Proteinase K-resistant material in ARR/VRQ sheep brain affected with classical scrapie is composed mainly of VRQ prion protein.

Classical scrapie is a prion disease in sheep and goats. In sheep, susceptibility to disease is genetically influenced by single amino acid substitutions. Genetic breeding programs aimed at enrichment of arginine-171 (171R) prion protein (PrP), the so-called ARR allele, in the sheep population have been demonstrated to be effective in reducing the occurrence of classical scrapie in the field. Understanding the molecular basis for this reduced prevalence would serve the assessment of ARR adaptation. The prion formation mechanism and conversion of PrP from the normal form (PrP(C)) to the scrapie-associated form (PrP(Sc)) could play a key role in this process. Therefore, we investigated whether the ARR allele substantially contributes to scrapie prion formation in naturally infected heterozygous 171Q/R animals. Two methods were applied to brain tissue of 171Q/R heterozygous sheep with natural scrapie to determine the relative amount of the 171R PrP fraction in PrP(res), the proteinase K-resistant PrP(Sc) core. An antibody test differentiating between 171Q and 171R PrP fragments showed that PrP(res) was mostly composed of the 171Q allelotype. Furthermore, using a novel tool for prion research, endoproteinase Lys-C-digested PrP(res) yielded substantial amounts of a nonglycosylated and a monoglycosylated PrP fragment comprising codons 114 to 188. Following two-dimensional gel electrophoresis, only marginal amounts (<9%) of 171R PrP(res) were detected. Enhanced 171R(res) proteolytic susceptibility could be excluded. Thus, these data support a nearly zero contribution of 171R PrP in PrP(res) of 171R/Q field scrapie-infected animals. This is suggestive of a poor adaptation of classical scrapie to this resistance allele under these natural conditions.

Use of a new immunoassay to measure PrP Sc levels in scrapie-infected sheep brains reveals PrP genotype-specific differences.

The diagnosis of prion diseases, such as scrapie and BSE, has traditionally relied upon the identification of the disease-associated form of the prion protein, PrP(Sc), based on its resistance to digestion by proteinase K (PK). A more recent development is the conformation-dependent immunoassay (CDI), which distinguishes between PrP Sc and normal PrP (PrP C) based on their differing solubility in guanidine hydrochloride rather than resistance or sensitivity to PK. We have developed a CDI-formatted sandwich immunoassay for the measurement of PrP Sc in sheep brain, which discriminates between clinically affected scrapie cases (natural or experimental) and uninfected controls of the same PrP genotype. Using this method, we have shown for the first time that, in sheep, the PrP genotype has a significant influence on the amount of PrP Sc deposited in the brains of animals experimentally infected with scrapie.

Using rotifer population demographic parameters to assess impacts of the degradation products from trinitrotoluene phytoremediation.

The objective of this study was to evaluate the chronic lethal and sublethal aquatic toxicity effects associated with the phytoremediation of water contaminated with 2,4,6-trinitrotoluene (TNT) by the wetland plant species Myriophyllum aquaticum (parrot feather). Rotifers (Brachionus calyciflorus) feeding on an algal species (Nannochloropsis spp.) were used as the aquatic test organisms. Continuous flow laboratory microcosms were used to quantify effects on rotifer populations from TNT and the primary degradation product aminodinitrotoluene (ADNT) during and after phytoremediation. Rotifer demographic parameters from life tables, including survivorship, fecundity, reproductive values, net reproductive rate, generation time, intrinsic growth rate, and life expectancy, were used as measures of treatment effects. High-performance liquid chromatography analyses were performed to determine nitroaromatic concentrations. Results from this study have revealed significant differences in rotifer demographic parameters between microcosms with elevated initial TNT concentrations. Significant differences in demographic parameters also existed between the microcosms that did and did not receive phytoremediation treatment and the control microcosms. Study results have indicated that TNT phytoremediation via artificial wetlands not only may clean up hazardous waste at munitions sites but also may encourage the growth of aquatic populations such as rotifers.

Shearing during pregnancy--review of a policy to increase birthweight and survival of lambs in New Zealand pastoral farming systems.

Over the past decade, lambing percentages have risen in conjunction with a rise in the percentage of multiple lambs born. Multiple-born lambs are smaller than their singleton counterparts and are particularly susceptible to starvation-exposure. Any technique that can increase the birthweight or thermoregulatory capability, or both, of otherwise lightweight lambs has the potential to substantially increase survival of multiple-born lambs. In the United Kingdom under housed conditions, shearing during pregnancy has been shown to increase both the birthweight and thermoregulatory capability of newborn lambs. However, shearing during pregnancy under pastoral conditions has failed to consistently affect the newborn lamb's thermoregulatory capability. In contrast, under New Zealand's pastoral conditions shearing during pregnancy has been found to increase birthweight, but results have been inconsistent in both magnitude and birth-rank specificity. Increase in feed intake by the dam, types of shearing comb used and changes in gestation length do not explain the variation observed. When studies involving shearing during pregnancy are collated it becomes apparent that there are two criteria that must be met to achieve a birthweight response. Firstly, the dam must have the potential to respond and secondly, the dam must have the means to respond. Any increase in lamb survival through shearing during pregnancy would be predominantly through an increase in birthweight of otherwise lightweight lambs. However, in the few studies conducted to date, an increase in birthweight has not resulted in a statistically significant increase in survival. For an increase in birthweight to have a positive effect on lamb survival, lambs must be otherwise destined to be born within a birthweight range in which survival rate is below optimum (<4.0 kg), and the increase in birthweight observed must move a significant proportion of otherwise lightweight lambs into a higher range of survival rate.

P34(cdc2) kinase is associated with cortical microtubules from higher plant protoplasts.

The cell cycle regulatory enzyme p34(cdc2) kinase is known to be localized to the preprophase band, the spindle and the phragmoplast, but not to interphase cortical microtubules. This was investigated further by mechanically cleaving substrate-attached protoplasts to leave plasma membrane disks bearing microtubules freed of nuclear and cytosolic signal. Antibodies to PSTAIRE and to specific C-terminal peptides of cdc2a, were used in immunofluorescence, protein blotting and immunogold electron microscopy to demonstrate that antigen is located on the cortical microtubules of carrot, tobacco BY-2 and Arabidopsis cells.

"Caged cytoskeletons": a rapid method for the isolation of microtubule-associated proteins from synchronized plant suspension cells.

In the cytoskeleton method for isolating microtubule-associated proteins MAP65, DcKRP120-1 and DcKRP120-2, carrot cells are first converted to protoplasts but this method cannot be used to isolate mitotic MAPs as mitotic synchrony is eroded during lengthy cellulase treatment. Anti-microtubule cycle blocks would also be unsuitable. We report here a method for overcoming these problems. Cellulase degradation of tobacco BY-2 cells for only several minutes allows extraction of detergent-soluble proteins, leaving synchronized "caged cytoskeletons" for depolymerization and enabling affinity purification of MAPs on neurotubules. This rapid and simple method should be of general utility: it can be bulked up, avoids anti-microtubule blocks, and is applicable to other cell suspensions. The effectiveness of the caged cytoskeleton method is demonstrated by comparing known MAPs (the 65 kDa structural MAPs and the kinesin-related protein, TKRP125) in synchronized cells taken at the mitotic peak with those in unsynchronized cells.

Immunohistochemical detection of myogenic cells in muscles of fetal and neonatal lambs.

Previous studies have implied that myonuclei accumulation in a muscle is more important than myofibre number in the determination of muscle size in fetal/neonatal lambs. However, due to the lack of a reliable marker, the role of myogenic precursor nuclei (satellite cells) in myofibre hypertrophy in late fetal and postnatal life is not well understood. In this study, MyoD was shown to be a useful marker for actively proliferating satellite cells in both fetal and neonatal lambs. MyoD was used to determine whether there were differences in the number of actively proliferating satellite cells between single and twin fetuses/neonates, which may explain at least some of the difference in myofibre size observed near birth. Eighteen single-bearing and 9 twin-bearing Coopworth ewes were randomly assigned to one of three slaughter groups (100, 120 and 140 days of gestation). The remaining ewes were kept on pasture until 20 days postpartum at which time 4 single and 4 twin lambs were sacrificed. Twin fetuses/neonates had lower body weights and muscle weights compared to singles. Lower muscle weights in the twins were associated with smaller myofibre cross-sectional areas and lower total nuclei numbers and myogenic precursor cell numbers per muscle in selected hind-limb muscles. These results indicate that myofibre hypertrophy in late gestation and early postnatal life is related to myogenic precursor cell number which may have important implications for growth potential of the growth-restricted fetus.

The effect of season and monensin sodium on the digestive characteristics of autumn and spring pasture fed to sheep.

The effects of season of growth and monensin treatment on ruminal digestion of fresh-cut autumn and spring pasture were measured in a single group of ruminally fistulated castrated male sheep, housed indoors in metabolism crates. Responses were assessed in terms of ruminal volatile fatty acid molar proportions, ammonia concentration, pH, apparent digestibility of the pasture, and nitrogen balance of the animals. Blood plasma concentrations of insulin, glucose, beta-hydroxybutyrate, urea, and NEFA were also evaluated. Autumn pasture contained significantly lower proportions of water-soluble carbohydrate (P < 0.05), cellulose (P < 0.05), and lignin (P < 0.05) and increased pectin (P < 0.05), hemicellulose (P < 0.05), and crude protein (P < 0.10) concentrations when compared with spring pasture. Voluntary DMI by sheep of autumn pasture was lower (P < 0.01) than that of spring pasture and was significantly (P < 0.05) reduced by monensin treatment. Monensin treatment significantly decreased the ruminal molar proportions of acetic acid (P < 0.10) and butyric acid (P < 0.001) and increased the molar proportions of propionic acid (P < 0.001) and minor VFA (P < 0.01). Nitrogen retention of the sheep was significantly (P < 0.05) reduced by monensin treatment. Plasma glucose levels were increased (P < 0.10) by monensin treatment during the fourth 5-d collection period in both seasons. Chemical analysis suggested that the composition of autumn pasture was different from that of spring pasture and that this was manifested in vivo by increased DMI and digestibility of spring vs autumn pasture. Ruminal fermentation of autumn pasture also had an increased acetate-to-propionate ratio compared with spring pasture. Monensin treatment acted consistently across seasons by increasing the proportion of propionate and decreasing the proportion of acetate in ruminal fluid.

Metabolic responses to mid-pregnancy shearing that are associated with a selective increase in the birth weight of twin lambs.

Previous studies have shown that shearing pregnant ewes at mid- or late-pregnancy is associated with an increase in lamb birth weight. The present study was designed to investigate metabolic responses that may underlie this response. Single- and twin-bearing ewes were either unshorn or shorn at mid-pregnancy (Day 69 of pregnancy; P69), and insulin, glucose and epinephrine challenges were conducted on P109-111 and P132-134. Shearing increased the birth weight of twin lambs by over 1 kg (P < 0.001) without having any effect on singleton birth weight. This response was associated with a 10-20% reduction in the insulin response to a glucose challenge (P < 0.05) without a change in glucose clearance following either glucose or insulin challenges. The lipolytic response to epinephrine challenge increased as pregnancy progressed, but was not associated with the increased birth weight of twin lambs born to shorn ewes. By late pregnancy, a 25% reduction in maternal IGF-I concentration and a two- to threefold increase in maternal IGFBP-1 concentration (P < 0. 05) associated with shearing were observed. The increase in lamb birth weight associated with mid-pregnancy shearing may have been associated with an increase in the non-insulin dependent uptake of glucose by the placental-fetal unit.

Muscle growth, cell number, type and morphometry in single and twin fetal lambs during mid to late gestation.

Muscle growth, myofibre number, type and morphometry were studied in large hindlimb muscles of single and twin fetal lambs during mid to late gestation. Placental insufficiency, evident by lower total placentome weight and number per fetus, resulted in reduced fetal weights from 100 to 140 days gestation in twins compared with singletons (at 140 days: 5016 +/- 108 g v. 5750 +/- 246 g, respectively; P<0.05). However, competition between littermates did not consistently reduce muscle mass (15-22%) until 140 days gestation. Apparent myofibre number increased with age, indicating that the full complement of myofibres in some large hindlimb muscles may be achieved during early postnatal life. Litter size did not impact on apparent myofibre number in the semitendinosus, plantaris or gastrocnemius muscles. However, a transient effect on myofibre number in the adductor femoris muscle was observed from 80-120 days gestation. The phenotypic maturation of myofibres was unaffected by increasing litter size. Smaller muscle mass in twins was associated with smaller myofibre cross-sectional area in the semitendinosus, adductor femoris and gastrocnemius muscles at 140 days gestation. A similar trend was observed for the plantaris muscle. These results indicate that while competition between littermates for nutrients in late gestation can impact on both fetal and muscle mass, the fetus has the capacity to buffer against the effects of restricted nutrient supply on myofibre hyperplasia and phenotypic maturation, but myofibre hypertrophy is compromised.

Morphometric analysis of myofibre development in the adductor femoris muscle of single and twin fetal lambs.

Cellular development of the adductor femoris muscle from twin and single fetuses was studied at 140 days gestation to evaluate the effect of moderate fetal growth retardation on myofibre development. Twin fetuses had lower bodyweights (13%) and disproportionately small adductor femoris muscle weights (22%) compared with single fetuses. Reduced muscle mass was associated with smaller myofibre cross-sectional areas (CSA) and lower DNA content (22%), indicative of fewer myonuclei and retarded myofibre hypertrophy. Myofibre number and the phenotypic maturation of the myofibres were similar between twins and singletons. These results indicate that even modest growth restriction during fetal life can negatively influence myofibre hypertrophy, highlighting the importance of fetal nutrition for muscle growth. Large muscles, such as the adductor femoris, have intrafascicularly terminating myofibres, which necessitates accurate sampling of the muscle when investigating possible perturbations in morphological characteristics (e.g. between singletons and twins). The second objective of the present study was to investigate the impact of the sampling site on the morphological parameters of the adductor femoris muscle. The apparent total myofibre number decreased from the proximal to the distal region of the adductor femoris muscle. The apparent number of slow-twitch fibres also decreased from the proximal to the medial region, but was not different between the medial and distal regions of the muscle. Similarly, myofibre CSA differed between the medial and distal regions. These results indicate that, particularly with large muscles, such as the adductor femoris, which has intrafascicularly terminating myofibres, single site sampling for the determination of morphological fibre characteristics may generate misleading results and that careful selection of the sampling area may be necessary.

Development of Weather-Dependent Flow Requirements for River Temperature Control.

/ Streamflow influences biological processes, habitat, and ecological integrity of streams in a number of vital ways. The establishment of weather-dependent minimum flows is essential to the protection of the aquatic environment and wildlife habitat from the adverse impacts of high water temperatures and to the effective utilization of the assimilative capacity of instream flows. In this study, weather-dependent flow requirements for summer river temperature control are derived from quantitative temperature-flow relationships. Correlation and regression of historical data and an analytical solution to the basic heat balance equation are employed to quantify the impacts of stream flow on river temperatures. Five-year continuous field measurements from the Platte River, Nebraska, USA, are used to illustrate the practical application in water-quality management. The methods and results demonstrate the feasibility of temperature control through streamflow management to meet water temperature standards and protect the aquatic biota by setting the appropriate weather-related minimum river discharges. The flow requirements are evaluated by a comparison with a critical discharge. Information provided in this paper will assist in planning streamflow regulation, design of river and reservoir operations, and application of water-quality criteria in environmental management.KEY WORDS: River discharges; Flow management; Habitat; Water quality; Weather; Wildlifehttp://link.springer-ny.com/link/service/journals/00267/bibs/24n4p529.html

Cloning and expression of a cDNA encoding a Vorticella convallaria spasmin: an EF-hand calcium-binding protein.

The stalked, ciliated protozoan Vorticella convallaria possesses a highly contractile cytoskeleton consisting of spasmonemes and myonemes. The major component of these contractile organelles is the calcium-binding protein(s) called spasmin. Cloning and characterization of spasmin would help elucidate this contractile system. Therefore, enriched spasmoneme protein preparations from these contractile stalks were used to produce a monoclonal antibody to spasmin. A monoclonal antibody, 1F5, was obtained that immunolocalized specifically to the spasmonemes and the myonemes and recognized a 20-kD calcium-binding protein in spasmoneme protein preparations. A putative spasmin cDNA was obtained from a V. convallaria cDNA library and the derived amino acid sequence of this cDNA revealed an acidic, 20-kD protein with calcium-binding helix-loop-helix domains. The physical properties of the putative spasmin were assessed by characterization of a recombinantly-produced spasmin protein. The recombinant spasmin protein was shown to bind calcium using calcium gel-shift assays and was recognized by the anti-spasmin antibody. Therefore, a V. convallaria spasmin was cloned and shown to be a member of the EF-hand superfamily of calcium-binding proteins.

Placental development and fetal growth in growth hormone-treated ewes.

The effects of recombinant bovine growth hormone (bGH) treatment of pregnant ewes on maternal metabolism, placental development and fetal growth were examined in two studies. In a preliminary study (experiment one), single-bearing ewes were treated by twice-daily subcutaneous injection for 7 days with bGH (n = 8) at a dose of 0.15 mg/kg LW/day or with saline (n = 8) between days 101 and 107 of gestation inclusive. In experiment two, single- and twin-bearing ewes were treated for 14 days with bGH (0.15 mg/kg L W/day) (n = 10) or saline (n = 10) between days 70 and 83 or days 98 and 111 of gestation inclusive. Ewes were killed on the day following termination of bGH treatment and fetal and placental measurements recorded. Maternal plasma concentrations of GH, IGF-I and insulin were higher (P < 0.001) in bGH-treated ewes relative to saline-treated ewes in both experiments. Consistent across experiments was an increase (P < 0.05) in the weight of the myoendometrium in bGH-treated ewes. Treatment with bGH also increased the total weight of the gravid uterus (P < 0.05) in both experiments. Weights of the uterine fluids were increased by bGH in experiment one (P < 0.05), but an effect of the same magnitude could not be repeated in experiment two. In experiment one, there was a tendency towards increased mean fetal body weights after growth hormone treatment, although the effect was non-significant. In experiment two, treatment with bGH was associated with significantly (P < 0.05) higher fetal weights, but only at the later stage of gestation (day 112). This effect was additive with that of fetal rank. Exogenous bGH treatment had little discernible effect on measures of placental size. It is concluded that administration of exogenous bGH to pregnant ewes can stimulate fetal growth, but only after about day 100 of gestation. This response seems most likely to reflect changes in maternal nutrient partitioning or placental function, rather than placental size. These studies suggest a role for growth hormone of maternal or placental origin in the regulation of fetal growth.

Model framework and principles of emergency management.

If not handled quickly and effectively, animal health emergencies can create significant problems for governments and industry. Placing strong emphasis on preventing a disease incursion in the first instance through the judicious use of quarantine and inspection measures will continue to be the major preventive strategy. However, the discovery of new and emerging diseases, some of which may already be present in a country or region, necessitates a rigorous emergency outbreak response mechanism premised on thorough preparation for an emergency. A generic approach to emergency management is the contemporary answer to preparing for, and dealing with, unpredictable events. In essence, generic emergency management plans encompass a set of core principles and key issues which are relevant to a particular industry or commodity. These principles and issues are then reflected in a model emergency management plan which guides participants through the various stages of preparing for, and responding to, an emergency situation and which forms the basis of more detailed farm/enterprise manuals and individual job cards. The authors illustrate the integrated nature of the model emergency management plan and the interrelationship with the core principles and key issues of emergency management planning.

Leadership 99. Interview by Terese Hudson.

Cost control, customer service and collaboration among health care sectors rank as top concerns with panelists in H&HN's annual Leadership Report. Efforts to improve community health are a priority, too, but are often frustrated by financial and other constraints. The 16 panelists represent managed care, physicians, and hospitals and health systems.