Induction of parturition in a large number of pregnant dairy goats and its benefits as a management tool in a commercial scale goat operation.

At LFB USA, Inc., transgenic goats are utilized for the production of recombinant human protein therapeutics in their milk through the rPRO™ Technology platform. This retrospective analysis and report describes the results of induced parturition and its use as a management tool in this large herd of dairy goats. Over a three-year period, 342 does received pronuclear microinjected (MI) embryos transferred into the oviductal lumen via midline laparotomy (day 1). To initiate the induction process, does were given intramuscular injections (IM) of 10 mg each of prostaglandin (Lutalyse®) and dexamethasone to induce parturition on days 144-148 of pregnancy. Mean and Standard Deviation (±SD) time to parturition was 36.7 (±6.5) hours. Does were given these injections at 4pm on Sundays with an expected kidding time of late Monday into Tuesday morning. Of the 342 does, 333 or 97% had kidded by 3pm the following Tuesday, and 313 or 91% kidded in the 18 h between 9pm Monday and 3pm on Tuesday or between 29 and 47 h post induction. By the end of Tuesday, most kids had received colostrum and were transferred to the nursery. The incidences of kid mortality and retained placenta were 2.5% and 1.5%, respectively, clearly achieving a priority at this commercial operation for generating a high percentage of live kids (97.5%) of marked value being produced. The use of induced parturition allowed this large dairy operation to designate two 9-h time blocks in which to concentrate parturition times within the herd. This facilitated strategic scheduling to optimize availability of staff, in order to assist with parturition, separate kids from the dam at birth, and ensure adequate and prompt feeding of colostrum. Predicting the time of kidding in this way can serve as an effective management tool, especially to help reduce kid mortality and prevent disease spread by restricting suckling of colostrum.

Johne's disease: a successful eradication programme in a dairy goat herd.

This retrospective analysis and report describes the successful eradication and posteradication surveillance programme for Johne's disease (Mycobacterium avium subspecies paratuberculosis (MAP)) in a closed herd of dairy goats. In 1994, MAP's presence in the goat herd was first suspected through individual annual serological screening and then subsequently confirmed through faecal culture and histopathology in 1997 when implementation of a more aggressive programme of testing and eradication of the diseased animals began. This programme included frequent serological screening of all adult goats using ELISA and agar gel immunodiffusion assays. Faecal cultures for bacteria were performed on suspect or positive animals and for all goats found dead or euthanased, and tissues were submitted for histopathology and acid-fast staining. Additional disease eradication measures included maintaining a closed herd and minimising faecal-oral transmission of MAP. Following a more aggressive testing regimen and euthanasia of goats with positive faecal culture, the herd was first considered free of MAP in 2003 and has remained free to the present day.

Differences in licensee, police and public opinions regarding interventions to reduce alcohol-related harm associated with licensed premises.

OBJECTIVES: To determine the level of support by licensees, police and the general public for interventions to reduce alcohol-related harm associated with licensed premises and to identify differences between the three groups. METHODS: Participants were 108 licensees of premises licensed to sell alcohol; 132 police officers; 200 members of the public. Questionnaires were administered either through work settings or by mail. Respondents' levels of agreement with interventions to reduce alcohol-related harm associated with licensed premises: responsible service of alcohol; security and crowd control; policing; patron transport; and linking of alcohol-related harm to licensed premises and communication. RESULTS: Police and members of the public were significantly more likely than licensees to agree with strategies under licensee control, such as subsidising patron transport and training staff to deal with intoxicated patrons. Police were more likely than licensees and members of the public to agree with strategies requiring community action and changes to liquor licensing laws. Licensees had significantly lower levels of agreement than the other groups about licensees' responsibility to reduce alcohol-related harm as a consequence of drinking at their premises. CONCLUSIONS: While there was good agreement between police officers and members of the public about strategies for reducing alcohol-related harm at licensed premises, licensees held divergent views about strategies within their control. Licensees were less likely than police and members of the public to agree they were responsible for reducing alcohol-related harm resulting from drinking at their premises.

Co-ordinated expression of amino acid metabolism in response to N and S deficiency during wheat grain filling.

Increasing demands for productivity together with environmental concerns about fertilizer use dictate that the future sustainability of agricultural systems will depend on improving fertilizer use efficiency. Characterization of the biological processes responsible for efficient fertilizer use will provide tools for crop improvement under reduced inputs. Transcriptomic and metabolomic approaches were used to study the impact of nitrogen (N) and sulphur (S) deficiency on N and S remobilization from senescing canopy tissues during grain filling in winter wheat (Triticum aestivum). Canopy tissue N was remobilized effectively to the grain after anthesis. S was less readily remobilized. Nuclear magnetic resonance (NMR) metabolite profiling revealed significant effects of suboptimal N or S supply in leaves but not in developing grain. Analysis of amino acid pools in the grain and leaves revealed a strategy whereby amino acid biosynthesis switches to the production of glutamine during grain filling. Glutamine accumulated in the first 7 d of grain development, prior to conversion to other amino acids and protein in the subsequent 21 d. Transcriptome analysis indicated that a down-regulation of the terminal steps in many amino acid biosynthetic pathways occurs to control pools of amino acids during leaf senescence. Grain N and S contents increased in parallel after anthesis and were not significantly affected by S deficiency, despite a suboptimal N:S ratio at final harvest. N deficiency resulted in much slower accumulation of grain N and S and lower final concentrations, indicating that vegetative tissue N has a greater control of the timing and extent of nutrient remobilization than S.

The multiple baseline design for evaluating population-based research.

There is a need for pragmatic and rigorous research designs to evaluate the effectiveness of population-based health interventions. The randomized controlled trial (RCT) has limitations in its practicality, ethical appropriateness, and cost when evaluating population-based interventions. Like RCTs, the multiple baseline design can demonstrate that a change in behavior has occurred, the change is a result of the intervention, and the change is significant. Especially important practical advantages over the RCT are that this design requires fewer population groups and communities may act as their own controls. Advantages and methodologic limitations of the multiple baseline design are discussed, and where feasible, strategies to minimize the impact of its limitations are suggested. Recommendations for future research are included.

The POLARIS peptide of Arabidopsis regulates auxin transport and root growth via effects on ethylene signaling.

The rate and plane of cell division and anisotropic cell growth are critical for plant development and are regulated by diverse mechanisms involving several hormone signaling pathways. Little is known about peptide signaling in plant growth; however, Arabidopsis thaliana POLARIS (PLS), encoding a 36-amino acid peptide, is required for correct root growth and vascular development. Mutational analysis implicates a role for the peptide in hormone responses, but the basis of PLS action is obscure. Using the Arabidopsis root as a model to study PLS action in plant development, we discovered a link between PLS, ethylene signaling, auxin homeostasis, and microtubule cytoskeleton dynamics. Mutation of PLS results in an enhanced ethylene-response phenotype, defective auxin transport and homeostasis, and altered microtubule sensitivity to inhibitors. These defects, along with the short-root phenotype, are suppressed by genetic and pharmacological inhibition of ethylene action. PLS expression is repressed by ethylene and induced by auxin. Our results suggest a mechanism whereby PLS negatively regulates ethylene responses to modulate cell division and expansion via downstream effects on microtubule cytoskeleton dynamics and auxin signaling, thereby influencing root growth and lateral root development. This mechanism involves a regulatory loop of auxin-ethylene interactions.

Cloning and functional characterisation of a cis-muuroladiene synthase from black peppermint (Menthaxpiperita) and direct evidence for a chemotype unable to synthesise farnesene.

Using oligonucleotide primers designed to the known gene sequence of an (E)-beta-farnesene (EbetaF) synthase, two cDNA sequences (MxpSS1 and MxpSS2) were cloned from a black peppermint (Menthaxpiperita) plant. MxpSS1 encoded a protein with 96% overall amino acid sequence identity with the EbetaF synthase. Recombinant MxpSS1 produced in Escherichia coli, after removal of an N-terminal thioredoxin fusion, had a K(m) for FPP of 1.91+/-0.1 microM and k(cat) of 0.18 s(-1), and converted farnesyl diphosphate (FPP) into four products, the major two being cis-muurola-3,5-diene (45%) and cis-muurola-4(14),5-diene (43%). This is the first cis-muuroladiene synthase, to be characterised. MxpSS2 encoded a protein with only two amino acids differing from EbetaF synthase. Recombinant MxpSS2 protein showed no activity towards FPP. One of the two mutations, at position 531 (leucine in MxpSS2 and serine in EbetaF synthase) was shown, by structural modelling to occur in the J-K loop, an element of the structure of sesquiterpene synthases known to be important in the reaction mechanism. Reintroduction of the serine at position 531 into MxpSS2 by site-directed mutagenesis restored EbetaF synthase activity (K(m) for FPP 0.98+/-0.12 microM, k(cat) 0.1 s(-1)), demonstrating the crucial role of this residue in the enzyme activity. Analysis, by GC-MS, of the sesquiterpene profile of the plant used for the cloning, revealed that EbetaF was not present, confirming that this particular mint chemotype had lost EbetaF synthase activity due to the observed mutations.

Effect of serum concentration, method of trypsinization and fusion/activation utilizing transfected fetal cells to generate transgenic dairy goats by somatic cell nuclear transfer.

This work was performed within a commercial nuclear transfer program to investigate different methods for synchronizing donor cell cycle stage, for harvesting donor cells, and for fusion and activation of reconstructed caprine embryos. Primary fetal cells isolated from day 35 to day 40 fetuses were co-transfected with DNA fragments encoding both the heavy and light immunoglobulin chains of three different monoclonal antibodies and neomycin resistance. Four neomycin resistant cell lines for each antibody were selected, expanded, and aliquots were both cryopreserved for later use as karyoplast donors or used for further genetic characterization. Transfected fetal cells were cultured in 0.5% FBS to synchronize G0/G1 cell cycle stage cells, then re-fed with 10% FBS prior to use to allow donor cells to re-enter the cell cycle. Alternatively, transfected fetal cells were grown to confluence in 10% FBS to induce contact inhibition to synchronize G0/G1 cell cycle stage cells. Adherent monolayers of transfected fetal donor cells were harvested by either partial or complete trypsinization. Donor cells were simultaneously fused and activated with enulceated in vivo produced ovulated oocytes from superovulated does. Half of the fused couplets received an additional electrical activation pulse and non-fused couplets were re-fused. Four live offspring were produced from 587 embryos generated from cell lines cultured in 0.5% FBS, while one live offspring was produced from 315 embryos generated from cell lines cultured in 10% FBS (0.7% versus 0.3% embryos transferred, respectively, P > 0.05). Five offspring were produced from 633 embryos generated from cell lines harvested by partial trypsinization (0.8% embryos transferred), and no offspring were produced from 269 embryos generated from cell lines harvested by complete trypsinization. Four live offspring were produced from 447 embryos generated from re-fused couplets, and one live offspring was produced from 230 embryos generated from fused couplets that received an additional electrical activation pulse (0.9% versus 0.4% embryos transferred, respectively, P > 0.05). These results suggest that low-serum culture of transfected goat fetal cells and harvest by partial trypsinization may be more efficient methods for generating transgenic goats by somatic cell nuclear transfer. In addition, re-fusion of non-fused couplet or an additional activation step was successful for producing live offspring.

Analysis of detergent-resistant membranes in Arabidopsis. Evidence for plasma membrane lipid rafts.

The trafficking and function of cell surface proteins in eukaryotic cells may require association with detergent-resistant sphingolipid- and sterol-rich membrane domains. The aim of this work was to obtain evidence for lipid domain phenomena in plant membranes. A protocol to prepare Triton X-100 detergent-resistant membranes (DRMs) was developed using Arabidopsis (Arabidopsis thaliana) callus membranes. A comparative proteomics approach using two-dimensional difference gel electrophoresis and liquid chromatography-tandem mass spectrometry revealed that the DRMs were highly enriched in specific proteins. They included eight glycosylphosphatidylinositol-anchored proteins, several plasma membrane (PM) ATPases, multidrug resistance proteins, and proteins of the stomatin/prohibitin/hypersensitive response family, suggesting that the DRMs originated from PM domains. We also identified a plant homolog of flotillin, a major mammalian DRM protein, suggesting a conserved role for this protein in lipid domain phenomena in eukaryotic cells. Lipid analysis by gas chromatography-mass spectrometry showed that the DRMs had a 4-fold higher sterol-to-protein content than the average for Arabidopsis membranes. The DRMs were also 5-fold increased in sphingolipid-to-protein ratio. Our results indicate that the preparation of DRMs can yield a very specific set of membrane proteins and suggest that the PM contains phytosterol and sphingolipid-rich lipid domains with a specialized protein composition. Our results also suggest a conserved role of lipid modification in targeting proteins to both the intracellular and extracellular leaflet of these domains. The proteins associated with these domains provide important new experimental avenues into understanding plant cell polarity and cell surface processes.