental paradigms have become less mandatory for theatre, for, by, and about young people outside the school context. Instead, 'new generation' approaches in youth-specific performance are placing greater value on young people's own preferences in cultural activity. Yet this development is being tempered and further complicated by a cultural 'generationalism', particularly in larger arts organization as the youth sector becomes a more integral part of marketing strategies for the future. The resulting ambiguity in the representation, value, and positioning of young people and youth-specific arts in Australia's theatre industry is considered by focusing on Magpie2, a former youth-specific company attached to the State Theatre Company of South Australia. Magpie2 ceased operation in 1998 after experimenting with a 'new generation' approach to theatre for young people in the State Theatre realm. Both the artistic policy of Magpie2 Director, Benedict Andrews, and the critical reception of his two productions in 1997, Future Tense and Features of Blown Youth, demonstrate how competing systems of cultural value characterize the field of youth-specific theatre in Australia.

Genotype-by-management interactions for grain yield and grain protein concentration of wheat

The magnitude of genotype-by-management (G x M) interactions for grain yield and grain protein concentration was examined in a multi-environment trial (MET) involving a diverse set of 272 advanced breeding lines from the Queensland wheat breeding program. The MET was structured as a series of management-regimes imposed at 3 sites for 2 years. The management-regimes were generated at each site-year as separate trials in which planting time, N fertiliser application rate, cropping history, and irrigation were manipulated. irrigation was used to simulate different rainfall regimes. From the combined analysis of variance, the G x M interaction variance components were found to be the largest source of G x E interaction variation for both grain yield (0.117 +/- 0.005 t(2) ha(-2); 49% of total G x E 0.238 +/- 0.028 t(2) ha(-2)) and grain protein concentration (0.445 +/- 0.020%(2); 82% of total G x E 0.546 +/- 0.057%(2)), and in both cases this source of variation was larger than the genotypic variance component (grain yield 0.068 +/- 0.014 t(2) ha(-2) and grain protein 0.203 +/- 0.026%(2)). The genotypic correlation between the traits varied considerably with management-regime, ranging from -0.98 to -0.31, with an estimate of 0.0 for one trial. Pattern analysis identified advanced breeding lines with improved grain yield and grain protein concentration relative to the cultivars Hartog, Sunco and Meteor. It is likely that a large component of the previously documented G x E interactions for grain yield of wheat in the northern grains region are in part a result of G x M interactions. The implications of the strong influence of G x M interactions for the conduct of wheat breeding METs in the northern region are discussed. (C) 2001 Elsevier Science B.V. All rights reserved.

An investigation of symptom-specific muscle hyperreactivity in upper extremity cumulative trauma disorder

Objective: The study examined symptom-specific muscle hyperreactivity in patients with chronic pain with upper limb cumulative trauma disorder (CTD). Design: Four tasks were presented in counterbalanced order and included neutral, general stressor, personal stressor, and pain stressor tasks. Ratings of stressfulness and recordings of skin conductance level confirmed the effectiveness of the experimental manipulations in inducing stress experiences for all subject groups. Setting: The study was conducted in a university research center. Patients: Thirty patients with CTD were matched as closely as possible for age and gender to control groups of chronic low back pain, arthritis, and pain-Free subjects Outcome Measures: Surface electromyograph recordings were taken from the frontalis, forearm flexors, trapezius, and lower back during baseline and tasks. Results: The study found no evidence of greater muscle tension increases or extended duration of return to baseline for the CTD or low back pain patients at any of the muscle sites for any of the tasks in comparison to control groups. Conclusions: The results indicate that symptom-specific psychophysiological responses may be limited to certain subgroups rather than being characteristic of chronic musculoskeletal pain patients in general.

The GRIP domain is a specific targeting sequence for a population of trans-Golgi network derived tubulo-vesicular carriers

Vesicular carriers for intracellular transport associate with unique sets of accessory molecules that dictate budding and docking on specific membrane domains. Although many of these accessory molecules are peripheral membrane proteins, in most cases the targeting sequences responsible for their membrane recruitment have yet to be identified. We have previously defined a novel Golgi targeting domain (GRIP) shared by a family of coiled-coil peripheral membrane Golgi proteins implicated in membrane trafficking. We show here that the docking site for the GRIP motif of p230 is a specific domain of Golgi. membranes. By immunoelectron microscopy of HeLa cells stably expressing a green fluorescent protein (GFP)-p230(GRIP) fusion protein, we show binding specifically to a subset of membranes of the trans-Golgi network (TGN). Real-time imaging of live HeLa cells revealed that the GFP-p230(GRIP) was associated with highly dynamic tubular extensions of the TGN, which have the appearance and behaviour of transport carriers. To further define the nature of the GRIP membrane binding site, in vitro budding assays were performed using purified rat liver Golgi membranes and cytosol from GFP-p230(GRIP) transfected cells. Analysis of Golgi-derived vesicles by sucrose gradient fractionation demonstrated that GFP-p230(GRIP) binds to a specific population of vesicles distinct from those labelled for beta -COP or gamma -adaptin. The GFP-p230(GRIP) fusion protein is recruited to the same vesicle population as full-length p230, demonstrating that the GRIP domain is solely proficient as a targeting signal for membrane binding of the native molecule. Therefore, p230 GRIP is a targeting signal for recruitment to a highly selective membrane attachment site on a specific population of trans-Golgi network tubulovesicular carriers.

Ataxia telangiectasia mutated (ATM) kinase and ATM and Rad3 related kinase mediate phosphorylation of Brca1 at distinct and overlapping sites - In vivo assessment using phospho-specific antibodies

Recent studies have provided evidence that breast cancer susceptibility gene products (Brca1 and Brca2) suppress cancer, at least in part, by participating in DNA damage signaling and DNA repair. Brca1 is hyperphosphorylated in response to DNA damage and co-localizes with Rad51, a protein involved in homologous-recombination, and Nbs1.Mre11.Rad50, a complex required for both homologous-recombination and nonhomologous end joining repair of damaged DNA. Here, we report that there is a qualitative difference in the phosphorylation states of Brca1 between ionizing radiation (IR) and UV radiation. Brca1 is phosphorylated at Ser-1423 and Ser-1524 after IR and W; however, Ser-1387 is specifically phosphorylated after IR, and Ser-1457 is predominantly phosphorylated after W. These results suggest that different types of DNA-damaging agents might signal to Brca1 in different ways. We also provide evidence that the rapid phosphorylation of Brca1 at Ser-1423 and Ser-1524 after IR (but not after W) is largely ataxia telangiectasia mutated (ATM) kinase-dependent. The overexpression of catalytically inactive ATM and Rad3 related (ATR) kinase inhibited the UV-induced phosphorylation of Brca1 at these sites, indicating that ATR controls Brca1 phosphorylation in vivo after the exposure of cells to UV light. Moreover, ATR associates with Brca1; ATR and Brca1 foci co-localize both in cells synchronized in S phase and after exposure of cells to DNA-damaging agents. ATR can itself phosphorylate the region of Brca1 phosphorylated by ATM (Ser-Gln cluster in the C terminus of Brca1, amino acids 1241-1530), However, there are additional uncharacterized ATR phosphorylation site(s) between residues 521 and 757 of Brca1, Taken together, our results support a model in which ATM and ATR act in parallel but somewhat overlapping pathways of DNA damage signaling but respond primarily to different types of DNA lesion.

Response of a scleractinian coral, Stylophora pistillata, to iron and nitrate enrichment

The purpose of this study was to determine whether the addition of iron alone or in combination with nitrate affects growth and photosynthesis of the scleractinian coral, Stylophora pistillata, and its symbiotic dinoflagellates. For this purpose, we used three series of two tanks for a 3-week enrichment with iron (Fe), nitrate (N) and nitrate + iron (NFe). Two other tanks were kept as a control (C). Stock solutions of FeCl3 and NaNO3 were diluted to final concentrations of 6 nM Fe and 2 muM N and continuously pumped from batch tanks into the experimental tanks with a peristaltic pump. Results obtained showed that iron addition induced a significant increase in the areal density of zooxanthellae (ANOVA, p = 0.0013; change from 6.3 +/- 0.7 x 10(5) in the control to 8.5 +/- 0.6 x 10(5) with iron). Maximal gross photosynthetic rates normalized per surface area also significantly increased following iron enrichment (ANOVA, p = 0.02; change from 1.23 +/- 0.08 for the control colonies to 1.81 +/- 0.24 mu mol O-2 cm(-2) h(-1) for the iron-enriched colonies). There was, however, no significant difference in the photosynthesis normalized on a per cell basis. Nitrate enrichment alone (2 muM) did not significantly change the zooxanthellae density or the rates of photosynthesis. Nutrient addition (both iron and nitrogen) increased the cell-specific density of the algae (CSD) compared to the control (G-test, p = 0.3 x 10(-9)), with an increase in the number of doublets and triplets. CSD was equal to 1.70 +/- 0.04 in the Fe-enriched colonies, 1.54 +/- 0.12 in the N- and NFe-enriched colonies and 1.37 +/- 0.02 in the control. Growth rates measured after 3 weeks in colonies enriched with Fe, N and NFe were 23%, 34% and 40% lower than those obtained in control colonies (ANOVA. p = 0.011). (C) 2001 Elsevier Science B.V. All rights reserved.

A methodology for discipline-specific curriculum development

A methodology and framework for discipline-specific curriculum development in a local context is described. These activities, as part of the Thailand-Australia Science and Engineering Assistance Project, were in response to a needs analysis for curriculum assistance to a number of publicly-funded Thai universities in the engineering priority area of Materials Processing and Manufacturing. The paper outlines a strategy for the delivery of a centralised curriculum development workshop for academic staff follow-up visits and local curriculum activities with participating universities, and the presentation of technical short courses as guidance for such activity in other settings and/or discipline areas. This paper is part of a process of documentation so that others can apply the developed methodology and framework for curriculum development. While the paper is a report on curriculum activities in a particular setting, it is written in a manner that allows application of the methodology to other settings. The reader is advised that each curriculum activity needs to adopt a methodology and strategy to fit the particular circumstances being considered To assist in applying this approach elsewhere, a description of the various steps in the curriculum process, and typical responses to some of the more global issues, have been presented. Full details are available in the various TASEAP reports prepared by the authors. Specific detail has been omitted where this detail does not provide any information for generalized consumption.

In vitro evidence for a bacterial pathogenesis of equine laminitis

Utilizing an in vitro laminitis explant model, we have investigated how bacterial broth cultures and purified bacterial proteases activate matrix metalloproteinases (MMPs) and alter structural integrity of cultured equine lamellar hoof explants. Four Gram-positive Streptococcus spp. and three Gram-negative bacteria all induced a dose-dependent activation of MMP-2 and MMP-9 and caused lamellar explants to separate. MMP activation was deemed to have occurred if a specific MMP inhibitor, batimastat, blocked MMP activity and prevented lamellar separation. Thermolysin and streptococcal pyrogenic exotoxin B (SpeB) both separated explants dose-dependently but only thermolysin was inhibitable by batimastat or induced MMP activation equivalent to that seen with bacterial broths. Additionally, thermolysin and broth MMP activation appeared to be cell dependent as MMP activation did not occur in isolation. These results suggest the rapid increase in streptococcal species in the caecum and colon observed in parallel with carbohydrate induced equine laminitis may directly cause laminitis via production of exotoxin(s) capable of activating resident MMPs within the lamellar structure. Once activated, these MMPs can degrade key components of the basement membrane (BM) hemidesmosome complex, ultimately separating the BM from the epidermal basal cells resulting in the characteristic laminitis histopathology of hoof lamellae. While many different causative agents have been evaluated in the past, the results of this study provide a unifying aetiological mechanism for the development of carbohydrate induced equine laminitis. (C) 2001 Elsevier Science B.V. All rights reserved.

Mechanisms of drought-induced population decline in an endangered wallaby

The bridled nailtail wallaby is restricted to one locality in central Queensland, Australia. The population declined severely during a major drought between 1991 and 1995. We investigated age-specific covariates of survival and proximate causes of mortality from 1994 to 1997, using mark-recapture and radio-tagging techniques at two study sites. Using a matrix population model, we also modelled the effect of drought on age-specific survival and the intrinsic rate of population increase,;,. The only significant covariate of survival for adults was a measure of health unrelated to drought. Rainfall, food, predator activity, year, sex and habitat were not associated with variation in adult survival. Juvenile survival was negatively affected by drought, and predation was the proximate cause of most juvenile deaths. The matrix projection model showed that the observed juvenile survivorship during the drought was low enough to have produced a population decline, although fecundity and survival of other age classes was high throughout the study. (C) 2001 Elsevier Science Ltd. All rights reserved.